技术领域technical field
本发明属于生物医药领域；更具体地，本发明涉及抑制乳腺癌骨转移的方法和制剂。The invention belongs to the field of biomedicine; more specifically, the invention relates to a method and a preparation for inhibiting bone metastasis of breast cancer.
背景技术Background technique
乳腺癌是对女性健康构成很大威胁的一种恶性疾病。而乳腺癌向重要器官如骨、肺、脑、肝等的转移是乳腺癌致死的主要原因。临床上的数据显示大约70%的晚期乳腺癌病人会发生骨转移。乳腺癌发生骨转移可能会给病人带来疼痛、骨折和高钙血等症状，这使得病人生活质量非常之低。迄今为止，FDA只审批了两种药物-zoledronic acid和Denosumab用于骨转移的治疗，前者对患者生存率影响不大，后者价格高昂，因此开发出更有效、低廉的药物迫在眉睫。而这首先需要本发明人对骨转移的机制有更深更全面的了解。Breast cancer is a malignant disease that poses a great threat to women's health. The metastasis of breast cancer to important organs such as bone, lung, brain and liver is the main cause of breast cancer death. Clinical data show that about 70% of advanced breast cancer patients will develop bone metastasis. Bone metastasis of breast cancer may bring symptoms such as pain, fracture and hypercalcemia to the patient, which makes the quality of life of the patient very low. So far, the FDA has only approved two drugs - zoledronic acid and Denosumab for the treatment of bone metastases. The former has little effect on the survival rate of patients, while the latter is expensive. Therefore, it is imminent to develop more effective and inexpensive drugs. And this first requires the inventors to have a deeper and more comprehensive understanding of the mechanism of bone metastasis.
在正常生理状况下，成人的骨骼处于骨基质解离和骨基质重构的平衡状态。而这种平衡中关键的调控因子是破骨细胞和成骨细胞。当破骨细胞诱发的溶骨作用强于成骨细胞诱发的骨重构作用时，总的效应就表现为溶骨，反之则相反。临床上大多数晚期发生骨转移的乳腺癌病人呈现溶骨性的症状，小部分病人呈现成骨性的症状。Under normal physiological conditions, the adult skeleton is in a state of balance between bone matrix dissociation and bone matrix remodeling. The key regulators in this balance are osteoclasts and osteoblasts. When osteoclast-induced osteolysis is stronger than osteoblast-induced bone remodeling, the overall effect is osteolysis, and vice versa. Clinically, most breast cancer patients with advanced bone metastases present osteolytic symptoms, and a small number of patients present osteoblastic symptoms.
DLC1基因位于人染色体8p21，最初因其经常在肝细胞癌中被发现丢失而被命名为Deleted in Liver Cancer-1。DLC1是一种Rho家族GTPase蛋白(RhoGTPase)的GTP水解酶活性活化蛋白(RhoGTPase Activating Protein，RhoGAPs)。RhoGAPs通过与鸟嘌呤核苷酸交换因子(Guanosine NucleotideExchange Factors，GEFs)和鸟苷酸解离抑制因子(Guanine nucleotide dissociationInhibitor，GDI)配合，调接RhoGTPase的酶解活性。RhoGTPase是Ras超家族的成员之一，当RhoGTPase处于失活状态时，GDI与RhoGTPase—GDP形成稳定的复合物存在于胞质中，它在GEFs作用下释放GDP之后，结合GTP而被激活，继而作用于下游效应分子。RhoGAPs的生化功能则是激活RhoGTPase固有的GTP水解酶活性，水解GTP成为GDP，使GTPase复归于失活的状态。因此DLC1是Rho通路重要的负调控因子。The DLC1 gene is located on human chromosome 8p21 and was originally named Deleted in Liver Cancer-1 because it is often found to be lost in hepatocellular carcinoma. DLC1 is a GTP hydrolase activity activating protein (RhoGTPase Activating Protein, RhoGAPs) of a Rho family GTPase protein (RhoGTPase). RhoGAPs regulates the enzymatic activity of RhoGTPase by cooperating with Guanosine Nucleotide Exchange Factors (GEFs) and Guanine nucleotide dissociation Inhibitor (GDI). RhoGTPase is one of the members of the Ras superfamily. When RhoGTPase is in an inactive state, GDI and RhoGTPase-GDP form a stable complex in the cytoplasm. After releasing GDP under the action of GEFs, it binds to GTP and is activated. Act on downstream effector molecules. The biochemical function of RhoGAPs is to activate the inherent GTP hydrolase activity of RhoGTPase, hydrolyze GTP into GDP, and return GTPase to an inactive state. Therefore, DLC1 is an important negative regulator of the Rho pathway.
DLC1基因在NCBI记录的转录本共有四个isoform，isoform1长度是7479bp，编码1528个氨基酸；isoform2长度是6044bp，编码1091个氨基酸；isoform3长度是2484bp，编码463个氨基酸；isoform4长度是5786bp，编码1017个氨基酸。其中isoform2是DLC1常见的转录本。其翻译的蛋白质结构中除了所有RhoGAPs成员共有的一个保守RhoGAP催化序列外，其氨基端存在一个保守的SAM(Sterile Alpha Motif)结构域和FAT(Focal Adhesion Targeting)序列，其羧基端存在一个START(Steroidogenic Acute Regulatory(StAR)-relatedLipid Transfer)结构域。The transcripts of the DLC1 gene recorded in NCBI have four isoforms. The length of isoform1 is 7479bp, encoding 1528 amino acids; the length of isoform2 is 6044bp, encoding 1091 amino acids; the length of isoform3 is 2484bp, encoding 463 amino acids; amino acids. where isoform2 is the common transcript of DLC1. In the protein structure of its translation, in addition to a conserved RhoGAP catalytic sequence shared by all RhoGAPs members, there is a conserved SAM (Sterile Alpha Motif) domain and FAT (Focal Adhesion Targeting) sequence at its amino terminal, and a START ( Steroidogenic Acute Regulatory (StAR)-related Lipid Transfer) domain.
DLC1不但被发现经常在肝癌细胞中被丢失，后来发现它在膀胱、肺、乳腺、胃、肠、脑、前列腺、部位的癌症组织中也被下调，下调的机制或是基因的丢失或是启动子的甲基化或是两者兼有之。DLC1的缺失在肝细胞癌小鼠模型中可促进肿瘤生长，上调DLC1在食道癌、肝癌、肺癌或乳腺癌细胞系中的表达可抑制肿瘤的形成、癌细胞的增殖和减少肿瘤细胞在软琼脂形成的克隆数。DLC1抑制肝癌细胞增殖可能与它诱导癌细胞的凋亡有关。Not only was DLC1 found to be frequently lost in liver cancer cells, but it was also found to be down-regulated in cancer tissues of the bladder, lung, breast, stomach, intestine, brain, and prostate. sub-methylation or both. Loss of DLC1 promotes tumor growth in a mouse model of hepatocellular carcinoma, and upregulation of DLC1 expression in esophageal, liver, lung or breast cancer cell lines inhibits tumor formation, cancer cell proliferation and reduces tumor cell growth in soft agar The number of clones formed. DLC1 inhibits the proliferation of liver cancer cells may be related to its induction of apoptosis of cancer cells.
DLC1不但与癌细胞的增殖相关，一些报导还揭示了DLC1抑制癌细胞迁移和侵袭的能力。DLC1在食道癌、乳腺癌、肝癌细胞和淋巴瘤细胞系中显著抑制细胞的迁移能力。在皮肤鳞癌、前列腺癌、肺癌中，DLC1抑制了细胞的侵袭能力。但是关于DLC1是否真正调控癌细胞的远端转移，相关的研究较少，癌细胞的侵袭和远端转移实际上差别很大。而且，骨骼是癌细胞转移最为频繁的器官，目前大量研究表明，癌细胞向骨的转移能力，多决定于癌细胞和转移微环境相互作用的能力，尤其是对破骨/成骨细胞成熟分化的影响，而与癌细胞自身的迁移侵袭能力关系不大。DLC1 is not only related to the proliferation of cancer cells, some reports also reveal the ability of DLC1 to inhibit the migration and invasion of cancer cells. DLC1 significantly inhibited cell migration in esophageal cancer, breast cancer, liver cancer cell lines and lymphoma cell lines. In squamous cell carcinoma of the skin, prostate cancer, and lung cancer, DLC1 inhibits the invasion ability of cells. However, there are few related studies on whether DLC1 really regulates the distant metastasis of cancer cells, and the invasion and distant metastasis of cancer cells are actually very different. Moreover, bone is the organ where cancer cells metastasize most frequently. At present, a large number of studies have shown that the ability of cancer cells to metastasize to bone is mostly determined by the ability of the interaction between cancer cells and the metastatic microenvironment, especially for the maturation and differentiation of osteoclasts/osteoblasts. However, it has little to do with the migration and invasion ability of cancer cells themselves.
关于DLC1在肿瘤发生发展中行使功能的机制上主要有两种解释。其一，通过依赖于GAP domain的方式；其二，通过不依赖于GAP domain的方式。依赖于GAP domain的方式主要通过失活Rho通路来抑制Rho下游的效应分子来实现。比如在前列腺癌细胞中，DLC1通过抑制RhoA和RhoC来上调E-cadherin的表达，E-cadherin的上调则抑制了细胞的侵袭能力。在肝癌及其他癌细胞中DLC1也被证实是通过抑制Rho通路来发挥其抑癌基因的功能，尽管在不同的模型中Rho通路下游包含不同的效应分子如E-cadherin、VEGF、osteopontin、MMP-9。There are two main explanations for the mechanism of DLC1 function in tumorigenesis and development. First, by relying on the GAP domain; second, by not relying on the GAP domain. The GAP domain-dependent approach is mainly achieved by inactivating the Rho pathway to inhibit the effector molecules downstream of Rho. For example, in prostate cancer cells, DLC1 up-regulates the expression of E-cadherin by inhibiting RhoA and RhoC, and the up-regulation of E-cadherin inhibits the invasion ability of cells. In liver cancer and other cancer cells, DLC1 has also been confirmed to exert its function as a tumor suppressor gene by inhibiting the Rho pathway, although in different models, the downstream of the Rho pathway contains different effector molecules such as E-cadherin, VEGF, osteopontin, MMP- 9.
不依赖于GAP domain的方式则涉及SAM domain、处于SAM domain和GAP domain之间的motif以及START domain。此外，DLC1能通过处于SAMdomain和GAP domain之间的七个氨基酸序列(aa348–354)与Annexin2竞争结合S100A10促使其暴露泛素化位点而被降解的途径抑制细胞的侵袭能力。而处于SAM domain和GAP domain之间的另一段序列(aa448–500)被发现能与Talin和FAK结合，它们的结合可能介导了DLC1与粘着斑蛋白的共定位，继而影响细胞的迁移能力。DLC1还能由START domain介导它与caveolin-1结合。它们的结合对DLC1的GAP活性没有显著影响，但是却影响了肺癌细胞的迁移能力和肿瘤形成能力。The way that does not depend on the GAP domain involves the SAM domain, the motif between the SAM domain and the GAP domain, and the START domain. In addition, DLC1 can inhibit cell invasion through the pathway of seven amino acid sequences (aa348–354) between SAMdomain and GAP domain that compete with Annexin2 to bind to S100A10 to expose the ubiquitination site and be degraded. Another sequence (aa448–500) between the SAM domain and the GAP domain was found to bind to Talin and FAK, and their combination may mediate the co-localization of DLC1 and vinculin, thereby affecting the migration ability of cells. DLC1 can also bind to caveolin-1 mediated by START domain. Their combination had no significant effect on the GAP activity of DLC1, but affected the migration ability and tumor formation ability of lung cancer cells.
但是，鉴于不同组织器官形成的肿瘤具有不同的特点，无法预期以上文献报导的肿瘤类型外，还对于哪些类型的肿瘤具有抑制功能，因此本领域技术人员尚需进一步的研究。骨是乳腺癌最常见的转移位点，但乳腺癌骨转移的机制尚未完全阐明。However, in view of the different characteristics of tumors formed in different tissues and organs, it is impossible to predict which types of tumors have the inhibitory function besides the tumor types reported in the above literature, so those skilled in the art still need further research. Bone is the most common metastatic site of breast cancer, but the mechanism of breast cancer bone metastasis has not been fully elucidated.
发明内容Contents of the invention
本发明的目的在于提供抑制乳腺癌骨转移的方法和制剂。The object of the present invention is to provide a method and preparation for inhibiting bone metastasis of breast cancer.
在本发明的第一方面，提供一种DLC1蛋白或其编码基因或其上调剂的用途，用于制备抑制乳腺癌骨转移的组合物(如药物)。In the first aspect of the present invention, a use of the DLC1 protein or its coding gene or its up-regulator is provided for preparing a composition (such as a drug) for inhibiting bone metastasis of breast cancer.
在一个优选例中，所述的DLC1蛋白通过抑制Rho活性而抑制破骨细胞成熟和抑制PTHLH表达上调，从而抑制乳腺癌骨转移。In a preferred example, the DLC1 protein inhibits the maturation of osteoclasts and the up-regulation of PTHLH expression by inhibiting Rho activity, thereby inhibiting bone metastasis of breast cancer.
在另一优选例中，所述的组合物用于：In another preference, the composition is used for:
抑制Rho活性；Inhibit Rho activity;
下调Smad3中第204和208位上丝氨酸的磷酸化水平；Down-regulate the phosphorylation level of serine 204 and 208 in Smad3;
抑制PTHLH表达；Inhibit PTHLH expression;
抑制破骨细胞成熟。Inhibits osteoclast maturation.
在另一优选例中，所述的DLC1蛋白的上调剂包括：DLC1蛋白的功能片段；重组表达DLC1蛋白或其功能片段的质粒。In another preferred example, the DLC1 protein up-regulator includes: a functional fragment of the DLC1 protein; a plasmid for recombinantly expressing the DLC1 protein or a functional fragment thereof.
在另一优选例中，所述的重组表达DLC1蛋白或其功能片段的质粒包括：pBabepuro-DLC1。In another preferred example, the plasmid for recombinantly expressing DLC1 protein or its functional fragments includes: pBabepuro-DLC1.
在本发明的另一方面，提供一种DLC1蛋白或其编码基因的用途，用于筛选抑制乳腺癌骨转移的潜在物质。In another aspect of the present invention, a use of DLC1 protein or its coding gene is provided for screening potential substances for inhibiting bone metastasis of breast cancer.
在本发明的另一方面，提供一种筛选抑制乳腺癌骨转移的潜在物质的方法，包括以下步骤：In another aspect of the present invention, there is provided a method for screening potential substances that inhibit bone metastasis of breast cancer, comprising the following steps:
(a)将候选物质与表达DLC1蛋白的体系接触；(a) contacting the candidate substance with a system expressing the DLC1 protein;
(b)检测DLC1蛋白的表达或活性，若所述候选物质在统计学上提高(如提高20%以上；更佳地提高40%以上；更佳地提高60%以上)DLC1蛋白的表达或活性，则表明该候选物质是抑制乳腺癌骨转移的潜在物质。(b) detecting the expression or activity of the DLC1 protein, if the candidate substance is statistically increased (such as increased by more than 20%; more preferably increased by more than 40%; more preferably increased by more than 60%) the expression or activity of the DLC1 protein , indicating that the candidate substance is a potential substance for inhibiting bone metastasis of breast cancer.
在一个优选例中，步骤(a)包括：在测试组中，在表达DLC1蛋白的体系中添加候选物质；步骤(b)中，检测DLC1蛋白的表达或活性，并与对照组比较，其中所述的对照组是不添加所述候选物质的、表达DLC1蛋白的体系；若测试组中DLC1蛋白的表达或活性在统计学上提高，就表明该候选物质是抑制乳腺癌骨转移的潜在物质。In a preferred example, step (a) includes: in the test group, adding candidate substances to the system expressing DLC1 protein; in step (b), detecting the expression or activity of DLC1 protein and comparing it with the control group, wherein the The aforementioned control group is a system expressing DLC1 protein without adding the candidate substance; if the expression or activity of DLC1 protein in the test group is statistically increased, it indicates that the candidate substance is a potential substance for inhibiting bone metastasis of breast cancer.
在另一优选例中，步骤(a)中，所述体系中还表达Rho蛋白；步骤(b)中，检测DLC1蛋白与Rho蛋白的相互作用，与对照组(不添加候选物质的表达DLC1蛋白与Rho蛋白的体系)相比，若测试组候选物质促进两者相互作用且使得Rho蛋白的活性在统计学上更低(低于对照组，如降低20%以上；更佳地降低40%以上；更佳地降低60%以上)，则表明该候选物质是抑制乳腺癌骨转移的潜在物质。In another preferred example, in step (a), Rho protein is also expressed in the system; in step (b), the interaction between DLC1 protein and Rho protein is detected, and the expression DLC1 protein of the control group (without adding candidate substances) Compared with the system of Rho protein), if the candidate substance of the test group promotes the interaction between the two and makes the activity of Rho protein statistically lower (lower than the control group, such as reducing by more than 20%; more preferably reducing by more than 40%) ; more preferably reduced by more than 60%), it shows that the candidate substance is a potential substance for inhibiting bone metastasis of breast cancer.
在另一优选例中，步骤(a)中，所述体系中还表达PTHLH蛋白；步骤(b)中，检测DLC1蛋白对PTHLH RNA或蛋白表达的调控，与对照组(不添加候选物质的表达DLC1蛋白与PTHLH RNA或蛋白的体系)相比，若候选物质促进DLC1对PTHLH RNA或蛋白的下调作用且使得PTHLH RNA或蛋白的表达在统计学上更低(低于对照组，如降低20%以上；更佳地降低40%以上；更佳地降低60%以上)，则表明该候选物质是抑制乳腺癌骨转移的潜在物质。In another preference, in step (a), PTHLH protein is also expressed in the system; in step (b), the regulation of DLC1 protein to PTHLH RNA or protein expression is detected, and the control group (without adding the expression of candidate substance Compared with DLC1 protein and PTHLH RNA or protein system), if the candidate substance promotes the downregulation effect of DLC1 on PTHLH RNA or protein and makes the expression of PTHLH RNA or protein statistically lower (lower than the control group, such as a 20% reduction more preferably reduce more than 40%; more preferably reduce more than 60%), it shows that the candidate substance is a potential substance for inhibiting bone metastasis of breast cancer.
在另一优选例中，步骤(a)中，所述体系中还表达Smad3蛋白；步骤(b)中，检测DLC1蛋白对于SMAD3蛋白的磷酸化水平的影响，与对照组(不添加候选物质的表达DLC1蛋白与Smad3蛋白的体系)相比，若候选物质使得Smad3蛋白第204和208位丝氨酸的磷酸化水平在统计学上更低(低于对照组，如降低20%以上；更佳地降低40%以上；更佳地降低60%以上)，则表明该候选物质是抑制乳腺癌骨转移的潜在物质。In another preference, in step (a), Smad3 protein is also expressed in the system; in step (b), the influence of DLC1 protein on the phosphorylation level of SMAD3 protein is detected, compared with that of the control group (no addition of candidate substance Compared with the system of expressing DLC1 protein and Smad3 protein), if the candidate substance makes the phosphorylation level of serine 204 and 208 of Smad3 protein statistically lower (lower than the control group, such as reducing more than 20%; better reducing more than 40%; more preferably reduced by more than 60%), it shows that the candidate substance is a potential substance for inhibiting bone metastasis of breast cancer.
在另一优选例中，所述体系选自：溶液体系、细胞体系、组织体系、器官体系、或动物体系。较佳地，所述的体系是细胞体系；更佳地所述的细胞体系是乳腺癌细胞、乳腺癌骨转移细胞(如SCP细胞)等。In another preferred embodiment, the system is selected from: a solution system, a cell system, a tissue system, an organ system, or an animal system. Preferably, the system is a cell system; more preferably, the cell system is breast cancer cells, breast cancer bone metastasis cells (such as SCP cells) and the like.
在另一优选例中，所述的候选物质包括(但不限于)：针对所述DLC1蛋白设计的小分子化合物、重组表达载体等。In another preferred example, the candidate substances include (but are not limited to): small molecule compounds designed for the DLC1 protein, recombinant expression vectors, and the like.
在本发明的另一方面，提供一种DLC1蛋白或其编码基因的用途，用于制备乳腺癌骨转移预后的试剂。In another aspect of the present invention, a use of the DLC1 protein or its coding gene is provided for preparing a reagent for the prognosis of bone metastasis of breast cancer.
在本发明的另一方面，提供特异性识别DLC1蛋白或其编码基因或其编码基因的试剂的用途，用于制备乳腺癌骨转移预后的试剂或试剂盒。In another aspect of the present invention, use of a reagent for specifically recognizing DLC1 protein or its encoding gene or its encoding gene is provided for preparing a reagent or kit for the prognosis of breast cancer bone metastasis.
在一个优选例中，所述的特异性识别DLC1蛋白或其编码基因的试剂选自：In a preferred example, the reagent specifically recognizing DLC1 protein or its coding gene is selected from:
特异性扩增DLC1蛋白编码基因的引物；Primers for specifically amplifying the DLC1 protein coding gene;
特异性识别DLC1蛋白编码基因的探针；或A probe that specifically recognizes the gene encoding the DLC1 protein; or
特异性结合DLC1蛋白的抗体或配体。Antibodies or ligands that specifically bind to the DLC1 protein.
在另一优选例中，对于乳腺癌患者，当检测结果显示DLC1蛋白低表达，预示骨转移发生的风险较高。In another preferred example, for breast cancer patients, when the detection result shows low expression of DLC1 protein, it indicates that the risk of bone metastasis is high.
在本发明的另一方面，提供一种用于乳腺癌骨转移预后的试剂盒，所述的试剂盒中含有：特异性识别DLC1蛋白或其编码基因的试剂。In another aspect of the present invention, a kit for the prognosis of breast cancer bone metastasis is provided, which contains: a reagent specifically recognizing the DLC1 protein or its coding gene.
在本发明的另一方面，提供Rho相关卷曲螺旋形成蛋白激酶(ROCK)抑制剂在制备预防、缓解或治疗乳腺癌骨转移疾病的药物中的用途。In another aspect of the present invention, a use of a Rho-associated coiled-coil-forming protein kinase (ROCK) inhibitor in the preparation of a drug for preventing, alleviating or treating breast cancer bone metastases is provided.
在另一优选例中，所述的ROCK抑制剂不是Y27632。In another preferred example, the ROCK inhibitor is not Y27632.
在另一优选例中，所述的Rho相关卷曲螺旋形成蛋白激酶抑制剂是Fasudil。In another preferred example, the Rho-associated coiled-coil forming protein kinase inhibitor is Fasudil.
本发明的其它方面由于本文的公开内容，对本领域的技术人员而言是显而易见的。Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein.
附图说明Description of drawings
图1、乳腺癌细胞系中DLC1表达水平与其骨转移能力呈负相关关系，但是和肺转移能力没有关联。(A)不同骨转移能力的MDA-MB-231亚系中DLC1表达水平。(B)不同肺转移能力的MDA-MB-231亚系中DLC1表达水平。Figure 1. The expression level of DLC1 in breast cancer cell lines was negatively correlated with its bone metastatic ability, but not with lung metastatic ability. (A) DLC1 expression levels in MDA-MB-231 sublines with different bone metastatic abilities. (B) DLC1 expression levels in MDA-MB-231 sublines with different lung metastatic abilities.
图2、DLC1过表达对SCP2骨转移能力的影响。(A)qPCR和Western blot分析验证SCP2的DLC1过表达稳转系的建立。(B)通过心脏注射把SCP2的DLC1过表达稳转系细胞移植进小鼠体内，然后利用小动物活体成像分析（BLI）对SCP2细胞的骨转移进行定量检测。(C)BLI和骨转移H/E染色分析的代表图。标尺，100μm。(D)心脏注射DLC1过表达SCP2细胞后小鼠的生存率。Figure 2. The effect of DLC1 overexpression on the ability of SCP2 to metastasize to bone. (A) qPCR and Western blot analysis verified the establishment of a DLC1 overexpression stable transgenic line of SCP2. (B) DLC1-overexpressing stable cells of SCP2 were transplanted into mice by cardiac injection, and bone metastases of SCP2 cells were quantified by small animal in vivo imaging analysis (BLI). (C) Representative images of BLI and H/E staining analysis of bone metastases. Scale bar, 100 μm. (D) Survival rate of mice after cardiac injection of DLC1-overexpressing SCP2 cells.
图3、DLC1敲低对SCP28骨转移能力的影响。(A)qPCR和Western blot分析验证SCP28的DLC1敲低稳转系的建立。(B)通过小动物活体成像分析（BLI）对小鼠体内SCP28向四肢骨转移的定量检测。(C)BLI、X光检测和骨转移H/E染色分析的代表图。标尺，100μm。(D)DLC1敲低对SCP28造成的溶骨区域面积的影响。(E)心脏注射DLC1敲低SCP28细胞后小鼠的生存率。Figure 3. The effect of DLC1 knockdown on the ability of SCP28 to metastasize to bone. (A) qPCR and Western blot analysis verified the establishment of DLC1 knockdown stable transduction line of SCP28. (B) Quantitative detection of SCP28 transfer to limb bones in mice by small animal in vivo imaging analysis (BLI). (C) Representative images of BLI, X-ray detection, and H/E staining analysis of bone metastases. Scale bar, 100 μm. (D) Effect of DLC1 knockdown on the area of osteolytic area caused by SCP28. (E) Survival rate of mice after cardiac injection of DLC1 knockdown SCP28 cells.
图4、Dlc1过表达对4T1骨转移能力的影响。(A)qPCR和Western blot分析验证4T1的Dlc1过表达稳转系的建立。(B)注射4T1后的小鼠四肢骨转移灶数量的检测。(C)代表性的H/E图片。标尺，100μm。(D)心脏注射Dlc1过表达4T1细胞后小鼠的生存率。Fig. 4. The effect of Dlc1 overexpression on bone metastasis ability of 4T1. (A) qPCR and Western blot analysis verified the establishment of Dlc1 overexpression stable transduction lines in 4T1. (B) Detection of the number of bone metastases in the limbs of mice injected with 4T1. (C) Representative H/E pictures. Scale bar, 100 μm. (D) Survival rate of mice after cardiac injection of Dlc1-overexpressing 4T1 cells.
图5、DLC1敲低或者过表达对乳腺癌细胞系肺转移能力没有显著影响。(A)通过微静脉注射把SCP28的DLC1敲低稳转系细胞移植进小鼠体内，然后利用小动物活体成像分析（BLI）对SCP28细胞的肺转移进行定量检测。(B)BLI分析和H/E染色分析的代表图。标尺，100μm。(C)微静脉注射DLC1敲低SCP28细胞后小鼠的生存率。(D)尾静脉注射Dlc1过表达4T1细胞后肺转移灶的代表性H/E图片。标尺，100μm。(E)Dlc1过表达对4T1肺转移灶数量的影响。(F)微静脉注射Dlc1过表达4T1细胞后小鼠的生存率。Figure 5. DLC1 knockdown or overexpression has no significant effect on lung metastasis ability of breast cancer cell lines. (A) The DLC1-knockdown stable transgenic cells of SCP28 were transplanted into mice by venous injection, and then the lung metastasis of SCP28 cells was quantitatively detected by small animal in vivo imaging analysis (BLI). (B) Representative images of BLI analysis and H/E staining analysis. Scale bar, 100 μm. (C) Survival rate of mice after venous injection of DLC1 knockdown SCP28 cells. (D) Representative H/E pictures of lung metastases after tail vein injection of Dlc1-overexpressing 4T1 cells. Scale bar, 100 μm. (E) Effect of Dlc1 overexpression on the number of 4T1 lung metastases. (F) Survival rate of mice after venous injection of Dlc1-overexpressing 4T1 cells.
图6、DLC1敲低或者过表达对SCP2或SCP28细胞Rho通路活性的影响。(A)DLC1敲低或者过表达对SCP2及SCP28细胞中Rho或CDC42蛋白活性、及其下游分子磷酸化的影响。(B)DLC1敲低或者过表达对SCP2及SCP28细胞中应力纤维形成的影响。标尺，10μm。Figure 6. The effect of DLC1 knockdown or overexpression on Rho pathway activity in SCP2 or SCP28 cells. (A) Effects of DLC1 knockdown or overexpression on Rho or CDC42 protein activity and phosphorylation of downstream molecules in SCP2 and SCP28 cells. (B) Effect of DLC1 knockdown or overexpression on stress fiber formation in SCP2 and SCP28 cells. Scale bar, 10 μm.
图7、GTPase通路介导了DLC1在乳腺癌骨转移过程中的功能。(A)DLC1GAP失活突变体R718E丧失了抑制Rho和CDC42的功能。(B-D)R718E突变体在乳腺癌骨转移中的功能分析。(B)小动物活体成像分析(BLI)检测裸鼠四肢骨转移瘤的生长；(C)BLI分析和H/E染色的代表图。标尺，50μm。(D)心脏注射表达R718E的SCP28细胞后小鼠的生存率。Figure 7. The GTPase pathway mediates the function of DLC1 in the process of breast cancer bone metastasis. (A) DLC1GAP inactivation mutant R718E lost the function of inhibiting Rho and CDC42. (B-D) Functional analysis of the R718E mutant in breast cancer bone metastasis. (B) Small animal in vivo imaging analysis (BLI) to detect the growth of bone metastases in the extremities of nude mice; (C) Representative images of BLI analysis and H/E staining. Scale bar, 50 μm. (D) Survival rate of mice after cardiac injection of R718E-expressing SCP28 cells.
图8、DLC1在乳腺癌细胞的内皮细胞粘附和跨内皮侵袭过程中的功能。(A)DLC1敲低或过表达对SCP28和SCP2细胞与骨内皮细胞HBMEC-60之间粘附的影响。(B)DLC1敲低或过表达对SCP28和SCP2跨骨内皮细胞侵袭的影响。(C)DLC1敲低或过表达对SCP28和SCP2与肺内皮细胞ST1.6R之间粘附的影响。(D)DLC1敲低或过表达对SCP28和SCP2跨肺内皮细胞侵袭的影响。Figure 8. The function of DLC1 in the process of endothelial cell adhesion and transendothelial invasion of breast cancer cells. (A) The effect of DLC1 knockdown or overexpression on the adhesion between SCP28 and SCP2 cells and bone endothelial cells HBMEC-60. (B) Effect of DLC1 knockdown or overexpression on the invasion of SCP28 and SCP2 across bone endothelial cells. (C) The effect of DLC1 knockdown or overexpression on the adhesion between SCP28 and SCP2 to lung endothelial cells ST1.6R. (D) Effect of DLC1 knockdown or overexpression on invasion of SCP28 and SCP2 across lung endothelial cells.
图9、DLC1在乳腺癌细胞诱导破骨细胞分化成熟过程中的功能。(A)经TGFβ处理的SCP28细胞与小鼠原代骨髓细胞共培养，诱导骨髓细胞分化成成熟破骨细胞。图示为DLC1在SCP28中的过表达对其诱导能力的影响。(B)SCP2细胞经TGFβ处理后取其条件培养液（CM），并分析CM诱导骨髓细胞向破骨细胞分化的能力。图示为DLC1敲低对SCP2CM诱导能力的影响。(C)SCP2CM诱导破骨细胞分化分析中TRAP染色代表图，箭头所指为TRAP染色阳性的成熟破骨细胞。(D)TRAP染色分析SCP28在小鼠体内形成的骨转移瘤部位癌-骨界面破骨细胞密度的代表图。(E)DLC1敲低对体内骨转移瘤中癌-骨界面单位长度破骨细胞数量的影响。标尺，100μm.*P<0.05;**P<0.01。Figure 9. The function of DLC1 in the induction of osteoclast differentiation and maturation of breast cancer cells. (A) TGFβ-treated SCP28 cells were co-cultured with primary mouse bone marrow cells to induce bone marrow cells to differentiate into mature osteoclasts. The diagram shows the effect of overexpression of DLC1 in SCP28 on its inducibility. (B) The conditioned medium (CM) of SCP2 cells treated with TGFβ was used to analyze the ability of CM to induce bone marrow cells to differentiate into osteoclasts. The figure shows the effect of DLC1 knockdown on the inducibility of SCP2CM. (C) Representative image of TRAP staining in the osteoclast differentiation assay induced by SCP2CM, and the arrows indicate mature osteoclasts that are positive for TRAP staining. (D) Representative image of osteoclast density at the cancer-bone interface at the site of bone metastases formed by SCP28 in mice analyzed by TRAP staining. (E) Effect of DLC1 knockdown on the number of osteoclasts per unit length at the cancer-bone interface in bone metastases in vivo. Scale bar, 100 μm. *P<0.05; **P<0.01.
图10、DLC1通过抑制Rho通路在乳腺癌细胞诱导破骨细胞成熟过程中发挥功能。(A)DLC1敲低的SCP28经TGFβ刺激，并用Rho抑制剂（C3）或者ROCK抑制剂（Y27632）处理后其条件培养液(CM)，分析CM诱导骨髓细胞向破骨细胞分化的能力变化。(B)DLC1过表达的SCP2经TGFβ刺激，并用RhoA突变体RhoA（63L）、C3或Y27632处理后取其CM，分析CM诱导破骨细胞分化能力的变化。同时，用C3或Y27632直接处理骨髓分化系统，作为实验的阴性对照。(C)源自过表达DLC1或R718E的SCP2细胞体内骨转移瘤的H/E或TRAP染色图片。(D)癌-骨界面单位长度破骨细胞数量分析。标尺，100μm.*P<0.05;**P<0.01。Figure 10. DLC1 plays a role in the induction of osteoclast maturation by breast cancer cells by inhibiting the Rho pathway. (A) The conditioned medium (CM) of DLC1-knockdown SCP28 was stimulated with TGFβ and treated with Rho inhibitor (C3) or ROCK inhibitor (Y27632), and the ability of CM to induce bone marrow cells to differentiate into osteoclasts was analyzed. (B) DLC1-overexpressed SCP2 was stimulated by TGFβ, and treated with RhoA mutant RhoA (63L), C3 or Y27632, and its CM was collected to analyze the changes in the ability of CM to induce osteoclast differentiation. At the same time, the myeloid differentiation system was directly treated with C3 or Y27632 as a negative control for the experiment. (C) H/E or TRAP staining pictures of bone metastases derived from SCP2 cells overexpressing DLC1 or R718E in vivo. (D) Analysis of the number of osteoclasts per unit length at the cancer-bone interface. Scale bar, 100 μm. *P<0.05; **P<0.01.
图11、DLC1影响TGFβ对其部分靶基因表达的调控。(A)被DLC1逆转的受TGFβ调控的基因在DLC1过表达后的表达水平热图。(B)被TGFβ上调或下调的下游靶基因群（指纹基因）在DLC1过表达与否的情况下整体水平受TGFβ调控的GSEA分析。(C)C3处理能逆转DLC1对TGFβ调控PTHLH表达的影响。图示为用C3或TGFβ处理DLC1过表达或对照细胞后利用Western blotting检测PTHLH在细胞内（CM）的水平。(D)用C3或TGFβ处理DLC1敲低或对照细胞后利用Western blotting检测PTHLH在胞内或胞外的水平。Figure 11. DLC1 affects the regulation of TGFβ on the expression of some of its target genes. (A) Heat map of expression levels of TGFβ-regulated genes reversed by DLC1 after DLC1 overexpression. (B) GSEA analysis of the downstream target gene group (fingerprint genes) up-regulated or down-regulated by TGFβ, whether the overall level is regulated by TGFβ under the condition of DLC1 overexpression or not. (C) C3 treatment can reverse the effect of DLC1 on TGFβ regulation of PTHLH expression. The diagram shows the intracellular (CM) level of PTHLH detected by Western blotting after DLC1 overexpressed or control cells were treated with C3 or TGFβ. (D) After treating DLC1 knockdown or control cells with C3 or TGFβ, the intracellular or extracellular levels of PTHLH were detected by Western blotting.
图12、PTHLH介导了DLC1在骨转移过程中的功能。(A)PTHLH抑制剂6-TG能掩盖DLC1调控癌细胞体内骨转移的功能。图示为用6-TG处理小鼠后SCP2DLC1过表达细胞骨转移的小动物活体成像分析（BLI）分析。(B)代表性的BLI图片。(C)骨转移瘤中PTHLH和SMAD3磷酸化(Ser425)水平的组织免疫化学分析。标尺，50μm。(D)6-TG和DLC1过表达对骨转移动物实验的小鼠生存率的影响。(E)Western blotting证实4T1中Pthlh和Dlc1双表达稳定细胞系建立成功(样品为细胞经TGFβ处理的条件培养液)。(F)Pthlh和Dlc1双表达后4T1细胞造成的溶骨转移区域面积的定量分析。Figure 12. PTHLH mediates the function of DLC1 in bone metastasis. (A) PTHLH inhibitor 6-TG can mask the function of DLC1 in regulating bone metastasis in cancer cells in vivo. The picture shows small animal in vivo imaging analysis (BLI) analysis of bone metastases of SCP2DLC1 overexpressing cells after mice were treated with 6-TG. (B) Representative BLI pictures. (C) Histoimmunochemical analysis of PTHLH and SMAD3 phosphorylation (Ser425) levels in bone metastases. Scale bar, 50 μm. (D) The effect of 6-TG and DLC1 overexpression on the survival rate of mice in bone metastasis animal experiments. (E) Western blotting confirmed the successful establishment of a stable cell line with double expression of Pthlh and Dlc1 in 4T1 (the sample is the conditioned medium of cells treated with TGFβ). (F) Quantitative analysis of the area of osteolytic metastases caused by 4T1 cells after dual expression of Pthlh and Dlc1.
图13、TGFβ-Smad通路介导了DLC1对PTHLH表达的调控。(A)其他各种通路的抑制剂不影响DLC1对PTHLH表达的影响。图示为用各种抑制剂处理SCP2细胞后分析PTHLH的表达水平。(B)在SCP2细胞中敲低SMAD4。图示为SMAD4的敲低效率。(C)SMAD4敲低对PTHLH表达的影响。Figure 13. The TGFβ-Smad pathway mediates the regulation of DLC1 on the expression of PTHLH. (A) Inhibitors of various other pathways do not affect the effect of DLC1 on PTHLH expression. The graph shows the analysis of PTHLH expression levels after treatment of SCP2 cells with various inhibitors. (B) Knockdown of SMAD4 in SCP2 cells. The graph shows the knockdown efficiency of SMAD4. (C) Effect of SMAD4 knockdown on PTHLH expression.
图14、Smad3上Ser204和Ser208的磷酸化介导了DLC1对PTHLH表达的调控。(A)C3、TGF-β处理和DLC1的过表达对SMAD3上Ser204、Ser208和Ser425位点磷酸化的影响。(B)TGFβ对SCP2中RHOA活性的影响。(C)TGFβ处理、过表达正常SMAD3和Smad3Ser204和Ser208磷酸化位点突变体(EPSM)、SMAD4敲低和DLC1过表达对PTHLH启动子活性的影响。(D)通过染色质免疫共沉淀-定量PCR分析SMAD3与PTHLH启动子的相互作用。Figure 14. Phosphorylation of Ser204 and Ser208 on Smad3 mediates regulation of PTHLH expression by DLC1. (A) Effects of C3, TGF-β treatment and DLC1 overexpression on the phosphorylation of Ser204, Ser208 and Ser425 on SMAD3. (B) Effect of TGFβ on RHOA activity in SCP2. (C) Effects of TGFβ treatment, overexpression of normal SMAD3 and Smad3 Ser204 and Ser208 phosphorylation site mutants (EPSM), SMAD4 knockdown and DLC1 overexpression on PTHLH promoter activity. (D) Analysis of SMAD3 interaction with PTHLH promoter by chromatin immunoprecipitation-quantitative PCR.
图15、临床数据显示DLC1表达水平与乳腺癌骨转移相关。(A)通过NKI临床数据库计算出的DLC1高表达样本组和DLC1低表达样本组的骨转移风险分析。(B)通过NKI临床数据计算出的DLC1高表达组和DLC1低表达组的肺转移风险分析。(C)乳腺癌癌旁组织、无转移预后的癌组织、有肺或骨转移预后的乳腺癌组织中DLC1的表达水平。(D)乳腺癌肿瘤样本中DLC1和PTHLH表达水平的关联。(E)乳腺癌原位癌、淋巴结转移瘤和骨转移瘤的DLC1表达水平的免疫组化分析。Figure 15. Clinical data showing that the expression level of DLC1 is correlated with bone metastasis of breast cancer. (A) Risk analysis of bone metastasis in the DLC1 high expression sample group and DLC1 low expression sample group calculated by the NKI clinical database. (B) Risk analysis of lung metastasis in DLC1 high expression group and DLC1 low expression group calculated by NKI clinical data. (C) The expression level of DLC1 in breast cancer adjacent tissues, cancer tissues without metastasis prognosis, and breast cancer tissues with lung or bone metastasis prognosis. (D) Correlation of DLC1 and PTHLH expression levels in breast cancer tumor samples. (E) Immunohistochemical analysis of DLC1 expression levels in carcinoma in situ of breast cancer, lymph node metastases, and bone metastases.
图16、靶向Rho-ROCK通路治疗乳腺癌骨转移的疗效分析。(A)小动物活体成像分析（BLI）检测用PBS、Fasudil或Y27632处理小鼠后对对SCP2骨转移的影响。(B)代表性的BLI图片和H/E图片。(C)X光检测注射PBS或Fasudil对4T1骨转移生长的影响。Figure 16. Analysis of the curative effect of targeting the Rho-ROCK pathway in the treatment of bone metastasis of breast cancer. (A) Small animal in vivo imaging analysis (BLI) to detect the effect of treating mice with PBS, Fasudil or Y27632 on bone metastasis of SCP2. (B) Representative BLI pictures and H/E pictures. (C) X-ray detection of the effect of injection of PBS or Fasudil on the growth of 4T1 bone metastases.
具体实施方式Detailed ways
本发明人经过广泛而深入的研究，通过对具有不同骨转移能力的细胞系和临床乳腺癌肿瘤样本的基因表达分析，首次发现一种GTPases激活蛋白DLC1蛋白的表达水平与骨转移倾向负相关，为有效预防、控制或治疗乳腺癌骨转移提供了新靶标。After extensive and in-depth research, the inventors found for the first time that the expression level of a GTPases activating protein DLC1 protein was negatively correlated with the tendency of bone metastasis by analyzing the gene expression of cell lines with different bone metastasis abilities and clinical breast cancer tumor samples. It provides a new target for effective prevention, control or treatment of breast cancer bone metastasis.
DLC1蛋白及Rho-ROCK通路及其相互作用DLC1 protein and Rho-ROCK pathway and their interaction
本发明揭示了一个基于与乳腺癌等骨转移密切相关的新靶标。本发明人通过基因敲低和过表达分析，发现DLC1抑制乳腺癌细胞的骨转移能力。而这种功能的行使依赖于它失活Rho-ROCK通路(Rho/Rho相关卷曲螺旋形成蛋白激酶(Rho associated coiled coil forming protein kinase，ROCK))，继而抑制诱导的Smad3linker region磷酸化，对后者的抑制阻碍了诱导的PTHLH的转录。最终阻碍骨转移中的“恶性循环”。The present invention reveals a new target based on a close relationship with bone metastasis such as breast cancer. Through gene knockdown and overexpression analysis, the present inventors found that DLC1 inhibited the bone metastasis ability of breast cancer cells. The exercise of this function depends on its inactivation of the Rho-ROCK pathway (Rho/Rho-related coiled-coil forming protein kinase (Rho associated coiled coil forming protein kinase, ROCK)), and then inhibits Induced phosphorylation of the Smad3linker region, inhibition of the latter blocked Induced transcription of PTHLH. Ultimately hindering the "vicious circle" in bone metastasis.
DLC1并非对所有靶器官的转移都会有调控的作用，对肺转移的研究显示肺转移过程中没有调控作用。进一步的研究表明DLC1通过调节Rho调控的Smad3linker region的磷酸化而下调了PTHLH的表达。同时本发明人发现如果DLC1被沉默，乳腺肿瘤细胞接受TGFβ刺激后Rho活性会上调，Rho活性增强则会上调Smad3linker region的磷酸化，从而促进PTHLH的表达。DLC1的沉默对TGFβ这种自激活的机制可能相当重要，因为当DLC1过表达时，即使乳腺肿瘤细胞接受TGFβ刺激，Rho活性也未见显著上调。而在体内的实验中本发明人发现在小鼠骨转移瘤中不但发现PTHLH表达显著被DLC1抑制，骨转移瘤中Smad3上第425位丝氨酸磷酸化水平也被DLC1抑制，在本发明人的体外实验中肿瘤细胞内的Smad3表达水平并未被DLC1下调。骨转移瘤中Smad3上第425位丝氨酸磷酸化水平被DLC1抑制的原因很可能是由于DLC1抑制了破骨细胞的成熟而导致更少的骨基质中TGFβ释放造成的。PTHLH和TGFβ是肿瘤骨转移“恶性循环”中重要的媒介蛋白，DLC1对它们的调控像车铩一样给“恶性循环”减速。因此这一发现不但首次阐明DLC1不依赖于调节细胞迁移的抑制肿瘤转移的新机制，同时也阐明了“恶性循环”过程中存在减速的机制。本发明人发现，Smad3linker region(Ser204/208)的磷酸化为TGFβ诱导PTHLH转录所必需，同时Smad3上该位点的磷酸化也是TGFβ诱导其他靶基因转录所必需。DLC1 does not have a regulatory effect on the metastasis of all target organs, and studies on lung metastasis have shown that there is no regulatory effect in the process of lung metastasis. Further studies showed that DLC1 downregulated the expression of PTHLH by regulating the phosphorylation of the Rho-regulated Smad3linker region. At the same time, the inventors found that if DLC1 is silenced, the Rho activity of breast tumor cells will be up-regulated after being stimulated by TGFβ, and the enhanced Rho activity will up-regulate the phosphorylation of Smad3linker region, thereby promoting the expression of PTHLH. The silencing of DLC1 may be very important for the self-activation mechanism of TGFβ, because when DLC1 is overexpressed, even when breast tumor cells are stimulated by TGFβ, the Rho activity is not significantly up-regulated. In vivo experiments, the inventors found that not only the expression of PTHLH was significantly inhibited by DLC1 in bone metastases of mice, but also the phosphorylation level of Serine 425 on Smad3 in bone metastases was also inhibited by DLC1. In the experiment, the expression level of Smad3 in tumor cells was not down-regulated by DLC1. The reason why the phosphorylation level of serine 425 on Smad3 in bone metastases is inhibited by DLC1 is probably because DLC1 inhibits the maturation of osteoclasts, resulting in less TGFβ release in bone matrix. PTHLH and TGFβ are important mediator proteins in the "vicious circle" of tumor bone metastasis, and the regulation of them by DLC1 slows down the "vicious circle" like a car. Therefore, this discovery not only elucidated for the first time a new mechanism of DLC1 inhibiting tumor metastasis independent of regulating cell migration, but also clarified the mechanism of deceleration in the "vicious cycle" process. The inventors found that the phosphorylation of Smad3linker region (Ser204/208) is necessary for TGFβ to induce PTHLH transcription, and the phosphorylation of this site on Smad3 is also necessary for TGFβ to induce the transcription of other target genes.
基于本发明人的新发现，本发明提供DLC1蛋白在乳腺癌骨转移疾病中的作用机理和用途，用于制备预防、改善或治疗乳腺癌骨转移疾病的药物，或用于筛选预防、改善或治疗乳腺癌骨转移疾病的潜在物质。Based on the new discovery of the present inventors, the present invention provides the mechanism and application of DLC1 protein in breast cancer bone metastases, for the preparation of drugs for preventing, improving or treating breast cancer bone metastases, or for screening for preventing, improving or treating breast cancer bone metastases. Potential substances for the treatment of bone metastatic disease from breast cancer.
任何适合的DLC1蛋白均可用于本发明。所述的DLC1蛋白包括它们的序列全长形式或其生物活性片段。优选的，所述的DLC1蛋白的氨基酸序列可以与NCBI Reference Sequence:NM_006094.3所示的序列基本上相同。但是鉴于DLC1蛋白在不同动物中的保守性，来自于其它动物的DLC1蛋白也被包含在本发明中。由本发明的研究结果也可见，人源的DLC1蛋白在鼠(如大鼠、小鼠)体内的表达特征接近，发挥相同的功能，可见其在人、鼠或其它动物中的通用性。Any suitable DLC1 protein may be used in the present invention. The DLC1 proteins include their full-length sequences or their biologically active fragments. Preferably, the amino acid sequence of the DLC1 protein may be substantially identical to the sequence shown in NCBI Reference Sequence: NM_006094.3. However, in view of the conservation of DLC1 proteins in different animals, DLC1 proteins from other animals are also included in the present invention. It can also be seen from the research results of the present invention that the human-derived DLC1 protein has similar expression characteristics in mice (such as rats and mice) and performs the same function, which shows its versatility in humans, mice or other animals.
经过一个或多个氨基酸残基的取代、缺失或添加而形成的DLC1蛋白的氨基酸序列也包括在本发明中，只要其保留全长蛋白的功能。DLC1蛋白或它们的生物活性片段包括一部分保守氨基酸的替代序列，所述经氨基酸替换的序列并不影响其活性或保留了其部分的活性。适当替换氨基酸是本领域公知的技术，所述技术可以很容易地被实施并且确保不改变所得分子的生物活性。这些技术使本领域人员认识到，一般来说，在一种多肽的非必要区域改变单个氨基酸基本上不会改变生物活性。The amino acid sequence of the DLC1 protein formed by substitution, deletion or addition of one or more amino acid residues is also included in the present invention, as long as it retains the function of the full-length protein. The DLC1 protein or their biologically active fragments include a part of conservative amino acid replacement sequence, and the amino acid replacement sequence does not affect its activity or retains part of its activity. Appropriate substitution of amino acids is a well-known technique in the art, which can be readily performed and ensures that the biological activity of the resulting molecule is not altered. These techniques allow those skilled in the art to recognize that, in general, changes to single amino acids in non-essential regions of a polypeptide do not substantially alter biological activity.
任何一种DLC1蛋白的生物活性片段都可以应用到本发明中。在这里，DLC1蛋白的生物活性片段的含义是指作为一种多肽，其仍然能保持全长蛋白的全部或部分功能。通常情况下，所述的生物活性片段至少保持50%的全长蛋白的活性。在更优选的条件下，所述活性片段能够保持全长蛋白的60%、70%、80%、90%、95%、99%、或100%的活性。Any biologically active fragment of DLC1 protein can be used in the present invention. Here, the biologically active fragment of the DLC1 protein refers to a polypeptide that can still maintain all or part of the functions of the full-length protein. Usually, the biologically active fragment retains at least 50% of the activity of the full-length protein. Under more preferred conditions, the active fragment can maintain 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the activity of the full-length protein.
DLC1蛋白的调节剂Modulator of DLC1 protein
如本文所用，所述的“上调剂”、“促进剂”、“激动剂”、“激活剂”可互换使用。所述的“上调”、“促进”包括了蛋白活性的“上调”、“促进”或蛋白表达的“上调”、“促进”。任何可上调DLC1蛋白的表达或活性状态、促进DLC1蛋白成熟和剪切、提升DLC1蛋白的稳定性、延长DLC1蛋白的有效作用时间、或调节(提高)DLC1蛋白的转录或翻译的物质均可用于本发明，作为制备抑制乳腺癌骨转移的药物的有效物质。As used herein, the "up-regulator", "accelerator", "agonist" and "activator" can be used interchangeably. The "up-regulation" and "promotion" include "up-regulation" and "promotion" of protein activity or "up-regulation" and "promotion" of protein expression. Any substance that can up-regulate the expression or activity state of the DLC1 protein, promote the maturation and cleavage of the DLC1 protein, increase the stability of the DLC1 protein, prolong the effective action time of the DLC1 protein, or regulate (improve) the transcription or translation of the DLC1 protein can be used The present invention is used as an effective substance for preparing medicine for inhibiting bone metastasis of breast cancer.
作为本发明的优选方式，所述的DLC1的激动剂、促进剂或上调剂包括(但不限于)：人工重组DLC1蛋白、可表达DLC1蛋白的重组表达质粒、激活或促进DLC1功能或活性的特异性多肽、可促进DLC1与Rho通路蛋白发生特异性结合或相互作用的物质。As a preferred mode of the present invention, the agonists, promoters or upregulators of DLC1 include (but not limited to): artificial recombinant DLC1 protein, recombinant expression plasmids capable of expressing DLC1 protein, specific agents for activating or promoting DLC1 function or activity Sexual polypeptides, substances that can promote specific binding or interaction between DLC1 and Rho pathway proteins.
鉴于上述的DLC1蛋白的“上调剂”、“促进剂”、“激动剂”、“激活剂”对于DLC1蛋白的促进作用，它们可被用于制备抑制乳腺癌骨转移的组合物。In view of the promotion effect of the above-mentioned "upregulator", "promoter", "agonist" and "activator" of DLC1 protein on DLC1 protein, they can be used to prepare a composition for inhibiting bone metastasis of breast cancer.
筛选抑制乳腺癌骨转移的潜在物质的方法Method for screening potential substances inhibiting bone metastasis of breast cancer
在得知了所述的DLC1蛋白对于乳腺癌骨转移的发生或发展的影响作用后，可以采用本领域熟知的多种方法来筛选抑制乳腺癌骨转移的潜在物质。可从所述的潜在物质中找到可用于制备抑制乳腺癌骨转移的药物的物质。After knowing the effect of the DLC1 protein on the occurrence or development of breast cancer bone metastasis, various methods well known in the art can be used to screen for potential substances that inhibit breast cancer bone metastasis. Substances that can be used to prepare drugs for inhibiting bone metastasis of breast cancer can be found from the potential substances.
因此，本发明提供一种筛选抑制乳腺癌骨转移的潜在物质的方法，包括以下步骤：(a)将候选物质与表达DLC1蛋白的体系接触；(b)检测DLC1蛋白的表达或活性，若所述候选物质在统计学上提高DLC1蛋白的表达或活性，则表明该候选物质是抑制乳腺癌骨转移的潜在物质。Therefore, the present invention provides a method for screening potential substances that inhibit bone metastasis of breast cancer, comprising the following steps: (a) contacting the candidate substance with a system expressing DLC1 protein; (b) detecting the expression or activity of DLC1 protein, if the If the candidate substance can statistically increase the expression or activity of the DLC1 protein, it indicates that the candidate substance is a potential substance for inhibiting bone metastasis of breast cancer.
鉴于所述的DLC1蛋白与Rho蛋白发生相互作用与乳腺癌骨转移的相关性，所述的筛选方法可以通过观察两者的相互作用进行。该方法包括：检测DLC1蛋白与Rho蛋白相互作用情况，若所述候选物质使得两者相互作用且使得Rho蛋白的活性在统计学上更低，则表明该候选物质是预防、改善或治疗乳腺癌骨转移的潜在物质。In view of the correlation between the interaction between DLC1 protein and Rho protein and bone metastasis of breast cancer, the screening method can be carried out by observing the interaction between the two. The method includes: detecting the interaction between the DLC1 protein and the Rho protein, and if the candidate substance makes the two interact and makes the activity of the Rho protein statistically lower, it indicates that the candidate substance can prevent, improve or treat breast cancer Potential material for bone metastases.
鉴于所述的DLC1蛋白下调PTHLH表达与乳腺癌骨转移的相关性，所述的筛选方法可以通过观察DLC1蛋白对PTHLH的调控进行。该方法包括：检测DLC1蛋白对PTHLH mRNA或蛋白水平上的调控情况，若所述候选物质使得PTHLH mRNA或蛋白的表达水平在统计学上更低，则表明该候选物质是预防、改善或治疗乳腺癌骨转移的潜在物质。In view of the correlation between the down-regulation of PTHLH expression by DLC1 protein and the bone metastasis of breast cancer, the screening method can be carried out by observing the regulation of PTHLH by DLC1 protein. The method includes: detecting the regulation of DLC1 protein on PTHLH mRNA or protein level, if the candidate substance makes the expression level of PTHLH mRNA or protein statistically lower, it indicates that the candidate substance is effective in preventing, improving or treating breast cancer. Potential material for cancer bone metastasis.
鉴于所述的DLC1蛋白与Smad3蛋白发生相互作用与乳腺癌骨转移的相关性，所述的筛选方法可以通过观察两者的相互作用进行。该方法包括：检测DLC1蛋白与Smad3蛋白相互作用情况，若所述候选物质使得两者相互作用且使得Smad3蛋白第204和208位丝氨酸的磷酸化水平在统计学上更低，则表明该候选物质是预防、改善或治疗乳腺癌骨转移的潜在物质。In view of the correlation between the interaction between the DLC1 protein and the Smad3 protein and the bone metastasis of breast cancer, the screening method can be performed by observing the interaction between the two. The method comprises: detecting the interaction between the DLC1 protein and the Smad3 protein, and if the candidate substance makes the two interact and makes the phosphorylation levels of the 204th and 208th serines of the Smad3 protein statistically lower, it indicates that the candidate substance It is a potential substance for preventing, improving or treating bone metastasis of breast cancer.
在本发明中，所述的体系选自(但不限于)：溶液体系、细胞体系、亚细胞体系、组织体系、器官体系、或动物体系。In the present invention, the system is selected from (but not limited to): solution system, cell system, subcellular system, tissue system, organ system, or animal system.
作为本发明的优选实施例，所述的细胞体系选自(但不限于)：乳腺癌细胞、乳腺癌骨转移细胞(如SCP细胞)等。As a preferred embodiment of the present invention, the cell system is selected from (but not limited to): breast cancer cells, breast cancer bone metastasis cells (such as SCP cells) and the like.
所述的体系中可含有内源的或重组表达的Rho信号通路蛋白，用于在其中加入候选物质，观察候选物质对Rho信号通路蛋白表达或活性的影响。The system may contain endogenous or recombinantly expressed Rho signaling pathway proteins for adding candidate substances therein to observe the impact of candidate substances on the expression or activity of Rho signaling pathway proteins.
作为本发明的优选方式，所述的方法还包括：对获得的潜在物质进行进一步的细胞实验和/或动物试验，以进一步选择和确定对于预防、缓解或治疗乳腺癌骨转移真正有用的物质。As a preferred mode of the present invention, the method further includes: conducting further cell experiments and/or animal experiments on the obtained potential substances to further select and determine substances that are really useful for preventing, alleviating or treating bone metastasis of breast cancer.
另一方面，本发明还提供了采用所述筛选方法获得的抑制乳腺癌骨转移的潜在物质。这些初步筛选出的物质可构成一个筛选库，以便于人们最终可以从中筛选出能够对于预防或治疗乳腺癌骨转移有用的物质。On the other hand, the present invention also provides potential substances for inhibiting bone metastasis of breast cancer obtained by the screening method. These preliminarily screened substances can constitute a screening library, so that people can finally screen out substances useful for preventing or treating bone metastasis of breast cancer.
预后试剂或试剂盒Prognostic reagents or kits
DLC1虽然在很多类恶性肿瘤中被视作预后因子，然而它的表达水平与靶器官特异性转移的关系并没被人研究过。本发明人在分析已发表的数据和自行采集的临床乳腺癌肿瘤样本后发现，DLC1的表达水平与乳腺癌骨转移倾向呈现负相关的关系，与肺转移倾向不相关。提示DLC1的表达水平或许可以作为乳腺癌器官特异性转移的预后指标，进而为乳腺癌个体化治疗提供依据。Although DLC1 is regarded as a prognostic factor in many types of malignant tumors, the relationship between its expression level and target organ-specific metastasis has not been studied. After analyzing published data and self-collected clinical breast cancer tumor samples, the present inventors found that the expression level of DLC1 was negatively correlated with bone metastasis tendency of breast cancer, and not correlated with lung metastasis tendency. It is suggested that the expression level of DLC1 may be used as a prognostic indicator of breast cancer organ-specific metastasis, and then provide a basis for individualized treatment of breast cancer.
基于本发明人的上述新发现，可以将DLC1作为诊断乳腺癌骨转移的标志物：(i)进行乳腺癌骨转移的鉴别诊断和/或易感性分析；(ii)评估相关人群的乳腺癌骨转移治疗药物、药物疗效、预后，以及选择合适的治疗方法；(iii)早期评估相关人群乳腺癌骨转移患病风险，早期监测早期防治。比如，可分离出由DLC1基因表达异常而导致乳腺癌骨转移的人群，从而可进行更有针对性地治疗。Based on the above new findings of the present inventors, DLC1 can be used as a marker for the diagnosis of breast cancer bone metastasis: (i) differential diagnosis and/or susceptibility analysis of breast cancer bone metastasis; (ii) assessment of breast cancer bone metastasis in related populations Metastasis treatment drugs, drug efficacy, prognosis, and selection of appropriate treatment methods; (iii) Early assessment of the risk of breast cancer bone metastasis in relevant populations, early monitoring and early prevention and treatment. For example, the group of breast cancer bone metastases caused by abnormal expression of DLC1 gene can be isolated, so that more targeted treatment can be carried out.
因此，本发明提供了DLC1基因或蛋白的用途，用于制备乳腺癌骨转移的预后试剂或试剂盒。Therefore, the present invention provides the use of DLC1 gene or protein for preparing a prognostic reagent or kit for bone metastasis of breast cancer.
可采用各种本领域已知的技术来检测DLC1的存在与否以及表达或活性情况，这些技术均包含在本发明中。例如可用已有的技术如Southern印迹法、Western印迹法、DNA序列分析、PCR等，这些方法可结合使用。Various techniques known in the art can be used to detect the presence or absence, expression or activity of DLC1, and these techniques are included in the present invention. For example, existing techniques such as Southern blotting, Western blotting, DNA sequence analysis, PCR, etc. can be used, and these methods can be used in combination.
本发明还提供了用于在分析物中检测DLC1的存在与否以及表达情况的试剂。优选的，当进行基因水平的检测时，可以采用特异性扩增DLC1的引物；或特异性识别DLC1的探针来确定DLC1的存在与否；当进行蛋白水平的检测时，可以采用特异性结合DLC1蛋白的抗体或配体来确定DLC1蛋白的表达情况。The present invention also provides reagents for detecting the presence or absence and expression of DLC1 in an analyte. Preferably, when performing detection at the gene level, primers that specifically amplify DLC1 can be used; or probes that specifically recognize DLC1 can be used to determine the presence or absence of DLC1; when performing detection at the protein level, specific binding can be used Antibody or ligand for DLC1 protein to determine the expression of DLC1 protein.
针对DLC1的引物或特异性探针的设计是本领域人员熟知的技术，例如，制备一种探针，其可与DLC1基因上特定位点发生特异性结合，而不与DLC1基因以外的其它基因特异性结合，且所述探针带有可检测信号。The design of primers or specific probes for DLC1 is a technique well known to those skilled in the art, for example, preparing a probe that can specifically bind to a specific site on the DLC1 gene, but not to other genes other than the DLC1 gene specific binding, and the probe has a detectable signal.
利用特异性结合DLC1蛋白的抗体来检测分析物中DLC1蛋白表达情况的方法也是本领域人员熟知的技术。The method of detecting the expression of DLC1 protein in an analyte by using an antibody specifically binding to DLC1 protein is also well known to those skilled in the art.
本发明还提供了用于在分析物中检测DLC1的存在与否以及表达情况的试剂盒，该试剂盒包括：特异性扩增DLC1基因的引物；特异性识别DLC1基因的探针；或特异性结合DLC1蛋白的抗体或配体。The present invention also provides a kit for detecting the presence or absence and expression of DLC1 in an analyte, the kit comprising: a primer for specifically amplifying the DLC1 gene; a probe for specifically recognizing the DLC1 gene; or a specific Antibodies or ligands that bind the DLC1 protein.
此外，所述的试剂盒中还可包括用于提取DNA、PCR、杂交、显色等所需的各种试剂，包括但不限于：抽提液、扩增液、杂交液、酶、对照液、显色液、洗液等。In addition, the kit can also include various reagents required for DNA extraction, PCR, hybridization, color development, etc., including but not limited to: extraction solution, amplification solution, hybridization solution, enzyme, control solution , Chromogenic solution, lotion, etc.
此外，所述的试剂盒中还可包括使用说明书和/或核酸序列分析软件等。In addition, the kit may also include instructions for use and/or nucleic acid sequence analysis software and the like.
ROCK抑制剂的用途Uses of ROCK Inhibitors
本发明人在本项研究中发现，ROCK抑制剂(如Y27632和Fasudil)显著地抑制了乳腺癌骨转移瘤的生长。较佳地是Fasudil，其在相同的抑制效应下所花费的成本更小，值得关注。此外Fasudil也是美国FDA批准上市的药物，这些方面的优势使得Fasudil更适合被用来进行临床实验。In this study, the inventors found that ROCK inhibitors (such as Y27632 and Fasudil) significantly inhibited the growth of breast cancer bone metastases. The preferred one is Fasudil, which costs less under the same inhibitory effect and deserves attention. In addition, Fasudil is also a drug approved by the US FDA. These advantages make Fasudil more suitable for clinical trials.
其它与Fasudil具有类似工作机理的ROCK抑制剂也被包含在本发明中，用于制备抑制乳腺癌骨转移的药物。Other ROCK inhibitors with a similar working mechanism to Fasudil are also included in the present invention, and are used to prepare drugs for inhibiting bone metastasis of breast cancer.
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，2002中所述的条件，或按照制造厂商所建议的条件。Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Experimental methods not indicating specific conditions in the following examples are usually according to conventional conditions such as edited by J. Sambrook et al., Molecular Cloning Experiment Guide, Third Edition, Science Press, 2002, or according to the conditions described in the manufacturer suggested conditions.
材料和方法Materials and methods
一、实验材料1. Experimental materials
抗体、试剂Antibodies, Reagents
Mouse anti-human DLC1购自Becton，Dickinson and Company，Rabbitanti-human RHOA，RHOB，RHOC，phospho-PAK4(Ser474)，SMAD3购自CellSignaling Technology公司，anti-human PTHLH购自AOGMA公司，anti-humanphospho-SMAD3(Ser425)购自Abcam公司，anti-human phospho-Vimentin(Ser71)购自MBL International Corporation公司)，anti-humanβ-actin抗体购自Merck公司，anti-human GAPDH抗体购自Proteingroup公司，HRP标记的goat antimouse IgG，goat anti rabbit IgG，donkey anti goat IgG抗体购自Pierce公司；anti-human SMAD3pSer204和anti-human SMAD3pSer208由美国Rutgers大学提供。Mouse anti-human DLC1 was purchased from Becton, Dickinson and Company, Rabbitanti-human RHOA, RHOB, RHOC, phospho-PAK4 (Ser474), SMAD3 was purchased from CellSignaling Technology Company, anti-human PTHLH was purchased from AOGMA Company, anti-humanphospho-SMAD3 (Ser425) was purchased from Abcam Company, anti-human phospho-Vimentin (Ser71) was purchased from MBL International Corporation), anti-human β-actin antibody was purchased from Merck Company, anti-human GAPDH antibody was purchased from Proteingroup Company, HRP-labeled goat antimouse IgG, goat anti rabbit IgG, donkey anti goat IgG antibodies were purchased from Pierce; anti-human SMAD3pSer204 and anti-human SMAD3pSer208 were provided by Rutgers University, USA.
Rho抑制剂C3购自Cytoskeleton公司，CDC42抑制剂为促进细胞内化吸收的一段序列(GRKKRRQRRRPPQC)后接一段CDC42蛋白对应的17-32位氨基酸序列(Rajnicek,A.M等，J Cell Sci119,1723-1735(2006))，该小肽由上海吉尔生化公司合成。RAC1抑制剂NSC23766购自Santa Cruz公司，PTHLH抑制剂6-thioguanine(6-TG)购自Sigma。ROCK抑制剂Y27632和Fasudil购自Selleck公司。ERK抑制剂PD98059，P38抑制剂P38MAPKinase inhibitor，JNK抑制剂JNK inhibitor II和PI3K抑制剂LY294002购自Calbiochem公司。Rho inhibitor C3 was purchased from Cytoskeleton, and the CDC42 inhibitor is a sequence (GRKKRRQRRRPPQC) that promotes the internalization and absorption of cells followed by a sequence of 17-32 amino acids corresponding to the CDC42 protein (Rajnicek, A.M, etc., J Cell Sci119, 1723-1735 (2006)), the small peptide was synthesized by Shanghai Gil Biochemical Company. RAC1 inhibitor NSC23766 was purchased from Santa Cruz, and PTHLH inhibitor 6-thioguanine (6-TG) was purchased from Sigma. ROCK inhibitors Y27632 and Fasudil were purchased from Selleck. ERK inhibitor PD98059, P38 inhibitor P38MAPKinase inhibitor, JNK inhibitor JNK inhibitor II and PI3K inhibitor LY294002 were purchased from Calbiochem.
Human insulin购自Roche公司；Human EGF购自Invitrogen公司；hydrocortisone购自Merck公司；choleratoxin购自Enzo公司；BCA蛋白含量检测试剂盒和ECL发光液购自Pierce公司；protein size ladder购自Fermentas公司；4%Agarose gel购自Invitrogen公司；BD Matrigel Basement MembraneMatrix购自BD bioscience公司；Puromycin DiHCl Cell Culture-Tested购自Merck公司；Polyethylenimine购自Sigma公司；Tetracycline hydrochloride购自Sigma公司，Crystal Violet购自BBI公司；Para甲醛(PFA)购自Sigma公司；三氯乙酸(trichloroacetic acid，TCA)、丙酮、乙醇等购自上海国药公司。Human insulin was purchased from Roche Company; Human EGF was purchased from Invitrogen Company; hydrocortisone was purchased from Merck Company; choleratoxin was purchased from Enzo Company; BCA protein content detection kit and ECL luminescent liquid were purchased from Pierce Company; protein size ladder was purchased from Fermentas Company; %Agarose gel was purchased from Invitrogen; BD Matrigel Basement MembraneMatrix was purchased from BD bioscience; Puromycin DiHCl Cell Culture-Tested was purchased from Merck; Polyethyleneimine was purchased from Sigma; Tetracycline hydrochloride was purchased from Sigma, and Crystal Violet was purchased from BBI; Formaldehyde (PFA) was purchased from Sigma; trichloroacetic acid (TCA), acetone, ethanol, etc. were purchased from Shanghai Sinopharm Company.
分子克隆相关试剂：T4DNA连接酶、Pfu聚合酶、RT-PCR、Real time PCRkit购自Takara；限制性内切酶购自NEB。Reagents related to molecular cloning: T4DNA ligase, Pfu polymerase, RT-PCR, and Real time PCR kit were purchased from Takara; restriction enzymes were purchased from NEB.
细胞培养相关试剂：DMEM培养液、F12培养液、basal medium199、basalmedium200、胎牛血清、青霉素和链霉素、胰酶等购自Invitrogen。Reagents related to cell culture: DMEM culture medium, F12 culture medium, basal medium199, basalmedium200, fetal bovine serum, penicillin and streptomycin, pancreatin, etc. were purchased from Invitrogen.
二、实验方法2. Experimental method
1、质粒构建1. Plasmid construction
人DLC1及DLC1第718位精氨酸突变为谷氨酸GAP Domain失活突变体(R718E)的表达质粒(pBabepuro-DLC1和pBabepuro-DLC1(R718E))由美国NorthCarolina大学提供。持续激活的RhoA突变体表达质粒RhoA(63L)由复旦大学Dr.Lan Ma提供。Smad报告基因表达质粒(SBE)和SMAD4knockdown表达质粒由美国普林斯顿大学康毅滨实验室提供。PTHLH isoform3promoter(-309bp～100bp)插入pGL3-basic vector的MluI和XhoI位点构建PTHLH isoform3promoter报告基因。将该报告质粒的Smad3结合序列AGACAG(-57～-52bp)突变成序列GATACC后得到了PTHLH isoform3promoter mutant报告质粒。将小鼠Dlc1CDS区克隆入载体pMSCV-puro(购自ClonTech)的XhoI和HpaI位点构建了Dlc1表达质粒。将小鼠Pthlh CDS区克隆入载体pMSCV-hygro(购自ClonTech)的Bgl II和Xho I位点构建了Pthlh表达质粒。The expression plasmids (pBabepuro-DLC1 and pBabepuro-DLC1(R718E)) of human DLC1 and the mutated arginine at position 718 of DLC1 to glutamic acid GAP Domain inactivation mutant (R718E) were provided by North Carolina University, USA. The continuously activated RhoA mutant expression plasmid RhoA (63L) was provided by Dr. Lan Ma of Fudan University. Smad reporter gene expression plasmid (SBE) and SMAD4knockdown expression plasmid were provided by Kang Yibin Laboratory of Princeton University. PTHLH isoform3promoter (-309bp～100bp) was inserted into MluI and XhoI sites of pGL3-basic vector to construct PTHLH isoform3promoter reporter gene. The PTHLH isoform3promoter mutant reporter plasmid was obtained after the Smad3 binding sequence AGACAG (-57～-52bp) of the reporter plasmid was mutated into the sequence GATACC. The mouse Dlc1 CDS region was cloned into the XhoI and HpaI sites of the vector pMSCV-puro (purchased from ClonTech) to construct the Dlc1 expression plasmid. The mouse Pthlh CDS region was cloned into the Bgl II and Xho I sites of the vector pMSCV-hygro (purchased from ClonTech) to construct the Pthlh expression plasmid.
用于制备KD1DLC1knockdown稳转系的shRNA表达质粒采用pSuper-Retro-puro作为载体(购自OligoEngine)，经HindIII和BglII双酶切后接入酶切片段。根据RNA干扰设计原则合成目的序列如下：The shRNA expression plasmid used to prepare the KD1DLC1knockdown stable transduction line used pSuper-Retro-puro as a vector (purchased from OligoEngine), and was inserted into the restriction fragment after double digestion with HindIII and BglII. According to the principle of RNA interference design, the target sequence was synthesized as follows:
用于制备KD1DLC1knockdown稳转系：For the preparation of KD1DLC1knockdown stable transformation line:
KD1：正向序列(5’→3’)：gatctccCCTTGACTGGAATATGTAAttcaagagaTTACATATTCCAGTCAAGGtttttggaaa(SEQ ID NO:1)；KD1: forward sequence (5'→3'): gatctccCCTTGACTGGAATATGTAAttcaagagaTTACATATTCCAGTCAAGGtttttggaaa (SEQ ID NO: 1);
KD1：反向序列(5’→3’)：agcttttccaaaaaCCTTGACTGGAATATGTAAtctcttgaaTTACATATTCCAGTCAAGGgga(SEQ ID NO:2)。KD1: reverse sequence (5'→3'): agcttttccaaaaaCCTTGACTGGAATATGTAAtctcttgaaTTACATATTCCAGTCAAGGgga (SEQ ID NO: 2).
用于制备KD2DLC1knockdown稳转系：For the preparation of KD2DLC1knockdown stable transformation line:
KD2：正向序列(5’→3’)：gatctccCCCGATTGCAAATAGTGATttcaagagaATCACTATTTGCAATCGGGtttttggaaa(SEQ ID NO:3)；KD2: forward sequence (5'→3'): gatctccCCCGATTGCAAATAGTGATttcaagagaATCACTATTTGCAATCGGGtttttggaaa (SEQ ID NO: 3);
KD2：反向序列(5’→3’)：agcttttccaaaaaCCCGATTGCAAATAGTGATtctcttgaaATCACTATTTGCAATCGGGgga(SEQ ID NO:4)。KD2: reverse sequence (5'→3'): agcttttccaaaaaCCCGATTGCAAATAGTGATtctcttgaaATCACTATTTGCAATCGGGgga (SEQ ID NO: 4).
2、细胞培养2. Cell culture
MCF10系列(10A，10AT，CA1a，CA1h)按照DMEM/F12加10μg/ml Insulin，20ng/ml EGF，0.5μg/ml Hydrocortisone，100ng/ml Cholera Toxin，5%马血清，100U/mL青霉素和100μg/mL链霉素的培养液置于37℃的CO2培养箱中培养。RAW264.7、C2C12、MDA-MB-231细胞及各种SCPs(single-cell-derivedprogenies)：SCP2、SCP4、SCP6、SCP46、SCP28、SCP26、SCP25、SCP21、1833、4173、4175、4142、4180、3481细胞，由普林斯顿大学康毅滨实验室提供，使用DMEM加10%胎牛血清、100U/mL青霉素和100μg/mL链霉素的培养液置于37℃的CO2培养箱中培养。各稳转系传代10代以内的细胞扩增后进行动物实验。肺内皮细胞ST1.6R按照basal medium199加20%胎牛血清和50μg/mL endothelial growth factor supplements(Sigma)的培养液置于37℃的CO2培养箱中培养。骨内皮细胞HBMEC-60以basal medium200加20%胎牛血清和1×low serum growth supplements(Invitrogen)做培养液，置于37℃的CO2培养箱中培养。MCF10 series (10A, 10AT, CA1a, CA1h) according to DMEM/F12 plus 10μg/ml Insulin, 20ng/ml EGF, 0.5μg/ml Hydrocortisone, 100ng/ml Cholera Toxin, 5% horse serum, 100U/mL penicillin and 100μg/ml Place the culture medium of mL streptomycin in a CO2 incubator at 37°C. RAW264.7, C2C12, MDA-MB-231 cells and various SCPs (single-cell-derived progenies): SCP2, SCP4, SCP6, SCP46, SCP28, SCP26, SCP25, SCP21, 1833, 4173, 4175, 4142, 4180, 3481 cells, provided by Yibin Kang’s laboratory at Princeton University, were cultured in a CO2 incubator at 37°C using DMEM plus 10% fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin. Animal experiments were carried out after the cells of each stable transduction line were expanded within 10 generations. Lung endothelial cells ST1.6R were cultured in a CO2 incubator at 37°C according to the culture medium of basal medium199 plus 20% fetal bovine serum and 50 μg/mL endothelial growth factor supplements (Sigma). Bone endothelial cells HBMEC-60 were cultured in basal medium200 plus 20% fetal bovine serum and 1×low serum growth supplements (Invitrogen) in a CO2 incubator at 37°C.
3、RT-PCR3. RT-PCR
①总RNA提取①Total RNA extraction
一般而言，细胞用1ml Trizol裂解。震荡30s。加0.2ml氯仿，剧烈摇动30s，室温2min。13000rpm，4℃离心，15min。吸上层无色水相，移入另一EP管中。加等体积异丙醇，-20℃，30min。13000rpm，4℃离心，10min。弃上清，加75%乙醇1ml，振荡。13000rpm，4℃离心，5min。弃上清，吸尽残留液体，室温干燥5min。沉淀溶于40-100μl DEPC水。取出1μl测其OD260/OD280。-80℃保存。Generally, cells are lysed with 1ml Trizol. Vibrate for 30s. Add 0.2ml of chloroform, shake vigorously for 30s, room temperature for 2min. Centrifuge at 13000rpm at 4°C for 15min. Aspirate the upper colorless aqueous phase and transfer to another EP tube. Add an equal volume of isopropanol, -20°C, 30min. Centrifuge at 13000rpm at 4°C for 10min. Discard the supernatant, add 1ml of 75% ethanol, and shake. Centrifuge at 13000rpm at 4°C for 5min. Discard the supernatant, suck up the residual liquid, and dry at room temperature for 5 min. The pellet was dissolved in 40-100 μl DEPC water. Take out 1μl to measure its OD260/OD280. Store at -80°C.
②逆转录合成cDNA② cDNA synthesis by reverse transcription
反应体系如下：5×RT Buffer2μl；RNase Free dH2O补足至10μl；dNTPMixture(各2.5mM)2μl；RNase Inhibitor0.25μl；Reverse Transcriptase0.25μl；Oligo dT0.5μl；Random6primer0.5μl；RNA500ng；反应条件为：42℃10min，95℃2min。The reaction system is as follows: 5×RT Buffer2μl; RNase Free dH2 O supplemented to 10μl; dNTPMixture (2.5mM each) 2μl; RNase Inhibitor0.25μl; Reverse Transcriptase0.25μl; Oligo dT0.5μl; Random6primer0.5μl; : 10 minutes at 42°C, 2 minutes at 95°C.
4、Real time PCR4. Real time PCR
PCR反应体系：ddH2O1.0μl；模板cDNA3.0μl；Premix Ex TaqTM5μl；上游引物(10μM)0.4μl；下游引物(10μM)0.4μl；ROX0.2μl。PCR reaction system: ddH2 O 1.0 μl; template cDNA 3.0 μl; Premix Ex TaqTM 5 μl; upstream primer (10 μM) 0.4 μl; downstream primer (10 μM) 0.4 μl; ROX 0.2 μl.
反应条件：Step1：50℃2min；Step2：95℃10min；Step3：95℃15sec；Step4：60℃1min；Step5：95℃15sec；Step6：60℃15sec；Step7：95℃15sec。其中Step3、Step4进行40个循环后再进行后续Step5。Reaction conditions: Step1: 50°C 2min; Step2: 95°C 10min; Step3: 95°C 15sec; Step4: 60°C 1min; Step5: 95°C 15sec; Step6: 60°C 15sec; Step7: 95°C 15sec. Among them, Step3 and Step4 are performed for 40 cycles, and then the follow-up Step5 is performed.
引物如下：Primers are as follows:
人DLC1 F：CCGTGCTTGATGTGCAGAAAG(SEQ ID NO:5)Human DLC1 F: CCGTGCTTGATGTGCAGAAAG (SEQ ID NO:5)
R：ACCAGTTGCCCGTAGCCAAT(SEQ ID NO:6)R: ACCAGTTGCCCGTAGCCAAT (SEQ ID NO:6)
小鼠Dlc1 F：CGGACACCATGATCCTAACA(SEQ ID NO:7)Mouse Dlc1 F: CGGACACCATGATCCTAACA (SEQ ID NO: 7)
R：ATACTGGGGGAAACCAGTCA(SEQ ID NO:8)R: ATACTGGGGGAAACCAGTCA (SEQ ID NO:8)
人PTHLH F：ACAGTTGGAGTAGCCGGTTG(SEQ ID NO:9)Human PTHLHF: ACAGTTGGAGTAGCCGGTTG (SEQ ID NO: 9)
R：CCCTTGTCATGGAGGAGCTG(SEQ ID NO:10)R: CCCTTGTCATGGAGGAGCTG (SEQ ID NO: 10)
人IL11 F：AGCGGACAGGGAAGGGTTA(SEQ ID NO:11)Human IL11 F: AGCGGACAGGGAAGGGTTA (SEQ ID NO: 11)
R：CTGTATCTGGCCACAGGCTC(SEQ ID NO:12)R: CTGTATCTGGCCACAGGCTC (SEQ ID NO: 12)
人CTGF F：CCTGCAGGCTAGAGAAGCAG(SEQ ID NO:13)Human CTGF F: CCTGCAGGCTAGAGAAGCAG (SEQ ID NO: 13)
R：TGGAGATTTTGGGAGTACGG(SEQ ID NO:14)R: TGGAGATTTTGGGAGTACGG (SEQ ID NO: 14)
人JAG1 F：ATGGGCCCCGAATGTAACAG(SEQ ID NO:15)Human JAG1 F: ATGGGCCCCGAATGTAACAG (SEQ ID NO: 15)
R：ATCACAGTACAGGCCTTGCC(SEQ ID NO:16)R: ATCACAGTACAGGCCTTGCC (SEQ ID NO: 16)
人Smad4 F：CCATCCAGCATCCACCAAGT(SEQ ID NO:17)Human Smad4 F: CCATCCAGCATCCACCAAGT (SEQ ID NO: 17)
R：GGCCCTGATGCTATCTGCAA(SEQ ID NO:18)R: GGCCCTGATGCTATCTGCAA (SEQ ID NO: 18)
人CDKN1A F：AGTCAGTTCCTTGTGGAGCC(SEQ ID NO:19)Human CDKN1A F: AGTCAGTTCCTTGTGGAGCC (SEQ ID NO: 19)
R：CATTAGCGCATCACAGTCGC(SEQ ID NO:20)R: CATTAGCGCATCACAGTCGC (SEQ ID NO:20)
用作ChIP-qPCR的human PTHLH引物Human PTHLH primers for ChIP-qPCR
F：CCCACCAGAGGAGGTAGACA(SEQ ID NO:21)F: CCCACCAGAGGAGGTAGACA (SEQ ID NO:21)
R：CCTTTCGTTCCAGAGCCACT(SEQ ID NO:22)R: CCTTTCGTTCCAGAGCCACT (SEQ ID NO:22)
用作mouse OPG和RANKL mRNA定量的引物参照参考文献Lee,J.H.,etal.The Journal of biological chemistry284,13725-13734(2009)和Bishop,K.A.等，The Journal of biological chemistry286,20880-20891(2011)。The primers used for the quantification of mouse OPG and RANKL mRNA refer to references Lee, J.H., et al. The Journal of biological chemistry 284, 13725-13734 (2009) and Bishop, K.A. et al., The Journal of biological chemistry 286, 20880-20891 (2011).
5、质粒转染与病毒感染5. Plasmid transfection and virus infection
H29(human retrovirus packing line with dox control)细胞由美国普林斯顿大学康毅滨实验室提供，按照普通培养液加Tetracycline(工作浓度：1μg/ml)培养。为了做动物体内化学发光分析，相关细胞系均用含GFP和firefly luciferase双重标记基因逆病毒(质粒由Dr.Yibin Kang提供)感染。PEI转染方法依照分子克隆。收集over-expression和knock down的逆病毒，感染细胞3-4次后，当感染过的细胞达到80%汇合率后，使用含不同浓度的抗生素培养基筛选稳转系，多克隆挑选后扩增，进行相应的RT-qPCR和Western Blotting验证。H29 (human retrovirus packing line with dox control) cells were provided by Kang Yibin Laboratory of Princeton University in the United States, and were cultured according to ordinary culture medium plus Tetracycline (working concentration: 1 μg/ml). For in vivo chemiluminescence analysis in animals, the relevant cell lines were infected with retroviruses containing double marker genes of GFP and firefly luciferase (plasmids provided by Dr. Yibin Kang). The PEI transfection method followed Molecular Cloning. Collect over-expression and knock down retroviruses, infect the cells 3-4 times, and when the infected cells reach 80% confluency, use antibiotic media with different concentrations to screen for stable transfection lines, and multi-clonal selection to amplify , for corresponding RT-qPCR and Western Blotting verification.
6、Conditioned media(CM)收集6. Conditioned media (CM) collection
细胞近融合后传代，计数2.5-5.0×106细胞接种，贴壁后PBS冲洗残留的FBS，换用无血清培养液(CM)，收集24-72h无血清培养液，1500rpm离心10min，用0.2μm的滤膜过滤，-80℃保存。Subculture after the cells are nearly confluent, count 2.5-5.0×106 cells to inoculate, rinse the residual FBS with PBS after attachment, replace with serum-free culture medium (CM), collect 24-72h serum-free culture medium, centrifuge at 1500rpm for 10min, and use 0.2 Filter through a μm filter and store at -80°C.
收集conditioned media(CM)上清，取1ml于EP管中。加入1/9体积的100%TCA，颠倒10次混匀。样品置于冰浴中大于1小时，15000g，离心20分钟。可见有棕黑色沉淀，倒掉上清，除去残余在管口的液体。加200μl预冷的丙酮，15000g，离心20分钟，重复3次。加入50μl Loading buffer，100℃加热5分钟，-80℃保存备用。Collect the conditioned media (CM) supernatant, take 1ml into the EP tube. Add 1/9 volume of 100% TCA and mix by inverting 10 times. Samples were placed in an ice bath for more than 1 hour, centrifuged at 15,000g for 20 minutes. It can be seen that there is a brown-black precipitate, pour off the supernatant, and remove the liquid remaining in the nozzle. Add 200μl pre-cooled acetone, 15000g, centrifuge for 20 minutes, repeat 3 times. Add 50μl Loading buffer, heat at 100°C for 5 minutes, and store at -80°C for later use.
7、免疫印迹(Western Blotting)7. Western Blotting
取等量的至少20μg蛋白，使用10%SDS-Page胶电泳分离，转膜，(CM沉淀的蛋白转膜后用丽春红Ponceau S染色确定蛋白量是否相同)，5%脱脂奶粉室温封闭1小时，相应一抗4℃孵育3h或过夜。TBST洗膜5分钟，共3次，加入HRP标记二抗室温孵育1小时，TBST洗5分钟，共3次。ECL显色。Take an equal amount of at least 20 μg of protein, use 10% SDS-Page gel electrophoresis to separate, transfer to membrane, (after transferring the protein precipitated by CM, stain with Ponceau S to determine whether the protein amount is the same), block with 5% skimmed milk powder at room temperature for 1 Hours, the corresponding primary antibody was incubated at 4°C for 3 hours or overnight. Wash the membrane with TBST for 5 minutes, 3 times in total, add HRP-labeled secondary antibody and incubate at room temperature for 1 hour, wash with TBST for 5 minutes, 3 times in total. ECL color development.
8、克隆形成实验(Colony formation assay)8. Colony formation assay
准备琼脂胶：用75℃水浴锅熔解4%soft agar，42℃恒温保持其熔解，用常规培养基将4%soft agar稀释至0.6%和0.3%的浓度。0.6%soft agar作为下层胶铺于24孔板，5000或10000个细胞接种于0.5ml0.3%soft agar内，然后铺于0.6%的下层胶之上。每隔四天向孔内添加100μl培养基，2周后计数含多于50个细胞的克隆，拍照，实验重复3次。Prepare agar gel: Melt 4% soft agar in a 75°C water bath, keep it melted at a constant temperature of 42°C, and dilute 4% soft agar to a concentration of 0.6% and 0.3% with a conventional medium. 0.6% soft agar was spread on 24-well plate as the lower layer glue, 5000 or 10000 cells were inoculated in 0.5ml 0.3% soft agar, and then spread on the 0.6% lower layer glue. Add 100 μl of medium to the wells every four days, count clones containing more than 50 cells after 2 weeks, take pictures, and repeat the experiment 3 times.
9、细胞划痕实验(Wound healing assay)9. Wound healing assay
细胞生长至汇合后换用含0.5%FBS的DMEM培养12h，用新的枪头轻轻的在单层培养细胞间划四条平行的痕迹，划痕横穿过孔，枪头尽量与板孔的底部垂直，不要倾斜。划痕后，用培养基轻轻的清洗板孔3次。随后每隔4或6小时拍照，统计细胞爬行的速度，实验重复3次。After the cells grow to confluence, replace them with DMEM containing 0.5% FBS and culture them for 12 hours. Use a new pipette tip to gently draw four parallel marks between the cultured cells in the monolayer. The bottom is vertical, not slanted. After scratching, gently wash the wells 3 times with culture medium. Afterwards, pictures were taken every 4 or 6 hours, and the speed of cell crawling was counted. The experiment was repeated 3 times.
10、细胞侵袭实验(Invasion assay)10. Cell invasion assay (Invasion assay)
准备：Matrigel基质胶使用前4h从-20℃转移至4℃待其自然溶化，移液吸头等均在实验前-20℃预冷。Preparation: Matrigel was transferred from -20°C to 4°C 4 hours before use to allow it to melt naturally, and pipette tips were pre-cooled at -20°C before the experiment.
制备Matrigel胶涂层(冰上操作)：用无血清的冷细胞培养基DMEM稀释Matrigel胶(终浓度3mg/ml)，取40μl稀释胶加到24well transwell上室中，37℃孵育transwell1-2h。Prepare Matrigel gel coating (operation on ice): Dilute Matrigel gel (final concentration 3 mg/ml) with serum-free cold cell culture medium DMEM, add 40 μl diluted gel to the upper chamber of 24well transwell, and incubate transwell at 37°C for 1-2h.
准备细胞悬液：细胞近融合后传代，计数0.5×105或者1.0×105细胞，上室加200μl细胞悬液，下室中加入350μl含10%FBS的细胞培养基，37℃孵育，12小时--24小时。Prepare cell suspension: subculture after cells are nearly confluent, count 0.5×105 or 1.0×105 cells, add 200 μl cell suspension to the upper chamber, add 350 μl cell culture medium containing 10% FBS to the lower chamber, incubate at 37°C, 12 Hours - 24 hours.
染色和计数：移去transwells，在新的24孔板中用PBS轻洗小室，然后一组小室置于新的含有500μl0.1%结晶紫的well中，30min后取出，PBS清洗。照相，另一组well小室用胰酶消化，于1000rpm离心4min,去上清，用100μlPBS重悬细胞，并用hemacytometer计数这一组well跨越小室的肿瘤细胞，实验重复3次。Staining and counting: Remove the transwells, lightly wash the chambers in a new 24-well plate with PBS, then place a group of chambers in a new well containing 500 μl 0.1% crystal violet, take it out after 30 minutes, and wash with PBS. In the photo, another group of well chambers was digested with trypsin, centrifuged at 1000rpm for 4min, the supernatant was removed, the cells were resuspended in 100 μl PBS, and the tumor cells in this group of well spanning chambers were counted with a hemacytometer. The experiment was repeated 3 times.
11、Active Rho和Cdc42检测11. Detection of Active Rho and Cdc42
当细胞生长至80%汇合率时，细胞用预冷的TBS清洗一次，然后用ActiveRho and Cdc42Detection Kits(Pierce，No.16116和16119)提供的裂解液裂解细胞，收集裂解液，于16000g在4℃离心15min，收集上清，依照kits提供的protocol检测活性Rho和Cdc42。When the cells grew to 80% confluence, the cells were washed once with pre-cooled TBS, and then the cells were lysed with the lysate provided by ActiveRho and Cdc42Detection Kits (Pierce, No.16116 and 16119). The lysate was collected and stored at 16000g at 4°C After centrifugation for 15 min, the supernatant was collected, and the active Rho and Cdc42 were detected according to the protocol provided by the kits.
12、Stress fiber染色12. Stress fiber dyeing
当细胞生长至40%汇合率时，细胞用含0.5%BSA的DMEM饥饿24h，之后吸掉原培养基，用PBS洗一次然后用10%福尔马林固定15min，用0.1%Triton X-100处理10min，然后再用5U/mL rhodamine phalloidin(Invitrogen)染色20min。染色样本用激光共聚焦显微镜观察，每个样本随机选取6个视野用作Stress fiber强度的统计，统计用软件Image-Pro Plus5.1进行。When the cells grew to 40% confluence, the cells were starved for 24 hours with DMEM containing 0.5% BSA, then the original medium was sucked off, washed once with PBS and then fixed with 10% formalin for 15 minutes, and then fixed with 0.1% Triton X-100 Treat for 10min, and then stain with 5U/mL rhodamine phalloidin (Invitrogen) for 20min. The stained samples were observed with a laser confocal microscope, and 6 fields of view were randomly selected for each sample for the statistics of the intensity of the stress fiber, and the statistics were performed with the software Image-Pro Plus5.1.
13、内皮粘附检测13. Detection of endothelial adhesion
当24孔板的内皮细胞长至100%汇合率时，用含10ng/mL TNFa的培养基继续培养细胞6h，之后用PBS洗涤内皮细胞两次，然后加入2×105荧光素酶标记的肿瘤细胞于内皮层之上，1小时后，用PBS洗涤加入到内皮层的肿瘤细胞两次，再用胰酶消化粘附在内皮的肿瘤细胞，最后检测消化下来的单位体积内肿瘤细胞的化学发光强度和记录细胞悬液体积。再由预先建立的肿瘤细胞数目-单位体积内肿瘤细胞的化学发光强度标准曲线计算出粘附的处理组和对照组肿瘤细胞数目。When the endothelial cells in the 24-well plate reached 100% confluence, continue to culture the cells for 6 h in the medium containing 10 ng/mL TNFa, then washed the endothelial cells twice with PBS, and then added 2×105 luciferase-labeled tumors The cells are on the endothelial layer. After 1 hour, the tumor cells added to the endothelial layer are washed twice with PBS, and then the tumor cells adhered to the endothelium are digested with trypsin, and finally the chemiluminescence of the digested tumor cells per unit volume is detected. Intensity and record cell suspension volume. Then, the number of adhered tumor cells in the treatment group and the control group was calculated from the pre-established standard curve of the number of tumor cells-the chemiluminescence intensity of the tumor cells in unit volume.
14、跨内皮迁移检测14. Transendothelial migration detection
将内皮细胞种植在transwell的小室内，当内皮细胞生长至100%汇合率时，用PBS将内皮洗涤一次，然后将5×104肿瘤细胞种植到铺内皮的小室，小室的下面加入350μl含10ng/mL EGF的M199或M200，培养24h后，消化小室外表面的肿瘤细胞，然后以上述细胞侵袭实验计数的方法计算各个well的跨越内皮的肿瘤细胞数目。Plant the endothelial cells in the transwell chamber. When the endothelial cells grow to 100% confluence, wash the endothelium once with PBS, then plant 5×104 tumor cells into the endothelial chamber, and add 350 μl containing 10ng M199 or M200/mL EGF, cultured for 24 hours, digested the tumor cells on the outer surface of the small cell, and then counted the number of tumor cells across the endothelium in each well by the above-mentioned cell invasion assay counting method.
15、Luciferase催化的化学发光检测15. Chemiluminescence detection catalyzed by Luciferase
目的基因报告质粒和Renilla luciferase参照质粒以LipofectamineTM2000媒介共转染靶细胞。24h后，吸尽培养基，向24孔板中每孔加入50μL裂解缓冲液，然后将24孔板在室温下轻摇1h，之后依次各取出10μL细胞裂解液与30μL firefly luciferin底物混合液或30μL renilla luciferase底物混合液混合，用Lumat LB9507luminometer(Berthold)记录样本的化学发光强度。Target gene reporter plasmid and Renilla luciferase reference plasmid were co-transfected into target cells with LipofectamineTM 2000 medium. After 24 hours, aspirate the medium, add 50 μL of lysis buffer to each well of the 24-well plate, then shake the 24-well plate at room temperature for 1 hour, and then take out 10 μL of cell lysate and 30 μL of firefly luciferin substrate mixture or 30 μL of renilla luciferase substrate mixture was mixed, and the chemiluminescence intensity of the sample was recorded with a Lumat LB9507 luminometer (Berthold).
16、基因表达芯片分析16. Gene expression microarray analysis
表达空载或DLC1的SCP2细胞分别用溶剂或者10ng/Ml TGFβ处理24h，之后用Trizol抽提total RNA，然后将四组RNA送上海芯片公司(ShanghaiBiotechnology Corporation)采用Affymetrix U133plus2.0arrays进行基因表达分析。如果表达空载的SCP2某个基因在TGFβ处理条件下和溶剂处理条件下基因表达水平比值>2或者<0.5，此基因就被定义为TGFβ-响应基因，在TGFβ-响应基因中，如果表达DLC1的SCP2细胞在TGFβ处理条件下与溶剂处理表达水平相似，则被归属于A组；如果表达DLC1的SCP2细胞与表达空载的SCP2细胞在TGFβ处理条件下的表达水平之比大于2，则被归属于B组。SCP2 cells expressing empty load or DLC1 were treated with solvent or 10ng/Ml TGFβ for 24h, and then the total RNA was extracted with Trizol, and then the four sets of RNA were sent to Shanghai Biotechnology Corporation for gene expression analysis using Affymetrix U133plus2.0arrays. If a gene expressing empty SCP2 has a gene expression level ratio of >2 or <0.5 under TGFβ-treated conditions and solvent-treated conditions, the gene is defined as a TGFβ-responsive gene. Among TGFβ-responsive genes, if DLC1 is expressed If the expression level of SCP2 cells under TGFβ treatment was similar to that of solvent treatment, they were assigned to group A; if the ratio of the expression level of DLC1-expressing SCP2 cells to that of empty SCP2 cells under TGFβ treatment was greater than 2, they were classified as group A. belongs to Group B.
17、染色质免疫共沉淀17. Chromatin immunoprecipitation
表达空载或DLC1的SCP2细胞分别用SMAD3(由Rutgers University Dr.Fang Liu提供)或者SMAD3突变体(EPSM)(由Rutgers University Dr.Fang Liu提供)转染，24h后，分别用溶剂或者10ng/mL TGFβ处理24h，之后用1%甲醛交联细胞10min，然后用125mM Glycine处理5min终止甲醛作用。用胰酶消化细胞20min，之后2000rpm离心4min，PBS洗涤一次后，加入8ml细胞裂解缓冲液冰上裂解20min，5000rpm离心5min后再次加入8ml细胞裂解缓冲液冰上裂解20min，5000rpm离心5min，弃上清，向沉淀加入200μL核裂解缓冲液，让细胞核裂解10min，再向裂解液中加入300μL稀释缓冲液，然后超声处理，使基因组被打断为约500bp左右的长度。接着，以13300rpm的转速离心10min，依次取上清100μL于两个1.5ml EP管，各加入400μL稀释缓冲液，之后加入smad3抗体和Rabbit IgG孵育过夜。第二天，将该混合液与用鲑鱼精子DNA和BSA封阻过的protein A beads孵育4h，之后800g离心2min，以透析缓冲液洗涤两次，接着用洗涤缓冲液洗涤两次，然后用200μL抽提缓冲液在67℃孵育30min，离心取上清，添加RNase A和0.3M NaCl在67℃孵育5h，之后用-80℃预冷的无水乙醇沉淀DNA，最后用DNAkit(TIANGEN)纯化DNA。SCP2 cells expressing empty load or DLC1 were transfected with SMAD3 (provided by Rutgers University Dr. Fang Liu) or SMAD3 mutant (EPSM) (provided by Rutgers University Dr. Fang Liu) respectively. After 24h, they were respectively treated with solvent or 10ng/ The cells were treated with mL TGFβ for 24 hours, then cross-linked with 1% formaldehyde for 10 minutes, and then treated with 125 mM Glycine for 5 minutes to terminate the formaldehyde effect. Digest the cells with trypsin for 20min, then centrifuge at 2000rpm for 4min, wash once with PBS, add 8ml of cell lysis buffer to lyse on ice for 20min, centrifuge at 5000rpm for 5min, then add 8ml of cell lysis buffer to lyse on ice for 20min, centrifuge at 5000rpm for 5min, discard Clear, add 200 μL of nuclear lysis buffer to the pellet, let the cell nuclei lyse for 10 minutes, then add 300 μL of dilution buffer to the lysate, and then sonicate to break the genome into a length of about 500 bp. Then, centrifuge at 13300rpm for 10min, take 100μL of the supernatant in two 1.5ml EP tubes, add 400μL of dilution buffer to each, and then add smad3 antibody and Rabbit IgG to incubate overnight. The next day, the mixture was incubated with protein A beads blocked with salmon sperm DNA and BSA for 4 h, then centrifuged at 800 g for 2 min, washed twice with dialysis buffer, then washed twice with washing buffer, and then washed with 200 μL Incubate the extraction buffer at 67°C for 30min, centrifuge to take the supernatant, add RNase A and 0.3M NaCl and incubate at 67°C for 5h, then precipitate the DNA with -80°C pre-cooled absolute ethanol, and finally purify the DNA with DNAkit (TIANGEN) .
18、破骨细胞成熟18. Osteoclast maturation
将5ng/mL TGFβ并之1.0μg/mL C3或者10μM Y27632，或者10μM6-TG处理空载组或处理组细胞72h，或者先用RhoA(63L)病毒(表达RhoA(63L)的质粒由复旦大学Dr.Lan Ma提供)感染表达空载或DLC1的SCP2细胞48h，之后用5ng/mL TGFβ处理病毒感染过的细胞72h，收集培养基待用。将4至6周大的小鼠引颈处死，立即取其后肢，去皮肉，无菌条件下用ɑ-MEM冲出骨髓，1000rpm离心4min，重悬细胞，将骨髓细胞置于24孔板培养，每孔细胞数目为1百万个。将上述培养基以10倍稀释的浓度添加到种植有骨髓细胞的24孔板中，并加入浓度为25ng/mL的M-CSF(PeproTech)，六天后按照TRAP染色试剂盒(Sigma387A)提供的protocol染色观察破骨细胞成熟，细胞染色呈现阳性并且含有不少于3个细胞核的细胞被定义为成熟的破骨细胞。Combine 5ng/mL TGFβ with 1.0μg/mL C3 or 10μM Y27632, or 10μM 6-TG to treat the empty group or treatment group cells for 72h, or first use RhoA(63L) virus (a plasmid expressing RhoA(63L) was provided by Dr. Fudan University provided by Lan Ma) to infect SCP2 cells expressing empty load or DLC1 for 48 hours, then treat the virus-infected cells with 5 ng/mL TGFβ for 72 hours, and collect the medium for use. Mice aged 4 to 6 weeks were killed by necking, and their hind limbs were removed immediately, the skin and flesh were removed, and the bone marrow was washed out with ɑ-MEM under sterile conditions, centrifuged at 1000rpm for 4 minutes, the cells were resuspended, and the bone marrow cells were cultured in a 24-well plate. The number of cells per well was 1 million. Add the above-mentioned medium at a concentration of 10 times dilution to a 24-well plate seeded with bone marrow cells, and add M-CSF (PeproTech) at a concentration of 25 ng/mL. Six days later, follow the protocol provided by the TRAP staining kit (Sigma387A) Staining was used to observe the maturation of osteoclasts. Cells with positive staining and no less than 3 nuclei were defined as mature osteoclasts.
此外，为近一步确定条件培养基作用的靶细胞，105RAW264.7单独或者与同样数目的被WNT3a诱导成熟的C2C12一起种植于48孔板，之后将5ng/mLTGFβ处理表达空载或Dlc1的4T1细胞(美国Karmanos癌症研究所Fred Miller博士提供)72h后的培养基以十倍稀释的终浓度添加于种植有RAW264.7的孔板内，以上述相同方法观察破骨细胞的成熟。In addition, in order to further determine the target cells of the conditioned medium, 105 RAW264.7 alone or with the same number of C2C12 induced by WNT3a to mature were planted in a 48-well plate, and then 5 ng/mL LTGFβ was treated with empty or Dlc1-expressing cells. The culture medium of 4T1 cells (provided by Dr. Fred Miller, Karmanos Cancer Institute, USA) after 72 hours was added to the well plate planted with RAW264.7 at a final concentration of ten-fold dilution, and the maturation of osteoclasts was observed in the same way as above.
19、动物饲养19. Animal feeding
4-8周的Balb/c nude裸鼠购自上海斯莱克动物中心，饲养于SPF级屏障系统内(22-25℃，40-60%的相对湿度，12h的昼夜循环以及食物和水自由摄取)。Balb/c nude mice aged 4-8 weeks were purchased from Shanghai Slack Animal Center and were raised in an SPF-grade barrier system (22-25°C, 40-60% relative humidity, 12h day-night cycle and free intake of food and water). ).
20、小鼠左心室注射20. Mouse left ventricle injection
麻醉：1%戊巴比妥钠腹腔注射麻醉，每只裸鼠按30-40mg/kg的量分别计算所需的麻醉药剂量。Anesthesia: Anesthetize by intraperitoneal injection of 1% sodium pentobarbital, and calculate the required anesthetic dose for each nude mouse according to the amount of 30-40 mg/kg.
75%酒精前胸壁消毒，用手触及心尖搏动最明显处，约于胸骨左旁3mm第二肋间，瞄准正中与身体呈45度进针，回抽，若能看到有鲜红的血液喷出，则表示针己进入左心室，将细胞悬液慢慢推入后，迅速拔针。Before disinfecting the chest wall with 75% alcohol, touch the most obvious part of the heart apex with your hands, about 3 mm to the left of the sternum in the second intercostal space, aim at the center and insert the needle at a 45-degree angle to the body, and pull back. If you can see bright red blood spurting out , indicating that the needle has entered the left ventricle, slowly push the cell suspension in, and then pull out the needle quickly.
注射细胞量为：按1.0×106细胞/ml，0.1ml/只。按75mg/kg的剂量眼底注射D-luciferin(裸鼠静脉注射与此相同)，每周Berthold Imaging System成像，拍照。注射后继续饲养，自由进食，观察裸鼠生活状态及一般情况；注射后24小时内应严密观察裸鼠的生命体征和状态。The amount of injected cells is: 1.0×106 cells/ml, 0.1ml/head. D-luciferin was injected into the fundus at a dose of 75mg/kg (intravenous injection of nude mice was the same), and Berthold Imaging System imaging was performed every week, and photographs were taken. After the injection, continue to feed, eat freely, observe the living conditions and general conditions of the nude mice; within 24 hours after the injection, the vital signs and status of the nude mice should be closely observed.
在肿瘤细胞注射第三天，根据实验需要，处理组注射Y27632(8mg/kg溶于100μL PBS中，1次/2天，腹腔注射)或Fasudil(50mg/kg溶于100μL PBS中，1次/1天，皮下注射)或6-TG(1.0mg/kg溶于100μL PBS中，1次/1天，皮下注射)，对照组注射溶剂，药物处理时间为4-8周。On the third day of tumor cell injection, according to the needs of the experiment, the treatment group was injected with Y27632 (8 mg/kg dissolved in 100 μL PBS, 1 time/2 days, intraperitoneal injection) or Fasudil (50 mg/kg dissolved in 100 μL PBS, 1 time/ 1 day, subcutaneous injection) or 6-TG (1.0mg/kg dissolved in 100μL PBS, 1 time/1 day, subcutaneous injection), the control group was injected with solvent, and the drug treatment time was 4-8 weeks.
21、小鼠静脉注射21. Intravenous injection of mice
汞灯照射小鼠使血管扩张，固定后进针。进针前针尖斜面向上，一手持住尾巴另手持针，以针管与静脉血管平行的方向缓慢进针，刺进2-3mm后可清楚看到针尖斜面，此时缓慢试着推注，见沿静脉一条白线进入小鼠身体，注射细胞量为：按2.0×106细胞/ml，0.1ml/只。注射完毕后，退针，按住入注部位快速拔针，并按压片刻止血。The mouse was irradiated with mercury lamp to dilate the blood vessels, and the needle was inserted after fixation. Before inserting the needle, the bevel of the needle tip is facing upwards. Hold the tail in one hand and the needle in the other. Slowly insert the needle in a direction parallel to the venous vessel. After puncturing 2-3mm, you can clearly see the bevel of the needle tip. At this time, slowly try to push the injection. A white line of vein enters the body of the mouse, and the injected cell volume is: 2.0×106 cells/ml, 0.1ml/mouse. After the injection, withdraw the needle, press the injection site and quickly pull out the needle, and press for a while to stop the bleeding.
22、脂肪垫原位注射22. In situ injection of fat pad
细胞密度2.0×107细胞/ml，与Matrigel1:1混合放置冰上。小鼠麻醉后解剖暴露脂肪垫，按10μL/只注射于腹部左侧4号脂肪垫。The cell density was 2.0×107 cells/ml, mixed with Matrigel 1:1 and placed on ice. After the mice were anesthetized, the fat pads were dissected and exposed, and 10 μL/mouse was injected into the No. 4 fat pad on the left side of the abdomen.
23、乳腺癌转移灶的检测23. Detection of breast cancer metastases
当注射进肿瘤细胞的小鼠出现肉眼可见的骨损伤时，小鼠取出做X线检查。之后解剖取出骨转移灶，固定后用10%中性EDTA于4℃处理两周，之后进行常规包埋切片做H&E和免疫组化染色。对于肺转移的病灶，则是在肿瘤细胞尾静脉注射后四-六周解剖小鼠摘取肺做常规包埋切片做H&E。When the mice injected with tumor cells showed visible bone damage, the mice were taken out for X-ray examination. Afterwards, bone metastases were dissected and removed, fixed and treated with 10% neutral EDTA at 4°C for two weeks, and routinely embedded for H&E and immunohistochemical staining. For lung metastases, the mice were dissected four to six weeks after the tail vein injection of tumor cells, and the lungs were removed for routine embedded sectioning for H&E.
24、临床样本分析24. Analysis of clinical samples
Bos等和van‘t Veer等(Bos,P.D.,et al.Nature459,1005-1009(2009)；Minn,A.J.,et al.Nature436,518-524(2005)；van‘t Veer,L.J.,et al.Nature415,530-536(2002))描述的样本根据其DLC1表达水平中值被分成两组：DLC1表达组和DLC沉默组。然后比较两组的Cox survival曲线。此外，从山东大学齐鲁医院获取了64例乳腺癌原位瘤标本，用Trizol提取了标本的总RNA，做了DLC1mRNA和PTHLH mRNA qPCR定量分析，进一步做了它们的表达相关性分析。还从上海长征医院获得了乳腺癌原位癌样本、乳腺癌淋巴结转移灶样本和乳腺癌骨转移灶样本。这些样本经过常规的IHC分析其DLC1的表达水平，结果以0，1，2，3表示。0表示DLC1没有表达；1表示DLC1低水平表达；2表示DLC1以中等水平表达；3表示DLC1高水平表达。Bos et al. and van't Veer et al. (Bos, P.D., et al. Nature459, 1005-1009 (2009); Minn, A.J., et al. Nature436, 518-524 (2005); van't Veer, L.J., et al The samples described in Nature 415, 530-536 (2002)) were divided into two groups according to their median DLC1 expression levels: DLC1 expression group and DLC silence group. Then compare the Cox survival curves of the two groups. In addition, 64 breast cancer in situ tumor specimens were obtained from Qilu Hospital of Shandong University, the total RNA of the specimens was extracted with Trizol, quantitative analysis of DLC1 mRNA and PTHLH mRNA by qPCR was carried out, and their expression correlation analysis was further carried out. Samples of carcinoma in situ of breast cancer, lymph node metastases of breast cancer and bone metastases of breast cancer were also obtained from Shanghai Changzheng Hospital. These samples were analyzed by conventional IHC for the expression level of DLC1, and the results were expressed as 0, 1, 2, 3. 0 means no expression of DLC1; 1 means low level expression of DLC1; 2 means medium level expression of DLC1; 3 means high level expression of DLC1.
25、数据分析25. Data Analysis
体外实验中对照组和实验组之间的差异使用双尾或单尾student’s t检验。P值小于0.05被认为具有显著性差异。BLI曲线采用ANOVA分析进行比较，动物或病人的survival曲线通过Log-rank test检验其差异显著性。Differences between control and experimental groups in in vitro experiments were analyzed using the two-tailed or one-tailed Student's t test. A P value of less than 0.05 was considered to have a significant difference. The BLI curves were compared by ANOVA analysis, and the survival curves of animals or patients were tested for significant difference by Log-rank test.
实施例1、DLC1是乳腺癌骨转移抑制因子Embodiment 1, DLC1 is breast cancer bone metastasis inhibitory factor
Yibin Kang等(Kang，Y.，et al.Cancer Cell3，537-549(2003))通过体内筛选和体外挑单克隆体内验证的方法从MDA-MB-231细胞筛选到具有不同器官转移倾向的细胞亚系，这些亚系的DLC1表达水平见图1。Yibin Kang et al. (Kang, Y., et al. Cancer Cell3, 537-549 (2003)) screened from MDA-MB-231 cells to cells with different organ metastasis tendencies through in vivo screening and in vitro single clone verification in vivo sublines, and the DLC1 expression levels of these sublines are shown in Figure 1.
从图1可以看出DLC1在各种亚系中的表达水平只与其骨转移能力反相关(图1A)，与其肺转移能力没有明显关联性(图1B)。因此本发明人聚焦于DLC1在乳腺癌骨转移中的功能与机制的研究。It can be seen from Figure 1 that the expression level of DLC1 in various sublines is only inversely correlated with its bone metastatic ability (Fig. 1A), but not significantly correlated with its lung metastatic ability (Fig. 1B). Therefore, the inventors focused on the research on the function and mechanism of DLC1 in bone metastasis of breast cancer.
首先本发明人在DLC1沉默的高骨转移细胞系SCP2中过表达DLC1(应用pBabepuro-DLC1)(图2A)，并将过表达DLC1和空载的稳转系通过左心室注射进雌性裸鼠体内观察DLC1过表达对骨转移的影响。在第6周，过表达组的平均BLI值比对照组小10倍以上(图2B)，病理切片显示过表达组的骨转移灶大小明显小于对照组(图3C)，过表达组的小鼠无骨转移生存率明显高于对照组(图2D)。First, the inventors overexpressed DLC1 (pBabepuro-DLC1) in the DLC1-silenced high bone metastases cell line SCP2 (Figure 2A), and injected the overexpressed DLC1 and empty-loaded stable transfection lines into female nude mice through the left ventricle To observe the effect of DLC1 overexpression on bone metastasis. At week 6, the average BLI value of the overexpression group was more than 10 times smaller than that of the control group (Fig. 2B), and pathological sections showed that the size of bone metastases in the overexpression group was significantly smaller than that of the control group (Fig. The bone metastasis-free survival rate was significantly higher than that of the control group (Fig. 2D).
之后，为探讨DLC1的沉默是否为乳腺癌细胞骨转移能力增加所需，本发明人用DLC1高表达的具有中等骨转移能力的SCP28细胞系构建了靶向DLC1不同序列区的两株knockdown稳转系KD1、KD2和空载稳转系(空载为pSuper-Retro-puro)(图3A)。用类似的方法将过DLC1knockdown和空载的稳转系通过左心室注射进雌性裸鼠体内观察DLC1knockdown对SCP28骨转移的影响。在第5周，KD1或KD2组的平均BLI值比对照组高出10倍以上(图3B)，病理切片和X射线显示KD1或KD2组的骨转移灶中肿瘤细胞对骨的侵害明显强于对照组(图3C和3D)，KD1或KD2组的小鼠无骨转移生存率明显低于对照组(图3E)。Later, in order to explore whether the silencing of DLC1 is required for the increased ability of breast cancer cells to metastasize to bone, the inventors used the SCP28 cell line with high expression of DLC1 and moderate bone metastases to construct two stable knockdown strains targeting different sequence regions of DLC1 Line KD1, KD2 and no-load stable rotation line (no-load is pSuper-Retro-puro) (Fig. 3A). In a similar way, DLC1knockdown and empty stable transfection lines were injected into female nude mice through the left ventricle to observe the effect of DLC1knockdown on SCP28 bone metastasis. At week 5, the average BLI value of the KD1 or KD2 group was more than 10 times higher than that of the control group (Fig. 3B). Pathological sections and X-rays showed that tumor cells in the bone metastases of the KD1 or KD2 group had significantly stronger invasion of bone than that of the control group. The bone metastasis-free survival rate of mice in the control group (Fig. 3C and 3D), KD1 or KD2 group was significantly lower than that in the control group (Fig. 3E).
其次，为进一步探讨DLC1在骨转移中的功能是否受免疫系统的影响，本发明人在源自小鼠的乳腺癌细胞4T1里过表达(转入pMSCV-puro)了小鼠的Dlc1(图4A)，然后用类似的方法观察过表达Dlc1对4T1骨转移的影响。本发明人发现，在第5周，过表达组的骨转移灶数目比对照组少5倍以上(图4B)，病理切片显示过表达组骨转移灶中肿瘤细胞对骨的侵害明显轻于对照组(图4C)，过表达组小鼠无骨转移生存率也明显高于对照组(图4D)。Secondly, in order to further explore whether the function of DLC1 in bone metastasis is affected by the immune system, the inventors overexpressed (transferred into pMSCV-puro) mouse Dlc1 in breast cancer cells 4T1 derived from mice (Fig. 4A ), and then use a similar method to observe the effect of overexpression of Dlc1 on 4T1 bone metastasis. The inventors found that at week 5, the number of bone metastases in the overexpression group was more than 5 times less than that in the control group (Figure 4B), and pathological sections showed that the tumor cells in the bone metastases in the overexpression group had significantly lighter invasion of bone than the control group. group (Figure 4C), and the bone metastasis-free survival rate of mice in the overexpression group was also significantly higher than that in the control group (Figure 4D).
鉴于DLC1是Rho通路重要的负调控因子，以往的报道表明DLC1在癌细胞向肺转移的过程中也起负调控作用(Ko，F.C.，et al.Nature communications4，1618(2013)和Goodison，S.，et al.Cancer Res65，6042-6053(2005))，因此本发明人同时也在SCP28和4T1中观察了DLC1在乳腺癌肺转移过程中的作用。结果显示无论是从BLI或是病理切片上分析(图5A、B、D、E)，还是从生存曲线上分析(图5C、F)，处理组和对照组小鼠的肺转移灶形成数目和大小基本相差无几。上述结果表明DLC1是乳腺癌器官(骨)特异性转移的负调控因子。Given that DLC1 is an important negative regulator of the Rho pathway, previous reports have shown that DLC1 also plays a negative regulatory role in the process of cancer cell metastasis to the lung (Ko, F.C., et al. Nature communications 4, 1618 (2013) and Goodison, S. , et al.Cancer Res65, 6042-6053 (2005)), so the inventor also observed the role of DLC1 in the process of breast cancer lung metastasis in SCP28 and 4T1. The results showed that whether it was analyzed from BLI or pathological sections (Fig. 5A, B, D, E), or analyzed from the survival curve (Fig. 5C, F), the number of lung metastases formed in the treatment group and the control group mice and Basically the same size. The above results indicate that DLC1 is a negative regulator of breast cancer organ (bone)-specific metastasis.
实施例2、Rho活性的抑制介导了DLC1在骨转移中的功能Example 2. Inhibition of Rho activity mediates the function of DLC1 in bone metastasis
DLC1虽然是Rho通路重要的负调控因子，但DLC1本身活性也受到其他蛋白的调控，在本发明人的细胞模型中也存在DLC1失活的可能，因此本发明人检测了DLC1对Rho通路及下游的一些蛋白的活性的影响。Although DLC1 is an important negative regulator of the Rho pathway, the activity of DLC1 itself is also regulated by other proteins. In the inventor's cell model, there is also the possibility of DLC1 inactivation. Therefore, the inventors tested the effect of DLC1 on the Rho pathway and its downstream activity of some proteins.
通过Rho活性及下游蛋白的磷酸化分析发现，DLC1在SCP2和SCP28中依然能失活Rho蛋白，并且对下游蛋白的激活也呈现抑制的现象(图6A)；在原位的条件下，DLC1也抑制了标志RhoA激活的应力纤维的形成(图6B)。According to the phosphorylation analysis of Rho activity and downstream proteins, DLC1 can still inactivate Rho protein in SCP2 and SCP28, and also inhibit the activation of downstream proteins (Figure 6A); under in situ conditions, DLC1 can also inactivate Rho protein Stress fiber formation, a marker of RhoA activation, was inhibited (Fig. 6B).
本发明人接着又探索了Rho活性的抑制是否介导DLC1在骨转移中的功能。本发明人之前在构建DLC1过表达的SCP2稳转系(应用pBabepuro-DLC1)时同时构建了一株含有DLC1GAP失活突变体过表达的SCP2稳转系(R718E)(应用pBabepuro-DLC1(R718E))，这些稳转系的成功建立通过检测Rho和Cdc42活性证实(图7A)。这两株细胞系和对照细胞系通过左心室注射的方式注入裸鼠体内，之后连续观察它们形成骨转移的情况。发现在第6周，对照组比过表达组(DLC1过表达的SCP2稳转系)BLI值高出15倍以上，但与GAP失活突变体组差别不大(图7B)，病理切片显示过表达组骨转移灶中肿瘤细胞对骨的侵害明显轻于GAP失活突变体组和对照组，但后二者之间无显著差异(图7C)，过表达组小鼠无骨转移生存率也明显高于GAP失活突变体组和对照组，后二者之间也无显著差异(图7D)。说明Rho活性的抑制介导了DLC1在骨转移中的功能。The inventors next explored whether inhibition of Rho activity mediates the function of DLC1 in bone metastasis. The present inventors constructed a stable SCP2 line (R718E) containing the overexpression of the DLC1GAP inactivation mutant (using pBabepuro-DLC1 (R718E)) while constructing the SCP2 stable transduction line (using pBabepuro-DLC1) of DLC1 overexpression. ), the successful establishment of these stable transgenic lines was confirmed by detecting the activities of Rho and Cdc42 (Fig. 7A). These two cell lines and a control cell line were injected into nude mice via left ventricle injection, and then they were serially observed for the formation of bone metastases. It was found that at week 6, the BLI value of the control group was more than 15 times higher than that of the overexpression group (DLC1 overexpressed SCP2 stable line), but it was not significantly different from the GAP inactivation mutant group (Figure 7B). The invasion of tumor cells in bone metastases in the expression group was significantly lighter than that of the GAP inactivation mutant group and the control group, but there was no significant difference between the latter two (Figure 7C), and the bone metastasis-free survival rate of mice in the overexpression group was also significantly higher than that of the GAP inactivation mutant group and the control group, and there was no significant difference between the latter two (Fig. 7D). It shows that the inhibition of Rho activity mediates the function of DLC1 in bone metastasis.
实施例3、DLC1对Rho通路的抑制阻碍破骨细胞的成熟Example 3. Inhibition of Rho pathway by DLC1 prevents maturation of osteoclasts
上述结果表明，DLC1通过Rho通路的抑制介导其在骨转移中负调控因子的角色。而在向肺转移的过程中DLC1的功能影响不显著，这使得本发明人进一步探索DLC1抑制乳腺癌器官特异性转移的机制。由于癌细胞与靶器官内皮的粘附是癌细胞形成转移的重要一步，本发明人首先检测了DLC1对SCP2和SCP28与骨内皮和肺内皮细胞的粘附以及它们跨内皮的迁移能力的影响。结果显示DLC1均抑制了乳腺癌细胞与骨和肺内皮的粘附(图8A和C)，而肿瘤细胞跨两类内皮的能力类似地被DLC1抑制(图8B和D)。The above results suggest that DLC1 mediates its role as a negative regulator in bone metastasis through inhibition of the Rho pathway. However, the function of DLC1 was not significantly affected in the process of lung metastasis, which led the inventors to further explore the mechanism of DLC1 inhibiting breast cancer organ-specific metastasis. Since the adhesion of cancer cells to the endothelium of target organs is an important step in the formation of metastasis of cancer cells, the inventors first examined the effect of DLC1 on the adhesion of SCP2 and SCP28 to bone endothelium and lung endothelial cells and their ability to migrate across the endothelium. The results showed that DLC1 inhibited both the adhesion of breast cancer cells to bone and lung endothelium (Figure 8A and C), while the ability of tumor cells to cross both types of endothelium was similarly inhibited by DLC1 (Figure 8B and D).
显然单纯从肿瘤细胞与内皮的粘附上不足以解释DLC1特异性地抑制乳腺癌细胞向骨的转移。因此，本发明人接着探讨DLC1对乳腺癌在骨形成克隆的影响。由于破骨细胞在癌细胞形成骨转移的过程中具有关键性的作用，本发明人检测了DLC1在乳腺癌细胞促进破骨细胞成熟过程中的作用。此外，鉴于TGFβ在骨转移中的关键作用，本发明人就用TGFβ处理乳腺癌细胞，然后用培养基去诱导小鼠骨髓中破骨细胞前体成熟。小鼠骨髓细胞在肿瘤细胞condtioned medium的培养显示，肿瘤细胞的DLC1显著抑制了破骨细胞的成熟(图9A-C)，抑制率大于50%；而在SCP28骨转移瘤中，DLC1沉默组瘤-骨界面单位长度上的破骨细胞数目则是对照组的3倍至5倍(图9D-E)。Obviously, the adhesion of tumor cells to endothelium alone is not enough to explain the specific inhibition of breast cancer cell metastasis to bone by DLC1. Therefore, the present inventors next explored the effect of DLC1 on the bone-forming clones of breast cancer. Since osteoclasts play a key role in the process of cancer cells forming bone metastases, the inventors detected the role of DLC1 in the process of breast cancer cells promoting osteoclast maturation. In addition, in view of the key role of TGFβ in bone metastasis, the inventors treated breast cancer cells with TGFβ, and then used culture medium to induce the maturation of osteoclast precursors in mouse bone marrow. The culture of mouse bone marrow cells in tumor cell conditioned medium showed that the DLC1 of tumor cells significantly inhibited the maturation of osteoclasts (Figure 9A-C), and the inhibition rate was greater than 50%. In SCP28 bone metastases, DLC1 silenced tumors - The number of osteoclasts per unit length of the bone interface was 3-5 times that of the control group (Fig. 9D-E).
之后，本发明人又继续探讨Rho通路的抑制是否介导了DLC1在破骨细胞成熟过程中的效应。本发明人用Rho通路的抑制剂C3和Y27632或者RhoA持续激活的突变体表达的反转病毒(RhoA(63L)亚克隆到pMSCV-hygro的Bgl II和Xho I位点，然后以此质粒转染H29)处理肿瘤细胞，然后用上述相同的实验方法检测处理过的肿瘤细胞对破骨细胞成熟的影响。RAP染色显示：Rho通路抑制剂C3和Y27632挽救了DLC1knockdown对破骨细胞成熟的促进(图10A)，它们对破骨细胞的成熟的影响类似于DLC1过表达对破骨细胞的成熟的影响(图10B)；在SCP2骨转移瘤中，相比于对照组DLC1的过表达减少了单位瘤-骨界面长度上的破骨细胞数目约9倍，但DLC1GAP失活突变体过表达对SCP2瘤-骨界面单位长度上的破骨细胞数目影响不显著(图10C-D)。这些结果表明Rho通路的抑制介导了DLC1在破骨细胞成熟过程中的效应。Afterwards, the inventors went on to explore whether the inhibition of Rho pathway mediates the effect of DLC1 in the process of osteoclast maturation. The present inventor subclones the retrovirus (RhoA (63L) expressed by inhibitors C3 and Y27632 of the Rho pathway or the continuously activated mutant of RhoA into the Bgl II and Xho I sites of pMSCV-hygro, and then transfects with this plasmid H29) treat the tumor cells, and then use the same experimental method as above to detect the effect of the treated tumor cells on the maturation of osteoclasts. RAP staining showed that: Rho pathway inhibitors C3 and Y27632 rescued the promotion of DLC1knockdown on osteoclast maturation (Fig. 10A), and their effects on osteoclast maturation were similar to the effects of DLC1 overexpression on osteoclast maturation (Fig. 10B); in SCP2 bone metastases, the overexpression of DLC1 reduced the number of osteoclasts in the length of the unit tumor-bone interface by about 9 times compared with the control group, but the overexpression of DLC1GAP inactivation mutant had no effect on SCP2 tumor-bone The number of osteoclasts per interface unit length was not significantly affected (Fig. 10C-D). These results suggest that inhibition of the Rho pathway mediates the effects of DLC1 during osteoclast maturation.
实施例4、DLC1抑制TGFβ诱导的PTHLH表达上调Example 4, DLC1 inhibits TGFβ-induced up-regulation of PTHLH expression
虽然DLC1抑制了肿瘤细胞诱导破骨细胞的成熟，但其机制未明。鉴于TGFβ在骨转移过程中具有关键的调控作用(Chen，Y.C.等，Breast Cancer Res12，215(2010))，而Rho通路和TGFβ通路也被报道有crosstalk(Kamaraju，A.K等，The Journal of biological chemistry280，1024-1036(2005))，因此本发明人做了四组样本处理：表达空载(pBabepuro，获自Addgene)或DLC1(pBabepuro-DLC1)的SCP2细胞分别用溶剂或者10ng/mL TGFβ处理24h，然后提取总RNA送芯片公司做基因表达芯片分析。表达芯片结果表明TGFβ诱导了其通路靶向基因的表达，同时还发现DLC1调控了两组受TGFβ调节表达的基因：A组基因，空载细胞中受TGFβ调节表达，表达水平在DLC1过表达和TGFβ处理条件下与溶剂处理表达水平相似；B组基因，空载细胞中受TGFβ调节表达，但表达DLC1的SCP2细胞与表达空载的SCP2细胞在TGFβ处理条件下的表达水平之比大于2(图11A-B)。在A组基因中发现排在第一位的是PTHLH，相比于对照组DLC1组中PTHLH表达被DLC1抑制了约3.2倍。本发明人用Western blot验证了DLC1在TGFβ处理条件下对PTHLH表达的调控，结果同基因芯片一致，并且还发现这种调控依赖于Rho通路(图11C-D)。Although DLC1 inhibits tumor cell-induced osteoclast maturation, the mechanism remains unclear. In view of the fact that TGFβ has a key regulatory role in the process of bone metastasis (Chen, Y.C. et al., Breast Cancer Res12, 215 (2010)), and the Rho pathway and TGFβ pathway have also been reported as crosstalk (Kamaraju, A.K et al., The Journal of biological chemistry280 , 1024-1036 (2005)), so the inventors did four groups of sample processing: SCP2 cells expressing empty load (pBabepuro, obtained from Addgene) or DLC1 (pBabepuro-DLC1) were treated with solvent or 10ng/mL TGFβ for 24h respectively , and then extract total RNA and send it to the microarray company for gene expression microarray analysis. The expression microarray results showed that TGFβ induced the expression of its pathway-targeted genes, and it was also found that DLC1 regulated two groups of genes whose expression was regulated by TGFβ: Group A genes, whose expression was regulated by TGFβ in empty cells, and whose expression level was between DLC1 overexpression and The expression level of TGFβ treatment was similar to that of solvent treatment; group B genes were regulated by TGFβ in empty cells, but the ratio of expression levels of DLC1-expressing SCP2 cells to empty-load SCP2 cells was greater than 2 ( Figure 11A-B). Among the genes in group A, it was found that PTHLH ranked first, and the expression of PTHLH was inhibited by DLC1 by about 3.2 times compared with the control group DLC1 group. The inventors used Western blot to verify the regulation of DLC1 on the expression of PTHLH under the condition of TGFβ treatment, and the result was consistent with the gene chip, and also found that this regulation was dependent on the Rho pathway (Fig. 11C-D).
鉴于PTHLH在骨转移中的重要性，本发明人进一步研究PTHLH是否介导了DLC1在骨转移中的角色，设计了两组实验：其一，用PTHLH抑制剂6-TG定期定量皮下注射被注入稳定表达DLC1和空载的SCP2的裸鼠，观察6-TG的应用对它们形成骨转移的影响；其二，本发明人在稳定表达Dlc1的4T1和空载细胞系里再稳定表达小鼠Pthlh(由载体pMSCV-hygro表达)和空载(pMSCV-hygro)，之后将它们通过左心室注射注入雌性小鼠体内，观察它们的骨转移形成。这两组实验结果显示，6-TG的应用使得稳定表达DLC1和空载的SCP2形成骨转移的能力几乎一致(图12A-D)，而Pthlh在已稳定表达DLC1和空载的4T1中的表达使得这两株4T1亚系形成骨转移的能力相近(图12E-F)。上述结果说明PTHLH介导了DLC1在乳腺癌骨转移过程中的功能。In view of the importance of PTHLH in bone metastasis, the inventors further studied whether PTHLH mediates the role of DLC1 in bone metastasis, and designed two sets of experiments: first, regular quantitative subcutaneous injections with PTHLH inhibitor 6-TG were injected Nude mice stably expressing DLC1 and empty SCP2, and observing the effect of 6-TG application on their formation of bone metastases; Second, the inventors stably express mouse Pthlh in 4T1 and empty cell lines stably expressing Dlc1 (expressed by vector pMSCV-hygro) and empty vehicle (pMSCV-hygro), and then they were injected into female mice via left ventricle injection to observe their bone metastases formation. The results of these two groups of experiments showed that the application of 6-TG made the ability of stably expressing DLC1 and empty SCP2 to form bone metastases almost identical (Figure 12A-D), while the expression of Pthlh in the stable expression of DLC1 and empty 4T1 The ability of these two 4T1 sublines to form bone metastases was made similar (Fig. 12E-F). The above results indicate that PTHLH mediates the function of DLC1 in the process of bone metastasis of breast cancer.
实施例5、DLC1通过调控Smad3linker region的磷酸化抑制TGFβ诱导的PTHLH表达上调Example 5. DLC1 inhibits the upregulation of PTHLH expression induced by TGFβ by regulating the phosphorylation of Smad3linker region
虽然DLC1调控了PTHLH的表达，但它是如何调控PTHLH的表达不得而知，为了解决这个问题，本发明人首先使用受TGFβ通路影响的其他多种通路的抑制剂处理SCP2，发现PTHLH的表达基本不受影响(图13A)。接着本发明人在SCP2中沉默了Smad4(应用SMAD4knockdown表达质粒)，发现PTHLH的表达在DLC1和空载表达的SCP2中相差不大且与无TGFβ处理条件下的表达水平相近(图13B-C)。这说明DLC1调控PTHLH的表达是Smad依赖的。Although DLC1 regulates the expression of PTHLH, it is unknown how it regulates the expression of PTHLH. In order to solve this problem, the inventors first used inhibitors of other pathways affected by the TGFβ pathway to treat SCP2, and found that the expression of PTHLH was basically Unaffected (Fig. 13A). Then the inventor silenced Smad4 in SCP2 (using the SMAD4knockdown expression plasmid), and found that the expression of PTHLH was not much different in DLC1 and empty-loaded SCP2 and was similar to the expression level under the condition of no TGFβ treatment (Fig. 13B-C) . This shows that DLC1 regulates the expression of PTHLH is Smad-dependent.
以前有文献报道Rho通路能调控Smad3的磷酸化，本发明人检测了DLC1对Smad3的磷酸化的影响。结果显示DLC1下调了Smad3上204和208位上丝氨酸的磷酸化水平(图14A)。鉴于Jungeun Lee等报道TGFβ也能激活Rho通路(Lee，J.，Moon等，J Biol Chem285，26618-26627(2010))，所以本发明人接着检测当SCP2接受TGFβ的刺激后，RhoA活性是否上调。当用10ng/ml的TGFβ处理24h后，通过active Rho kits(购自Pierce)检测发现，对照组RhoA活性显著上调，但过表达组未见RhoA活性发生显著改变。这说明DLC1在抑制TGFβ对RhoA活性的上调方面发挥着压倒性的作用(图14B)。然后本发明人用204和208位上丝氨酸突变为丙氨酸的Smad3的突变体(EPSM)或野生型Smad3表达质粒与PTHLH启动子报告质粒或含有Smad3结合序列突变的PTHLH启动子报告质粒共转染。转染结果显示204和208位上丝氨酸的磷酸化是Smad3通过Smad3结合序列媒介其激活PTHLH启动子活性所必需的(图14C)，因为当Smad3204和208位丝氨酸突变为丙氨酸后PTHLH启动子活性与DLC1同Smad3共转的活性相似，均远低于对照组仅Smad3转染后的PTHLH启动子活性。进一步的ChIP分析表明DLC1同Smad3或EPSM共转后因Smad3结合PTHLH启动子而被免疫沉淀下来的PTHLH启动子片段拷贝数与只转染EPSM的空载组几乎相同，都比只转染Smad3的空载组少了至少2.6倍。这暗示204和208位上丝氨酸的磷酸化加强了Smad3和PTHLH启动子的相互作用，这两个位点的磷酸化是PTHLH转录所必需的(图14D)。It was previously reported that the Rho pathway can regulate the phosphorylation of Smad3, and the inventors detected the effect of DLC1 on the phosphorylation of Smad3. The results showed that DLC1 down-regulated the phosphorylation levels of serines 204 and 208 on Smad3 ( FIG. 14A ). In view of Jungeun Lee et al. reported that TGFβ can also activate the Rho pathway (Lee, J., Moon et al., J Biol Chem285, 26618-26627 (2010)), so the inventors then detected whether RhoA activity was up-regulated when SCP2 was stimulated by TGFβ . After being treated with 10ng/ml TGFβ for 24h, it was found by active Rho kits (purchased from Pierce) that the activity of RhoA in the control group was significantly up-regulated, but there was no significant change in the activity of RhoA in the overexpression group. This indicates that DLC1 plays an overwhelming role in inhibiting the upregulation of RhoA activity by TGFβ (Fig. 14B). Then the inventor uses the mutant (EPSM) of Smad3 whose serine is mutated to alanine at positions 204 and 208 or the wild-type Smad3 expression plasmid and the PTHLH promoter reporter plasmid or the PTHLH promoter reporter plasmid containing the Smad3 binding sequence mutation. dye. The transfection results showed that the phosphorylation of serine at positions 204 and 208 was necessary for Smad3 to activate the activity of the PTHLH promoter through the mediation of the Smad3 binding sequence (Fig. The activity was similar to the activity of co-transfection of DLC1 and Smad3, both of which were much lower than the activity of PTHLH promoter after only Smad3 transfection in the control group. Further ChIP analysis showed that after DLC1 was co-transfected with Smad3 or EPSM, the copy number of the PTHLH promoter fragment that was immunoprecipitated due to Smad3 binding to the PTHLH promoter was almost the same as that of the blank group transfected with EPSM, and was higher than that of the group transfected only with Smad3. The no-load group was at least 2.6 times less. This suggested that phosphorylation of serines 204 and 208 enhanced the interaction between Smad3 and PTHLH promoter, and phosphorylation of these two sites was required for PTHLH transcription (Fig. 14D).
实施例6、临床乳腺癌样本中DLC1表达水平与PTHLH表达水平和骨转移预后负相关Example 6. The expression level of DLC1 in clinical breast cancer samples is negatively correlated with the expression level of PTHLH and the prognosis of bone metastasis
在细胞模型的基础上本发明人分析了DLC1调控PTHLH最终影响骨转移的机制，进一步分析了临床乳腺癌样本中DLC1表达水平与PTHLH表达水平和骨转移预后的关系。On the basis of cell models, the inventors analyzed the mechanism by which DLC1 regulates PTHLH and ultimately affects bone metastasis, and further analyzed the relationship between DLC1 expression level, PTHLH expression level and bone metastasis prognosis in clinical breast cancer samples.
首先本发明人利用已发表的NKI microarray数据(Bos，P.D.，et al.Nature459，1005-1009(2009)；Minn，A.J.，et al.Nature436，518-524(2005)；van‘tVeer，L.J.，et al.Nature415，530-536(2002))分析肿瘤样本中DLC1与骨和肺转移的关系，分析结果显示：样本中DLC1表达水平与骨转移预后成负相关的关系，与肺转移预后不相关(图15A-B)。First, the inventors used published NKI microarray data (Bos, P.D., et al. Nature459, 1005-1009 (2009); Minn, A.J., et al. Nature436, 518-524 (2005); van'tVeer, L.J., et al.Nature415, 530-536 (2002)) analyzed the relationship between DLC1 in tumor samples and bone and lung metastasis, and the analysis results showed that the expression level of DLC1 in samples was negatively correlated with the prognosis of bone metastasis, but not with the prognosis of lung metastasis (FIGS. 15A-B).
之后，本发明人从医院获得了64例原位乳腺癌肿瘤样本，本发明人抽取了其RNA通过qPCR分析发现与以骨转移形式复发的病人样本中DLC1表达水平显著低于未复发或以肺转移形式复发病人样本中的DLC1表达水平(图15C)。同时本发明人发现在这64例原位乳腺癌肿瘤样本中DLC1表达水平与PTHLH表达水平呈负相关(图15D)，相关系数是-0.32。Afterwards, the inventor obtained 64 cases of orthotopic breast cancer tumor samples from the hospital, and the inventor extracted its RNA and found that the expression level of DLC1 in the samples of patients with recurrence in the form of bone metastasis was significantly lower than that in patients with recurrence in the form of bone metastasis or in lung cancer. DLC1 expression levels in samples from relapsed patients in the metastatic form (Fig. 15C). At the same time, the inventors found that the expression level of DLC1 was negatively correlated with the expression level of PTHLH in these 64 orthotopic breast cancer tumor samples ( FIG. 15D ), and the correlation coefficient was -0.32.
此外，本发明人还分析了源自另一医院的9例原位乳腺癌和8例乳腺癌骨转移肿瘤样本中DLC1的表达，IHC结果显示骨转移瘤中DLC1的水平要远远低于原位乳腺癌(图15E)。In addition, the inventor also analyzed the expression of DLC1 in 9 cases of orthotopic breast cancer and 8 cases of breast cancer bone metastases from another hospital, and the results of IHC showed that the level of DLC1 in bone metastases was much lower than that of the original breast cancer (Fig. 15E).
实施例7、靶向ROCK通路治疗乳腺癌骨转移Example 7. Targeting the ROCK pathway to treat bone metastasis of breast cancer
上述实验结果证实DLC1抑制了ROCK介导的Smad3的linker region磷酸化，这导致PTHLH表达受到抑制，因此本发明人设想用靶向ROCK的药物治疗乳腺癌骨转移。本发明人将SCP2和4T1以左心室注射的方式分别注入裸鼠和Balb/c小鼠体内，然后用ROCK抑制剂Y27632或Fasudil治疗小鼠。在第4周的观察显示，相对于对照组骨转移瘤生长分别被Y27632和Fasudil抑制了103和92倍(图16A)。IHC显示抑制剂的应用很大程度上降低了转移灶PTHLH的表达和破骨细胞的数目(图16B-C)。X射线检查显示Fasudil组转移瘤对骨侵害的面积比对照组少了约1.9倍(图16D)。The above experimental results confirmed that DLC1 inhibited the phosphorylation of the linker region of Smad3 mediated by ROCK, which resulted in the inhibition of PTHLH expression, so the inventors contemplated using drugs targeting ROCK to treat bone metastasis of breast cancer. The inventors injected SCP2 and 4T1 into nude mice and Balb/c mice respectively by way of left ventricle injection, and then treated the mice with ROCK inhibitor Y27632 or Fasudil. Observations at week 4 showed that the growth of bone metastases was inhibited 103 and 92 times by Y27632 and Fasudil, respectively, relative to the control group ( FIG. 16A ). IHC showed that the application of the inhibitor greatly reduced the expression of PTHLH in metastases and the number of osteoclasts (Fig. 16B-C). X-ray examination showed that the bone invasion area of metastases in the Fasudil group was about 1.9 times less than that in the control group ( FIG. 16D ).
实施例8、以DLC1为靶点的抑制乳腺癌骨转移的潜在物质的筛选方法Example 8. Screening method for potential substances targeting DLC1 to inhibit bone metastasis of breast cancer
法1：Method 1:
细胞模型：表达DLC1的SCP2稳转细胞。Cell model: SCP2 stably transfected cells expressing DLC1.
测试组：用候选物质处理的上述细胞的培养物；Test group: a culture of the above-mentioned cells treated with the candidate substance;
对照组：不用候选物质处理的上述细胞的培养物。Control group: a culture of the above-mentioned cells not treated with the candidate substance.
检测两组中情况。如果与对照组相比，测试组中的DLC1蛋白的表达显著上调20%以上，则说明该候选物质是潜在的缓解抑制乳腺癌骨转移的物质。Check the situation in the two groups. If the expression of DLC1 protein in the test group is significantly up-regulated by more than 20% compared with the control group, it indicates that the candidate substance is a potential substance for alleviating and inhibiting bone metastasis of breast cancer.
法2：Method 2:
细胞模型：表达DLC1和Rho通路蛋白的SCP2稳转细胞。Cell model: SCP2 stably transfected cells expressing DLC1 and Rho pathway proteins.
测试组：用候选物质处理的上述细胞的培养物；Test group: a culture of the above-mentioned cells treated with the candidate substance;
对照组：不用候选物质处理的上述细胞的培养物。Control group: a culture of the above-mentioned cells not treated with the candidate substance.
检测两组中情况。如果与对照组相比，测试组中的DLC1蛋白的表达显著上调20%以上，并且使得Rho通路蛋白活性比对照组显著更低，则说明该候选物质是潜在的缓解抑制乳腺癌骨转移的物质。Check the situation in the two groups. If compared with the control group, the expression of DLC1 protein in the test group is significantly up-regulated by more than 20%, and the activity of the Rho pathway protein is significantly lower than that of the control group, it means that the candidate substance is a potential substance for alleviating and inhibiting bone metastasis of breast cancer .
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.
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| CN201310438956.5ACN104436162A (en) | 2013-09-24 | 2013-09-24 | Method and preparation for inhibiting bone metastasis due to breast cancer |
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| CN201310438956.5ACN104436162A (en) | 2013-09-24 | 2013-09-24 | Method and preparation for inhibiting bone metastasis due to breast cancer |
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| CN104436162Atrue CN104436162A (en) | 2015-03-25 |
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| CN201310438956.5APendingCN104436162A (en) | 2013-09-24 | 2013-09-24 | Method and preparation for inhibiting bone metastasis due to breast cancer |
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