技术领域technical field
本发明涉及疾病诊断预防技术领域，具体地，涉及肺癌诊断分子标记物lncRNALINC00516和试剂盒及其应用。The present invention relates to the technical field of disease diagnosis and prevention, in particular to lncRNALINC00516, a diagnostic molecular marker for lung cancer, a kit and applications thereof.
背景技术Background technique
肺癌发病率和死亡率在我国长期居各恶性肿瘤的首位，其发病时间短、转移快、预后不理想，其5年生存率仅为15％。肺癌容易发生各个不同脏器的转移，如脑转移、骨转移、肝转移等，并引起相应的症状，对病人生存造成极大的影响。大量研究和资料表明，早期诊断和早期治疗是防治肺癌和降低其死亡率的最有效方法，但由于缺乏理想的早期诊断方法，肺癌的早期诊断率仅14％左右。现有的传统肿瘤标志物(如癌胚抗原)的灵敏度和特异性均不够高。目前简单的胸透和痰液细胞学化验只能对肺癌进行早期诊断且敏感性非常低，关于肺癌转移诊断方式还缺乏有效、方便的检测手段。因此，开发一种微创而又特异灵敏的肺癌转移诊断标志物是当务之急。The morbidity and mortality of lung cancer rank first among all malignant tumors in my country for a long time. Its onset time is short, its metastasis is fast, its prognosis is not ideal, and its 5-year survival rate is only 15%. Lung cancer is prone to metastases to various organs, such as brain metastasis, bone metastasis, liver metastasis, etc., and causes corresponding symptoms, which have a great impact on the survival of patients. A large number of studies and data show that early diagnosis and early treatment are the most effective ways to prevent and reduce lung cancer and reduce its mortality. However, due to the lack of ideal early diagnosis methods, the early diagnosis rate of lung cancer is only about 14%. The sensitivity and specificity of existing traditional tumor markers (such as carcinoembryonic antigen) are not high enough. At present, simple chest X-ray and sputum cytology tests can only be used for early diagnosis of lung cancer and the sensitivity is very low. There is still a lack of effective and convenient detection methods for the diagnosis of lung cancer metastasis. Therefore, it is urgent to develop a minimally invasive and specific and sensitive diagnostic marker for lung cancer metastasis.
人体外周血的循环肿瘤细胞和肿瘤相关的DNA和RNA都可以被用作体外诊断。研究发现，来自血浆和血清中循环核酸相对比较稳定，一些肿瘤组织中会出现表达异常，且和肿瘤恶化有着密不可分的关系。lncRNA是一种长度大于200个核苷酸的RNA分子，没有编码蛋白质功能，部分可编辑一些肽类。lncRNA可通过表观遗传学、转录调控和转录后调控等多个层面调节细胞内基因的表达，参与机体各种生理病理过程。相关研究已发现lncRNA可与蛋白质、RNA或DNA形成复合物，调节肿瘤细胞的生长，与癌症转移密切相关。进入循环系统的lncRNA在正常条件下能被qRT-PCR检测到。Both circulating tumor cells and tumor-associated DNA and RNA from human peripheral blood can be used for in vitro diagnostics. Studies have found that circulating nucleic acids from plasma and serum are relatively stable, and abnormal expression occurs in some tumor tissues, and is inseparable from tumor progression. lncRNA is an RNA molecule longer than 200 nucleotides, which does not encode protein functions, and can partially edit some peptides. lncRNA can regulate the expression of intracellular genes through multiple levels of epigenetics, transcriptional regulation, and post-transcriptional regulation, and participate in various physiological and pathological processes of the body. Related studies have found that lncRNA can form a complex with protein, RNA or DNA, regulate the growth of tumor cells, and is closely related to cancer metastasis. LncRNAs entering the circulation system can be detected by qRT-PCR under normal conditions.
血液是最常见的临床检验材料，采集血液也是微小创伤性、无痛苦和极易于被人们接受的取材方法。因此，基于外周血筛选肺癌转移特异性lncRNA，并用作为分子标志物具有重要的检测诊断价值。Blood is the most common clinical test material, and blood collection is also a minimally invasive, painless and extremely acceptable method of drawing materials. Therefore, screening lung cancer metastasis-specific lncRNA based on peripheral blood and using it as a molecular marker has important detection and diagnosis value.
发明内容Contents of the invention
本发明的目的是为了克服现有技术的上述不足，提供一种肺癌或肺癌诊断分子标记物lncRNA LINC00516，所述lncRNA LINC00516在肺癌患者血清中特异性显著上调表达，LINC00516的表达与肺癌的病理类型呈显著正相关，能够作为肺癌诊断的分子标记物，尤其可作为肺癌或肺癌转移诊断的分子标记物，其灵敏度为81.1％、特异性为88.1％，具有较高的可靠性。The purpose of the present invention is to overcome the above-mentioned deficiencies in the prior art, and provide a lung cancer or lung cancer diagnostic molecular marker lncRNA LINC00516, said lncRNA LINC00516 is specifically significantly up-regulated in the serum of lung cancer patients, and the expression of LINC00516 is related to the pathological type of lung cancer It is significantly positively correlated, and can be used as a molecular marker for the diagnosis of lung cancer, especially for the diagnosis of lung cancer or lung cancer metastasis. The sensitivity is 81.1%, and the specificity is 88.1%, with high reliability.
本发明的另一目的在于提供所述lncRNA LINC00516在制备肺癌或肺癌转移诊断试剂盒和/或制剂中的应用。Another object of the present invention is to provide the application of the lncRNA LINC00516 in the preparation of a diagnostic kit and/or preparation for lung cancer or lung cancer metastasis.
本发明的另一目的在于提供一组检测lncRNA LINC00516的引物对和探针。Another object of the present invention is to provide a set of primer pairs and probes for detecting lncRNA LINC00516.
本发明的另一目的在于提供所述引物对和探针在制备肺癌或肺癌转移诊断试剂盒和/或制剂中的应用。Another object of the present invention is to provide the application of the primer pair and probe in the preparation of diagnostic kits and/or preparations for lung cancer or lung cancer metastasis.
本发明的另一目的在于提供一种肺癌或肺癌转移诊断试剂盒。Another object of the present invention is to provide a diagnostic kit for lung cancer or lung cancer metastasis.
为了实现上述目的，本发明是通过以下方案予以实现的：In order to achieve the above object, the present invention is achieved through the following schemes:
本发明人从肺癌患者的血液中分离和检测到肺癌转移特异性lncRNA LINC00516呈显著高表达，发现血液中的LINC00516来源于肺癌细胞，并分泌释放到血液中。此外，LINC00516的表达与肺癌的病理类型呈显著正相关，LINC00516对区分肺癌转移患者和肺癌不转移患者的灵敏度高达81.1％，特异性高达88.1％。因此，lncRNA LINC00516可作为一种新型的肺癌诊断和肺癌转移血液分子诊断标记物，能显著提高诊断的准确性、敏感性和特异性。此外，该分子标记物还可作为肺癌转移治疗药物的新治疗靶点。The inventors isolated and detected lung cancer metastasis-specific lncRNA LINC00516 was significantly highly expressed from the blood of lung cancer patients, and found that LINC00516 in the blood was derived from lung cancer cells and secreted and released into the blood. In addition, the expression of LINC00516 was significantly positively correlated with the pathological type of lung cancer. The sensitivity of LINC00516 to distinguish patients with lung cancer metastasis from those without lung cancer metastasis was as high as 81.1%, and the specificity was as high as 88.1%. Therefore, lncRNA LINC00516 can be used as a new blood molecular diagnostic marker for lung cancer diagnosis and lung cancer metastasis, which can significantly improve the diagnostic accuracy, sensitivity and specificity. In addition, this molecular marker can also be used as a new therapeutic target for drugs for the treatment of lung cancer metastasis.
因此，本发明请求保护核苷酸序列如SEQ ID NO:1所示的lncRNA LINC00516作为肺癌或肺癌转移诊断分子标记物的应用。Therefore, the present invention claims the use of the lncRNA LINC00516 whose nucleotide sequence is shown in SEQ ID NO:1 as a molecular marker for lung cancer or lung cancer metastasis diagnosis.
本发明还请求保护核苷酸序列如SEQ ID NO:1所示的lncRNA LINC00516作为肺癌转移诊断分子标记物的应用。The present invention also claims the application of the lncRNA LINC00516 whose nucleotide sequence is shown in SEQ ID NO:1 as a molecular marker for the diagnosis of lung cancer metastasis.
本发明还请求保护核苷酸序列如SEQ ID NO:1所示的lncRNA LINC00516在制备肺癌或肺癌转移诊断试剂盒和/或制剂中的应用。The present invention also claims the application of the lncRNA LINC00516 whose nucleotide sequence is shown as SEQ ID NO: 1 in the preparation of lung cancer or lung cancer metastasis diagnostic kits and/or preparations.
本发明还请求保护一组检测lncRNA LINC00516的引物对和探针，包括qRT-PCR正向引物、反向引物和Taqman探针，核苷酸序列依次如SEQ ID NO:2～4所示；所述Taqman探针的5’端结合有荧光物质FAM，3’端结合有淬灭物质TAMRA。The present invention also claims a set of primer pairs and probes for detecting lncRNA LINC00516, including qRT-PCR forward primers, reverse primers and Taqman probes, the nucleotide sequences of which are shown in SEQ ID NO: 2-4; The 5' end of the Taqman probe is combined with the fluorescent substance FAM, and the 3' end is combined with the quenching substance TAMRA.
LINC00516-F(SEQ ID NO:2)：5’-GCACAGAGCCTCGCCTT-3’；LINC00516-F (SEQ ID NO:2): 5'-GCACAGAGCCTCGCCTT-3';
LINC00516-R(SEQ ID NO:3)：5’-AGACAGTATCACAGGTTA-3’；LINC00516-R (SEQ ID NO:3): 5'-AGACAGTATCACAGGTTA-3';
Taqman探针(SEQ ID NO:4)：5’-FAM-TAATTCACAGTTCCACATGGCT-TAMRA-3’。Taqman probe (SEQ ID NO: 4): 5'-FAM-TAATTCACAGTTCCACATGGCT-TAMRA-3'.
本发明还请求保护上述引物对和探针在制备肺癌或肺癌转移诊断试剂盒和/或制剂中的应用。The present invention also claims the application of the above primer pair and probe in the preparation of diagnostic kits and/or preparations for lung cancer or lung cancer metastasis.
本发明还请求保护一种肺癌或肺癌转移诊断试剂盒，包含检测lncRNA LINC00516表达量的试剂。The present invention also claims a diagnostic kit for lung cancer or lung cancer metastasis, including a reagent for detecting the expression level of lncRNA LINC00516.
优选地，所述试剂包括荧光定量PCR反应体系；所述荧光定量PCR反应体系包括上述引物对和探针。Preferably, the reagent includes a fluorescent quantitative PCR reaction system; the fluorescent quantitative PCR reaction system includes the above-mentioned primer pair and probe.
优选地，所述荧光定量PCR反应体系由以下体积的各组分组成：10μL Taqman混合液，1μL Taqman探针，1μL qRT-PCR正向引物，1μL qRT-PCR反向引物，2μL cDNA模板，余量ddH2O补足至20μL。Preferably, the fluorescent quantitative PCR reaction system consists of the following volumes of components: 10 μL Taqman mixture, 1 μL Taqman probe, 1 μL qRT-PCR forward primer, 1 μL qRT-PCR reverse primer, 2 μL cDNA template, and Make up the amount of ddH2 O to 20 μL.
更优选地，所述Taqman混合液包括以下组分：2×Taqman DNA聚合酶、荧光染料、dNTP、镁离子和其他盐离子。More preferably, the Taqman mixture includes the following components: 2×Taqman DNA polymerase, fluorescent dye, dNTP, magnesium ions and other salt ions.
优选地，所述荧光定量PCR反应条件为：95℃15min；95℃15s，60℃45s，重复40个循环。Preferably, the fluorescent quantitative PCR reaction conditions are: 95° C. for 15 min; 95° C. for 15 s, 60° C. for 45 s, repeated for 40 cycles.
优选地，所述试剂盒和/或试剂还含有阳性对照液(即LINC00516标准品)、阴性对照液(即空白对照)。Preferably, the kit and/or reagents also contain a positive control solution (ie LINC00516 standard) and a negative control solution (ie blank control).
优选地，所述试剂盒还包含RNA逆转录反应体系；所述RNA逆转录反应体系由以下各组分组成：1μL Random Primer、1μg RNA template、1μL M-MLV RT、5μL dNTP Mix、5μLM-MLV 5×Reaction Buffer、0.5μL RNase Inhibitor和余量RNase Free H2O补足至25μL。Preferably, the kit also includes an RNA reverse transcription reaction system; the RNA reverse transcription reaction system consists of the following components: 1 μL Random Primer, 1 μg RNA template, 1 μL M-MLV RT, 5 μL dNTP Mix, 5 μL M-MLV Make up to 25 μL with 5×Reaction Buffer, 0.5 μL RNase Inhibitor and the rest RNase Free H2 O.
优选地，所述逆转录反应条件为40℃60min；70℃5min。Preferably, the reverse transcription reaction conditions are 40°C for 60 minutes; 70°C for 5 minutes.
上述试剂盒可用于定性或定量检测血清中LINC00516的表达量。The above kit can be used to qualitatively or quantitatively detect the expression level of LINC00516 in serum.
上述试剂盒的使用方法包括如下步骤：The using method of above-mentioned test kit comprises the following steps:
S1.采集血液，提供血浆中的总RNA；S1. Collect blood and provide total RNA in plasma;
S2.将总RNA逆转录成cDNA；S2. Reverse transcribing the total RNA into cDNA;
S3.利用上述qRT-PCR正向引物、反向引物和Taqman探针对步骤S2所得cDNA溶液进行荧光定量PCR检测，测定样品的Ct值；设肺癌患者CtLINC00516/CtU6值阈值为3.575，定义血液中检测Ct值大于等于3.575为阳性。S3. Use the above-mentioned qRT-PCR forward primer, reverse primer and Taqman probe to perform fluorescence quantitative PCR detection on the cDNA solution obtained in step S2, and measure the Ct value of the sample; set the CtLINC00516 /CtU6 threshold value of lung cancer patients to 3.575, define A Ct value greater than or equal to 3.575 in the blood is positive.
与现有技术相比，本发明具有以下有益效果：Compared with the prior art, the present invention has the following beneficial effects:
本发明所述lncRNA LINC00516在肺癌患者血清中特异性显著上调表达，LINC00516的表达与肺癌的病理类型呈显著正相关，能够作为肺癌诊断的分子标记物，尤其可作为肺癌转移诊断的分子标记物，其灵敏度为81.1％、特异性为88.1％，具有较高的可靠性。本发明所提供的试剂盒检测快速方便、准确率高，可以实现肺癌的早期诊断和预测，能广泛应用于医疗行业肺癌疾病的辅助诊断和治疗。The lncRNA LINC00516 of the present invention is specifically and significantly up-regulated in the serum of lung cancer patients, and the expression of LINC00516 is significantly positively correlated with the pathological type of lung cancer. It can be used as a molecular marker for the diagnosis of lung cancer, especially as a molecular marker for the diagnosis of lung cancer metastasis. Its sensitivity is 81.1%, specificity is 88.1%, and has high reliability. The detection kit provided by the invention is fast and convenient, has high accuracy, can realize early diagnosis and prediction of lung cancer, and can be widely used in auxiliary diagnosis and treatment of lung cancer in the medical industry.
附图说明Description of drawings
图1为lncRNA LINC00516分别在肺癌转移患者和正常人血液中的表达量。Figure 1 shows the expression levels of lncRNA LINC00516 in the blood of patients with lung cancer metastasis and normal people.
图2为高表达的lncRNA LINC00516促进肺癌细胞损伤修复的情况。Figure 2 shows the situation that highly expressed lncRNA LINC00516 promotes the damage repair of lung cancer cells.
图3为lncRNA LINC00516诊断肺癌转移的ROC曲线。Figure 3 is the ROC curve of lncRNA LINC00516 in the diagnosis of lung cancer metastasis.
具体实施方式Detailed ways
下面结合说明书附图及具体实施例对本发明作出进一步地详细阐述，所述实施例只用于解释本发明，并非用于限定本发明的范围。下述实施例中所使用的试验方法如无特殊说明，均为常规方法；所使用的材料、试剂等，如无特殊说明，为可从商业途径得到的试剂和材料。The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, which are only used to explain the present invention, and are not intended to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available reagents and materials unless otherwise specified.
实施例1肺癌转移特征性lncRNA LINC00516验证Example 1 Verification of lung cancer metastasis characteristic lncRNA LINC00516
本发明人团队经过前期的大量研究发现，SEQ ID NO:1所示lncRNA LINC00516与肺癌的诊断、转移相关，可作为肺癌诊断和肺癌转移血液分子诊断标记物。下面利用59例肺癌病人与95例正常人血液标本对LINC00516进行探针法PCR检测，从而对肺癌转移特征性lncRNA LINC00516进行验证。具体包括如下步骤：The team of the present inventors found through a lot of previous research that the lncRNA LINC00516 represented by SEQ ID NO: 1 is related to the diagnosis and metastasis of lung cancer, and can be used as a blood molecular diagnostic marker for lung cancer diagnosis and lung cancer metastasis. In the following, 59 cases of lung cancer patients and 95 cases of normal human blood samples were used to detect LINC00516 by probe method PCR, so as to verify the characteristic lncRNA LINC00516 of lung cancer metastasis. Specifically include the following steps:
1、提取血浆样本总RNA(TRIzol LS法)及RNA浓度的测定：1. Extraction of total RNA from plasma samples (TRIzol LS method) and determination of RNA concentration:
(1)样品均质化：每0.25mL样品加入0.75mL TRIzol LS试剂(TRIzol LS试剂与样品的体积比为3:1)；移液器上下吹匀几次，使样品均匀。注意：污染物质含量高的生物液体(如全血)应该先用无RNase水按1:1稀释。当样品少量或者样品体积<0.25mL，为方便提取RNA，需用无RNase水调整样品体积到0.25mL。(1) Sample homogenization: add 0.75mL TRIzol LS reagent per 0.25mL sample (the volume ratio of TRIzol LS reagent to sample is 3:1); pipette up and down several times to make the sample uniform. Note: Biological fluids with high levels of contaminants (such as whole blood) should first be diluted 1:1 with RNase-free water. When the sample is small or the sample volume is <0.25mL, in order to facilitate the extraction of RNA, it is necessary to adjust the sample volume to 0.25mL with RNase-free water.
(2)相分离：①室温静置5min，以利于匀浆样品中核蛋白体完全分离。②每0.75mLTRIzol LS试剂加入0.2mL氯仿。盖紧离心管的盖子。③震荡仪上震荡30s，室温静置2～15min。④4℃，12000rpm离心15min。离心后，混合物分离为3层，上层是澄清的水相，中间层，下层是红色的酚-氯仿相。RNA存在于水相里。水相体积约为70％均质时使用的TRIzol LS试剂量。(2) Phase separation: ①Leave at room temperature for 5 minutes to facilitate the complete separation of nucleosomes in the homogenized sample. ②Add 0.2mL chloroform for every 0.75mL TRIzol LS reagent. Cap the centrifuge tube tightly. ③ Shake on a shaker for 30 seconds, and let stand at room temperature for 2-15 minutes. ④ Centrifuge at 12,000 rpm for 15 minutes at 4°C. After centrifugation, the mixture separated into 3 layers, the upper layer was a clear aqueous phase, the middle layer, and the lower layer was a red phenol-chloroform phase. RNA exists in the aqueous phase. The volume of the aqueous phase is approximately 70% homogeneous in the amount of TRIzol LS reagent used.
(3)RNA沉淀：①水相转移到一个新管。每0.75mL TRIzol LS试剂加入0.5mL 100％异丙醇到水相。②混合异丙醇以从水相中沉淀RNA。上下颠倒10次，-20℃静置样品30min，以利于RNA沉淀。③4℃，12000rpm离心15min。注意：往往离心前RNA沉淀不可见，离心后在管侧面和底部形成一个凝胶样沉淀。(3) RNA precipitation: ① Transfer the aqueous phase to a new tube. Add 0.5 mL of 100% isopropanol to the aqueous phase per 0.75 mL of TRIzol LS reagent. ② Mix isopropanol to precipitate RNA from the aqueous phase. Invert upside down 10 times, and let the sample stand at -20°C for 30 minutes to facilitate RNA precipitation. ③ Centrifuge at 12000 rpm for 15 minutes at 4°C. Note: Often the RNA pellet is not visible before centrifugation and forms a gel-like pellet on the sides and bottom of the tube after centrifugation.
(4)RNA洗涤：①移走离心管里上清液，只留RNA沉淀。②每0.75mL TRIzol LS试剂加入1mL 75％乙醇洗涤RNA沉淀。涡旋震荡，混匀样品。③4℃，7500rpm离心5min，弃上清液。(4) RNA washing: ①Remove the supernatant in the centrifuge tube, leaving only the RNA precipitate. ② Add 1mL 75% ethanol to every 0.75mL TRIzol LS reagent to wash the RNA pellet. Vortex to mix the sample. ③ Centrifuge at 7500rpm for 5min at 4°C and discard the supernatant.
(5)重悬RNA：①真空或者空气干燥RNA沉淀5～10min，不要用真空离心干燥机干燥RNA沉淀。②用无RNase水(20～50μL)重悬RNA沉淀，移液器上下轻轻吹溶液几次。③在55～60℃的水浴槽中孵育10～15min。④继续下游操作，或保存于-40或-80℃冰箱中。注意：不要让RNA沉淀干燥太彻底，否则RNA沉淀溶解度降低，会使A260/280<1.6。(5) Resuspend RNA: ① Vacuum or air-dry the RNA pellet for 5-10 minutes, do not use a vacuum centrifugal dryer to dry the RNA pellet. ②Resuspend the RNA pellet with RNase-free water (20-50 μL), and gently blow the solution up and down with the pipette several times. ③Incubate in a water bath at 55-60°C for 10-15 minutes. ④Continue downstream operation, or store in -40 or -80°C refrigerator. Note: Do not allow the RNA pellet to dry too thoroughly, otherwise the solubility of the RNA pellet will decrease, making A260/280<1.6.
(6)RNA定量：①酶标仪Nanodrop 1000：启动Nucleic Acid模块后，按照屏幕提示用2μL无RNA酶水清洗基座；选择RNA40模式，用2μL对照空白溶液Blank；加入2μL RNA溶液Measure。②微孔板定量系统：共有24个孔，最右侧一列的三个孔为空白对照，剩余21个孔可以加入RNA样品进行检测。加样完毕后盖上玻片，放入载板中，用DNA_RNA quantification程序读值。(6) RNA quantification: ① Microplate reader Nanodrop 1000: After starting the Nucleic Acid module, follow the screen prompts to wash the base with 2 μL RNase-free water; select RNA40 mode, use 2 μL blank control solution Blank; add 2 μL RNA solution Measure. ② Microplate quantitative system: There are 24 wells in total, the three wells in the rightmost column are blank controls, and the remaining 21 wells can be added with RNA samples for detection. After loading the sample, cover the glass slide, put it into the carrier plate, and use the DNA_RNA quantification program to read the value.
(7)定量结果判读：纯RNA的A260/280＝2，在1.7～2之间为正常，(＜1.7时表明有蛋白质或酚污染；＞2.0时表明可能有异硫氰酸残存)。A230表示样品中存在一些污染物，如碳水化合物、盐(胍盐)等，较纯净的核酸A260/A230的比值大于2.0。(7) Interpretation of quantitative results: A260/280 of pure RNA = 2, between 1.7 and 2 is normal, (<1.7 indicates protein or phenol contamination; >2.0 indicates that there may be residual isothiocyanate). A230 indicates that there are some pollutants in the sample, such as carbohydrates, salt (guanidine salt), etc., and the ratio of A260/A230 of relatively pure nucleic acid is greater than 2.0.
2、总RNA的逆转录2. Reverse transcription of total RNA
(1)用MMLV Reverse Transcriptase(Promega)逆转录，操作如下：(1) Reverse transcription with MMLV Reverse Transcriptase (Promega), the operation is as follows:
表1反应体系Table 1 reaction system
置于PCR仪中，70℃5min，然后立即在冰上冷却5min。Place in a PCR instrument at 70°C for 5 minutes, then immediately cool on ice for 5 minutes.
(2)在上述反应体系中加入以下试剂进行逆转录反应：(2) Add the following reagents to the above reaction system for reverse transcription reaction:
表2逆转录反应体系Table 2 Reverse transcription reaction system
轻轻混匀后，置于PCR仪中，40℃60min；70℃5min。逆转录产物置于4℃短暂保存，或者进行后续操作。After mixing gently, place in a PCR instrument at 40°C for 60 minutes; 70°C for 5 minutes. The reverse transcription product was stored at 4°C for a short period of time, or carried out for subsequent operations.
3、实时定量PCR检测LINC00516的表达量3. Detection of the expression level of LINC00516 by real-time quantitative PCR
(1)实时定量PCR检测引物和探针(1) Real-time quantitative PCR detection primers and probes
LINC00516-F(SEQ ID NO:2)：5’-GCACAGAGCCTCGCCTT-3’；LINC00516-F (SEQ ID NO:2): 5'-GCACAGAGCCTCGCCTT-3';
LINC00516-R(SEQ ID NO:3)：5’-AGACAGTATCACAGGTTA-3’；LINC00516-R (SEQ ID NO:3): 5'-AGACAGTATCACAGGTTA-3';
Taqman探针(SEQ ID NO:4)：5’-FAM-TAATTCACAGTTCCACATGGCT-TAMRA-3’。Taqman probe (SEQ ID NO: 4): 5'-FAM-TAATTCACAGTTCCACATGGCT-TAMRA-3'.
(2)实时定量PCR：实时定量PCR反应使用罗氏480Probe Master实时荧光定量PCR系统，利用CFX96TMReal-Time PCR Detection Systems(C1000Touch PCR仪，Bio-rad)进行操作。定量PCR的扩增产物长度以80bp～150bp最为合适(可以延长至300bp)。反应体系如下：(2) Real-time quantitative PCR: real-time quantitative PCR reaction uses Roche The 480Probe Master real-time fluorescent quantitative PCR system was operated by CFX96TM Real-Time PCR Detection Systems (C1000Touch PCR instrument, Bio-rad). The length of the amplification product of quantitative PCR is most suitable for 80bp～150bp (can be extended to 300bp). The reaction system is as follows:
表3荧光定量PCR反应体系Table 3 Fluorescent quantitative PCR reaction system
反应条件：95℃15min；95℃15s，60℃45s，重复40个循环。Reaction conditions: 95°C for 15min; 95°C for 15s, 60°C for 45s, repeat 40 cycles.
(3)数据分析：本实验采用相对定量分析方法。每个样品做三个复孔，结果取三个复孔的平均CT值。△Ct＝目的lncRNA平均CT值-内参基因平均CT值；△△Ct＝△Ct转移-△Ct未转移；本试剂盒采用U6为内参，计算LINC00516的相对表达量。数据用SPSS20.0软件进行分析，以目标lncRNA相对表达量为自变量，组别为因变量，建立肺癌早期转移诊断的Logistic回归模型，回归模型的拟合度采用似然比检验，回归参数估计值采用非参数检验法。根据ROC曲线及曲线下面积(AUC)评估目标lncRNA诊断的敏感性和特异性。(3) Data analysis: This experiment adopts relative quantitative analysis method. Three replicate holes were made for each sample, and the average CT value of the three replicate holes was taken as the result. △Ct=average CT value of targetlncRNA -average CT value of internal referencegene ; This kit uses U6 as an internal reference to calculate the relative expression of LINC00516. The data was analyzed with SPSS20.0 software, with the relative expression of the target lncRNA as the independent variable and the group as the dependent variable, a Logistic regression model for the diagnosis of early metastasis of lung cancer was established, the fitting degree of the regression model was tested by likelihood ratio, and the regression parameters were estimated Values were tested using a nonparametric test. The sensitivity and specificity of the target lncRNA diagnosis were evaluated according to the ROC curve and the area under the curve (AUC).
4、肺癌转移特征性lncRNA LINC00516验证4. Validation of lung cancer metastasis characteristic lncRNA LINC00516
利用59例肺癌病人与95例正常人血液标本对LINC00516进行探针法PCR检测，发现肺癌病人血液标本中的LINC00516表达显著上调(P<0.0001)，结果如图1所示。此外，还发现在肺癌细胞中高表达LINC00516，细胞损伤修复能力增强，结果如图2所示。以上结果说明LINC00516是肺癌转移特征性lncRNA。Using 59 cases of lung cancer patients and 95 cases of normal human blood samples to detect LINC00516 by probe method PCR, it was found that the expression of LINC00516 in the blood samples of lung cancer patients was significantly up-regulated (P<0.0001), the results are shown in Figure 1. In addition, it was also found that the high expression of LINC00516 in lung cancer cells enhanced the ability of cell damage repair, the results are shown in Figure 2. The above results indicated that LINC00516 is a characteristic lncRNA of lung cancer metastasis.
5、利用血液LINC00516区分肺癌患者和健康人群5. Using blood LINC00516 to distinguish lung cancer patients from healthy people
从步骤1中获得血液标本，按照上述方法分析肺癌患者和正常人血液中LINC00516的表达水平。从结果图1可以看出，LINC00516可在肺癌患者血液中检出，转移患者比不转移患者高。Blood samples were obtained from step 1, and the expression levels of LINC00516 in the blood of lung cancer patients and normal people were analyzed according to the above method. It can be seen from the results in Figure 1 that LINC00516 can be detected in the blood of lung cancer patients, and the number of patients with metastasis is higher than that of patients without metastasis.
通过分析可知，肺癌不转移患者LINC00516可被检出的CtLINC00516/CtU6值在0.32～3.56之间，肺癌患者的Ct值在3.59～9.48之间。ROC曲线如图3所示。因此，设肺癌患者CtLINC00516/CtU6值阈值为3.575，定义血液中检测Ct值大于等于3.575为阳性。统计结果如表4所示，灵敏度达到81.1％，特异度达到88.1％，具有较高的可靠性。The analysis shows that the CtLINC00516 /CtU6 value of LINC00516 in non-metastatic lung cancer patients is between 0.32 and 3.56, and the Ct value of lung cancer patients is between 3.59 and 9.48. The ROC curve is shown in Figure 3. Therefore, the threshold value of CtLINC00516 /CtU6 in patients with lung cancer is set to be 3.575, and the detection of Ct value in blood is defined as positive if it is greater than or equal to 3.575. The statistical results are shown in Table 4, the sensitivity reaches 81.1%, the specificity reaches 88.1%, and has high reliability.
表4 LINC00516作为分子标记物检测的特点Table 4 The characteristics of LINC00516 as a molecular marker detection
实施例2Example 2
一种肺癌或肺癌转移诊断试剂盒，包含逆转录反应体系和荧光定量PCR反应体系；所述荧光定量PCR反应体系包括qRT-PCR正向引物、反向引物和Taqman探针；所述qRT-PCR正向引物、反向引物和Taqman探针的核苷酸序列分别依次如SEQ ID NO:2～4所示。所述Taqman探针的5’末端结合有荧光物质FAM，3’末端结合有淬灭物质TAMRA。A diagnostic kit for lung cancer or lung cancer metastasis, comprising a reverse transcription reaction system and a fluorescent quantitative PCR reaction system; the fluorescent quantitative PCR reaction system includes qRT-PCR forward primers, reverse primers and Taqman probes; the qRT-PCR The nucleotide sequences of the forward primer, the reverse primer and the Taqman probe are respectively shown in SEQ ID NO: 2-4. A fluorescent substance FAM is bound to the 5' end of the Taqman probe, and a quenching substance TAMRA is bound to the 3' end.
LINC00516-F(SEQ ID NO:2)：5’-GCACAGAGCCTCGCCTT-3’；LINC00516-F (SEQ ID NO:2): 5'-GCACAGAGCCTCGCCTT-3';
LINC00516-R(SEQ ID NO:3)：5’-AGACAGTATCACAGGTTA-3’；LINC00516-R (SEQ ID NO:3): 5'-AGACAGTATCACAGGTTA-3';
Taqman探针(SEQ ID NO:4)：5’-FAM-TAATTCACAGTTCCACATGGCT-TAMRA-3’。Taqman probe (SEQ ID NO: 4): 5'-FAM-TAATTCACAGTTCCACATGGCT-TAMRA-3'.
上述引物和探针可用于制备诊断其他肿瘤的试剂盒和/或试剂。The above primers and probes can be used to prepare kits and/or reagents for diagnosing other tumors.
所述荧光定量PCR反应体系由以下体积的各组分组成：10μL Taqman混合液，1μLTaqman探针，1μL qRT-PCR正向引物，1μL qRT-PCR反向引物，2μL cDNA模板，余量ddH2O补足至20μL。The fluorescent quantitative PCR reaction system is composed of the following components: 10 μL Taqman mixed solution, 1 μL Taqman probe, 1 μL qRT-PCR forward primer, 1 μL qRT-PCR reverse primer, 2 μL cDNA template, and the balance ddH2 O Make up to 20 μL.
所述Taqman混合液包括以下组分：2×Taqman DNA聚合酶、荧光染料、dNTP、镁离子和其他盐离子。The Taqman mixture includes the following components: 2×Taqman DNA polymerase, fluorescent dye, dNTP, magnesium ion and other salt ions.
所述荧光定量PCR反应条件为：95℃15min；95℃15s，60℃45s，重复40个循环。The fluorescent quantitative PCR reaction conditions are: 95° C. for 15 minutes; 95° C. for 15 s, 60° C. for 45 s, repeating 40 cycles.
所述试剂盒和/或试剂还含有阳性对照液(即LINC00516标准品)、阴性对照液(即空白对照)。The kit and/or reagent also contains a positive control solution (ie LINC00516 standard product) and a negative control solution (ie blank control).
所述RNA逆转录反应体系由以下各组分组成：1μL Random Primer、1μg RNAtemplate、1μL M-MLV RT、5μL dNTP Mix、5μL M-MLV 5×Reaction Buffer、0.5μL RNaseInhibitor和余量RNase Free H2O补足至25μL。The RNA reverse transcription reaction system consists of the following components: 1 μL Random Primer, 1 μg RNAtemplate, 1 μL M-MLV RT, 5 μL dNTP Mix, 5 μL M-MLV 5×Reaction Buffer, 0.5 μL RNase Inhibitor and the balance RNase Free H2 O to make up to 25 μL.
所述逆转录反应条件为40℃60min；70℃5min。The reverse transcription reaction conditions are 40° C. for 60 min; 70° C. for 5 min.
上述试剂盒可用于定性或定量检测血清中LINC00516的表达量。The above kit can be used to qualitatively or quantitatively detect the expression level of LINC00516 in serum.
上述试剂盒的使用方法包括如下步骤：The using method of above-mentioned test kit comprises the following steps:
S1.采集血液，提供血浆中的总RNA；S1. Collect blood and provide total RNA in plasma;
S2.将总RNA逆转录成cDNA；S2. Reverse transcribing the total RNA into cDNA;
S3.利用上述qRT-PCR正向引物、反向引物和Taqman探针对步骤S2所得cDNA溶液进行荧光定量PCR检测，测定样品的Ct值；设肺癌患者CtLINC00516/CtU6值阈值为3.575，定义血液中检测Ct值大于等于3.575为阳性。S3. Use the above-mentioned qRT-PCR forward primer, reverse primer and Taqman probe to perform fluorescence quantitative PCR detection on the cDNA solution obtained in step S2, and measure the Ct value of the sample; set the CtLINC00516 /CtU6 threshold value of lung cancer patients to 3.575, define A Ct value greater than or equal to 3.575 in the blood is positive.
最后所应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，对于本领域的普通技术人员来说，在上述说明及思路的基础上还可以做出其它不同形式的变化或变动，这里无需也无法对所有的实施方式予以穷举。凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明权利要求的保护范围之内。Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the scope of the present invention. For those of ordinary skill in the art, on the basis of the above descriptions and ideas, they can also make There is no need to and cannot exhaustively list all the implementation manners for other changes or changes in different forms. All modifications, equivalent replacements and improvements made within the spirit and principles of the present invention shall be included within the protection scope of the claims of the present invention.
序列表sequence listing
<110> 中山大学<110> Sun Yat-sen University
<120> 肺癌诊断分子标记物lncRNA LINC00516和试剂盒及其应用<120> lncRNA LINC00516, a molecular marker for lung cancer diagnosis, kit and its application
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| Application Number | Priority Date | Filing Date | Title |
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| CN201810982670.6ACN108998528B (en) | 2018-08-27 | 2018-08-27 | Lung cancer diagnosis molecular marker lncRNA LINC00516, kit and application thereof |
| Application Number | Priority Date | Filing Date | Title |
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| CN201810982670.6ACN108998528B (en) | 2018-08-27 | 2018-08-27 | Lung cancer diagnosis molecular marker lncRNA LINC00516, kit and application thereof |
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| CN108998528Atrue CN108998528A (en) | 2018-12-14 |
| CN108998528B CN108998528B (en) | 2021-09-21 |
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| CN201810982670.6AActiveCN108998528B (en) | 2018-08-27 | 2018-08-27 | Lung cancer diagnosis molecular marker lncRNA LINC00516, kit and application thereof |
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