CN115389764A - Application of protein FBXO7 in the preparation of endometrial cancer diagnostic markers - Google Patents

Abstract

The invention discloses an application of protein FBXO7 in preparing endometrial cancer diagnostic markers, which is characterized in that the protein FBXO7 is applied in preparing endometrial cancer diagnostic markers and/or therapeutic drugs, and further the protein FBXO7 mutant is applied in preparing endometrial cancer diagnostic reagents; further the application of the protein FBXO7 in the preparation of precise targeted therapeutic drugs for endometrial cancer and discloses the application of a mitochondrion inhibitor Mdivi-1 in the preparation of endometrial cancer with low expression and/or mutation of the FBXO7 protein, which has the advantages of being beneficial to the early diagnosis of endometrial cancer and being capable of designing a targeted therapy means aiming at the endometrial cancer.

Description

Translated fromChinese

蛋白质FBXO7在制备子宫内膜癌诊断标志物中的应用Application of protein FBXO7 in the preparation of endometrial cancer diagnostic markers

技术领域technical field

本发明属于生物医药领域，尤其是涉及一种蛋白质FBXO7在制备子宫内膜癌诊断标志物中的应用。The invention belongs to the field of biomedicine, and in particular relates to the application of a protein FBXO7 in preparing endometrial cancer diagnostic markers.

背景技术Background technique

子宫内膜癌，又称子宫体癌，是女性生殖系统最常见的恶性肿瘤之一。子宫内膜癌治疗的总体预后较好，但中晚期和复发转移的子宫内膜癌的预后极差，患者的平均生存期不到1年，因此早期精确的诊断便显得尤为重要。Endometrial cancer, also known as uterine body cancer, is one of the most common malignant tumors of the female reproductive system. The overall prognosis of endometrial cancer treatment is good, but the prognosis of middle-advanced and recurrent and metastatic endometrial cancer is extremely poor, and the average survival period of patients is less than 1 year, so early and accurate diagnosis is particularly important.

现有的子宫内膜癌的诊断主要依赖于诊断性刮宫进行组织病理学诊断，操作存在盲目性且有创伤，还容易遗漏微小的病灶，无法满足早期精准诊断的要求。此外，子宫内膜癌异质性显著，传统组织病理学评价可重复性较低，各肿瘤类型之间组织学特点常存在重叠，因此已经无法满足子宫内膜癌临床诊断与治疗的需要。而随着精准医疗的发展，根据患者特异生物分子（基因、蛋白等）特征实施的靶向治疗逐步成为研究热点，精准医疗也成为子宫内膜癌治疗的具有指导意义和广阔应用前景的新疗法。同时疾病的分类不再以传统病理为标准，而更加注重特异生物分子上的区别。The existing diagnosis of endometrial cancer mainly relies on diagnostic curettage for histopathological diagnosis. The operation is blind and invasive, and it is easy to miss tiny lesions, which cannot meet the requirements of early and accurate diagnosis. In addition, the heterogeneity of endometrial cancer is significant, the reproducibility of traditional histopathological evaluation is low, and the histological characteristics of various tumor types often overlap, so it can no longer meet the needs of clinical diagnosis and treatment of endometrial cancer. With the development of precision medicine, targeted therapy based on patient-specific biomolecular (gene, protein, etc.) . At the same time, the classification of diseases no longer takes the traditional pathology as the standard, but pays more attention to the distinction of specific biomolecules.

FBXO7是一种E3泛素连接酶，由522个氨基酸组成，蛋白大小约为62 kDa。其研究显示主要功能为维持细胞内线粒体的稳态和蛋白质的泛素化降解。目前国内外还没有公开任何关于在子宫内膜癌中将FBXO7蛋白作为诊治标志物的相关研究报道。FBXO7 is an E3 ubiquitin ligase consisting of 522 amino acids with a protein size of approximately 62 kDa. Its research shows that its main function is to maintain the homeostasis of intracellular mitochondria and the ubiquitination and degradation of proteins. At present, there is no published research report on the use of FBXO7 protein as a diagnostic and therapeutic marker in endometrial cancer at home and abroad.

发明内容Contents of the invention

本发明所要解决的技术问题是提供了一种与子宫内膜癌的患病率呈负相关的蛋白质FBXO7在制备子宫内膜癌诊断标志物中的应用。The technical problem to be solved by the present invention is to provide the application of a protein FBXO7 negatively correlated with the prevalence of endometrial cancer in the preparation of endometrial cancer diagnostic markers.

本发明解决上述技术问题所采用的技术方案为：一种蛋白质FBXO7在制备子宫内膜癌诊断标志物和/或治疗药物中的应用。The technical scheme adopted by the present invention to solve the above technical problems is: the application of a protein FBXO7 in the preparation of endometrial cancer diagnostic markers and/or therapeutic drugs.

进一步，所述的蛋白质FBXO7突变体在制备子宫内膜癌诊断试剂中的应用。Further, the application of the protein FBXO7 mutant in the preparation of endometrial cancer diagnostic reagents.

进一步，所述的蛋白质FBXO7在制备子宫内膜癌精准靶向治疗药物中的应用。Further, the application of the protein FBXO7 in the preparation of precise targeted therapy drugs for endometrial cancer.

一种线粒体分裂抑制剂Mdivi-1在制备FBXO7蛋白低表达和/或突变的子宫内膜癌中的应用。Application of a mitochondrial division inhibitor Mdivi-1 in the preparation of endometrial cancer with low expression and/or mutation of FBXO7 protein.

与现有技术相比，本发明的优点在于：本发明首次公开了蛋白质FBXO7在制备子宫内膜癌诊断标志物和/或治疗药物中的应用。对免疫组织化学染色图片进行分析，通过2位病理科专业人员打分评价FBXO7蛋白在子宫内膜癌患者样本和正常样本中的表达，蛋白质FBXO7在子宫内膜癌中表达明显下调，可用于辅助诊断子宫内膜癌。用细胞功能实验来显示FBXO7蛋白差异表达和突变对子宫内膜癌发生的影响，并通过靶向FBXO7蛋白相关下游信号分子通路来对子宫内膜癌进行治疗。相对诊断性刮宫进行组织病理学诊断，本技术有利于子宫内膜癌的早期诊断并能针对此设计靶向治疗的手段。Compared with the prior art, the present invention has the advantages that: the present invention discloses for the first time the application of protein FBXO7 in the preparation of endometrial cancer diagnostic markers and/or therapeutic drugs. The immunohistochemical staining pictures were analyzed, and the expression of FBXO7 protein in endometrial cancer patient samples and normal samples was scored by 2 pathology professionals. The protein FBXO7 expression was significantly down-regulated in endometrial cancer, which can be used for auxiliary diagnosis Endometrial cancer. Cell function experiments were used to show the effects of differential expression and mutation of FBXO7 protein on the occurrence of endometrial cancer, and to treat endometrial cancer by targeting FBXO7 protein-related downstream signaling molecular pathways. Compared with diagnostic curettage for histopathological diagnosis, this technology is beneficial to the early diagnosis of endometrial cancer and can design targeted therapy for this.

附图说明Description of drawings

图1为免疫组化验证FBXO7蛋白在人子宫内膜癌组织和正常子宫内膜组织中的表达情况。(A) 102例组织样本，包含子宫内膜癌组织97例与正常子宫内膜5例；选取FBXO7蛋白免疫组织化学染色的代表性图像（正常子宫内膜组织1例；子宫内膜癌组织2例；比例尺：20μm）；(B) 子宫内膜癌组织和正常子宫内膜组织免疫组化学染色结果评分（Positive;Low Positive; Negative）和统计堆积柱状图（*P<0.05）；Figure 1 is the immunohistochemical verification of the expression of FBXO7 protein in human endometrial cancer tissue and normal endometrial tissue. (A) 102 tissue samples, including 97 cases of endometrial cancer tissues and 5 cases of normal endometrium; representative images of FBXO7 protein immunohistochemical staining were selected (1 case of normal endometrial tissue; 2 cases of endometrial cancer tissue Example; scale bar: 20 μm); (B) Immunohistochemical staining scores (Positive; Low Positive; Negative) and stacked histogram of endometrial cancer tissue and normal endometrial tissue (*P<0.05);

图2为克隆形成实验验证FBXO7蛋白敲除对AN3 CA细胞增殖能力的影响。(A)Western Blotting实验验证FBXO7蛋白敲除以及回补野生型FBXO7蛋白和子宫内膜癌来源的FBXO7突变体蛋白的AN3 CA细胞稳定株的构建；(B) 克隆形成实验：敲除FBXO7蛋白的AN3CA细胞稳定株的增殖能力显著上升；而回补野生型FBXO7蛋白能逆转敲除FBXO7蛋白导致的细胞恶性表型，而回补子宫内膜癌来源的FBXO7突变体蛋白无法逆转；(C)克隆形成实验量化统计图（n.s:无统计学差异;*P<0.05）；Figure 2 is a colony formation experiment to verify the effect of FBXO7 protein knockout on the proliferation ability of AN3 CA cells. (A) Western Blotting experiment to verify the construction of FBXO7 protein knockout and complementation of wild-type FBXO7 protein and endometrial cancer-derived FBXO7 mutant protein AN3 CA cell stable strain; (B) Colony formation experiment: knockout of FBXO7 protein The proliferation ability of the stable AN3CA cell line was significantly increased; while replenishing the wild-type FBXO7 protein could reverse the malignant phenotype of the cells caused by knocking out the FBXO7 protein, but replenishing the FBXO7 mutant protein derived from endometrial cancer could not reverse it; (C) clone Form an experimental quantitative statistical map (n.s: no statistical difference; *P<0.05);

图3为FBXO7蛋白对INF2蛋白的泛素化降解。(A) 野生型FBXO7蛋白能够对INF2蛋白进行降解; (B) 野生型FBXO7蛋白能够对INF2蛋白进行泛素化修饰; (C) 野生型FBXO7蛋白能够对INF2蛋白进行降解，而子宫内膜癌来源的FBXO7突变体则失去降解INF2蛋白的功能；Figure 3 shows the ubiquitination degradation of INF2 protein by FBXO7 protein. (A) Wild-type FBXO7 protein can degrade INF2 protein; (B) Wild-type FBXO7 protein can ubiquitinate INF2 protein; (C) Wild-type FBXO7 protein can degrade INF2 protein, while endometrial cancer The derived FBXO7 mutant loses the function of degrading INF2 protein;

图4为应用Mdivi-1处理AN3 CA野生型细胞株和敲除FBXO7蛋白的AN3 CA细胞株。(A) Western Blotting检测Mdivi-1处理后AN3 CA细胞株蛋白表达变化，发现Mdivi-1并不影响INF2、FBXO7和Drp1的蛋白水平；(B) 克隆形成实验：Mdivi-1显著抑制敲除FBXO7蛋白的AN3 CA细胞稳定株的增殖能力；(C) 克隆形成实验量化统计图（*P<0.05）。Figure 4 shows the AN3 CA wild-type cell line treated with Mdivi-1 and the AN3 CA cell line knocked out of the FBXO7 protein. (A) Western Blotting detected the protein expression changes of AN3 CA cell line after Mdivi-1 treatment, and found that Mdivi-1 did not affect the protein levels of INF2, FBXO7 and Drp1; (B) Colony formation experiment: Mdivi-1 significantly inhibited the knockout of FBXO7 Proliferation ability of AN3 CA cell stable strain of protein; (C) Statistical graph of colony formation experiment quantification (*P<0.05).

具体实施方式Detailed ways

以下结合附图实施例对本发明作进一步详细描述。The present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments.

具体实施例specific embodiment

一、实验方法1. Experimental method

1、子宫内膜癌组织样本免疫组织化学染色1. Immunohistochemical staining of endometrial cancer tissue samples

子宫内膜癌组织芯片购买于陕西爱维拉生物科技有限公司，包含97例子宫内膜癌组织样本和5例子宫内膜正常组织样本。Endometrial cancer tissue chips were purchased from Shaanxi Avela Biotechnology Co., Ltd., including 97 endometrial cancer tissue samples and 5 endometrial normal tissue samples.

纳入标准：1.样本病理诊断明确，确诊为子宫内膜癌；2.患者未接受术前放、化疗治疗；3.患者既往无其他系统的恶性肿瘤。Inclusion criteria: 1. The pathological diagnosis of the sample is clear, and it is diagnosed as endometrial cancer; 2. The patient has not received preoperative radiotherapy and chemotherapy; 3. The patient has no previous malignant tumors of other systems.

本发明涉及的所有人体组织标本，均经过医学伦理的审核，只限实验室研究。All human tissue samples involved in the present invention have been reviewed by medical ethics and are limited to laboratory research.

(1) 抗原修复：将50×的柠檬酸钠抗原修复液用去离子水稀释到1×并摇晃均匀，将1×柠檬酸钠抗原修复液倒进高压锅中，合上锅盖，待锅内液体沸腾，将装有切片的玻片架置于高压锅中，使柠檬酸钠抗原修复液完全浸没切片，旋紧锅盖，待高压锅气阀开始均匀冒气后开始计时，8 min后关闭电磁炉电源，结束加热。将高压锅置于流动自来水下降温，打开锅盖，使石蜡切片冷却至室温。(1) Antigen restoration: Dilute 50× sodium citrate antigen restoration solution with deionized water to 1× and shake evenly, pour 1× sodium citrate antigen restoration solution into the pressure cooker, close the lid, and wait for the When the liquid boils, place the slide rack with the slices in the pressure cooker, completely submerge the slices in the sodium citrate antigen retrieval solution, tighten the lid of the pot, and start timing when the gas valve of the pressure cooker starts to emit gas evenly, and turn off the power of the induction cooker after 8 minutes , end heating. Place the pressure cooker under flowing tap water to lower the temperature, open the lid, and let the paraffin sections cool down to room temperature.

(2) 洗涤：首先使用去离子水充分洗涤切片5 min，接着用PBS磷酸盐缓冲液洗涤切片3 min，重复洗涤3次。(2) Washing: First, wash the slices thoroughly with deionized water for 5 min, then wash the slices with PBS phosphate buffered saline for 3 min, and repeat the washing 3 times.

(3) 封闭内源性过氧化物酶：将10%过氧化氢水溶液用去离子水稀释至3%浓度，每次现配现用，将石蜡切片放于湿盒内，在载玻片上组织所在位置滴加适量3%过氧化氢水溶液，合上湿盒的盖子，37℃封闭10 min。(3) Block endogenous peroxidase: Dilute 10% hydrogen peroxide aqueous solution with deionized water to a concentration of 3%, and use it each time, put the paraffin section in a wet box, and organize the Add an appropriate amount of 3% hydrogen peroxide aqueous solution dropwise at the location, close the lid of the wet box, and seal at 37°C for 10 min.

(4) 洗涤：用PBS磷酸盐缓冲液洗涤切片3 min×3次，甩掉载玻片上的液体；(4) Washing: wash the sections with PBS phosphate buffered saline for 3 min×3 times, and shake off the liquid on the slide;

(5) 血清封闭：洗涤后的切片摆放于湿盒内，每张切片上滴加适量10%驴血清封闭液，室温下封闭15 min后，甩掉切片上的封闭液；(5) Serum blocking: the washed slices were placed in a wet box, and an appropriate amount of 10% donkey serum blocking solution was added dropwise to each slice, and after blocking at room temperature for 15 minutes, the blocking solution on the slices was shaken off;

(6) 一抗孵育：擦净组织周围的封闭液，用免疫组织化学染色专用疏水笔围绕组织画圈，将切片置于湿盒内，滴加适当浓度的FBXO7抗体稀释液(抗体品牌：Proteintech；货号：10696-1-AP；稀释比：1:100)，使抗体稀释液浸没组织，置于4℃冰箱避光过夜；(6) Primary antibody incubation: wipe off the blocking solution around the tissue, draw a circle around the tissue with a special hydrophobic pen for immunohistochemical staining, place the section in a wet box, and drop an appropriate concentration of FBXO7 antibody diluent (antibody brand: Proteintech ; product number: 10696-1-AP; dilution ratio: 1:100), immerse the tissue in the antibody diluent, and place it in a 4°C refrigerator overnight in the dark;

(7) 洗涤：用PBS磷酸盐缓冲液洗涤切片3 min×3次；(7) Washing: wash the slices with PBS phosphate buffered saline for 3 min×3 times;

(8) 二抗孵育：擦干组织周围的液体，在组织上滴加适当浓度的HRP标记驴抗兔二抗，使抗体完全覆盖组织，37℃湿盒避光孵育1 h；(8) Secondary antibody incubation: wipe off the liquid around the tissue, drop an appropriate concentration of HRP-labeled donkey anti-rabbit secondary antibody on the tissue, so that the antibody completely covers the tissue, and incubate at 37°C in a wet box for 1 h;

(9) 洗涤：用PBS磷酸盐缓冲液洗涤切片3 min×3次；(9) Washing: wash the slices with PBS phosphate buffered saline for 3 min×3 times;

(10) DAB显色：将DAB显色液滴加到载玻片上组织所在位置，将载玻片置于倒置显微镜上观察组织染色情况，待染色强度达到最佳时甩掉显色液，使用去离子水洗涤3 min×3次。 (10) DAB color development: add DAB color development solution to the position of the tissue on the slide, place the slide glass on an inverted microscope to observe the staining of the tissue, discard the color development solution when the staining intensity reaches the best, and use Wash with deionized water for 3 min×3 times.

(11) 苏木素复染：在载玻片上组织所在位置滴加适量改良Lillie-Mayer苏木素染液，染色2 min，甩掉染色液，将装有载玻片的玻片架置于流动自来水下洗涤10 min以返蓝。(11) Hematoxylin counterstaining: Add an appropriate amount of modified Lillie-Mayer hematoxylin staining solution to the position of the tissue on the slide, stain for 2 minutes, shake off the staining solution, and wash the slide rack with slides under running tap water 10 minutes to return to blue.

(12) 脱水：将装有切片的玻片架依次浸泡于75%乙醇溶液5 min×1次；95%乙醇溶液中浸泡5 min×1次；无水乙醇溶液中浸泡5 min×3次；(12) Dehydration: Soak slide racks with slices in 75% ethanol solution for 5 min×1 time; soak in 95% ethanol solution for 5 min×1 time; soak in absolute ethanol solution for 5 min×3 times;

(13) 透明：将切片置于二甲苯溶液中浸泡5 min×2次； (13) Transparent: soak the slices in xylene solution for 5 min×2 times;

(14) 封片：在载玻片上组织所在位置滴加适量中性树胶，用镊子夹取一张盖玻片轻轻覆盖在载玻片上，置于通风橱中晾干成片； (14) Seal the slide: add an appropriate amount of neutral gum to the position of the tissue on the slide, use tweezers to pick up a cover glass and gently cover it on the slide, and place it in a fume hood to dry into pieces;

(15) 评分：将切片置于倒置显微镜上，依次在低倍镜、高倍镜下观察组织形态、细胞核染色、目的蛋白质染色情况等，2位专业病理科专业人员在不知情实验分组的情况下对图片进行打分。 (15) Scoring: Place the slices on an inverted microscope, and observe the tissue morphology, nuclear staining, and target protein staining under low-power and high-power lenses in turn. Two professional pathologists did not know the experimental grouping Rate the picture.

2. FBXO7蛋白敲除以及回补野生型FBXO7蛋白和子宫内膜癌来源的FBXO7突变体蛋白的AN3 CA细胞稳定株筛选和鉴定2. Screening and identification of stable strains of AN3 CA cells with FBXO7 protein knockout and complementation of wild-type FBXO7 protein and FBXO7 mutant protein derived from endometrial cancer

(1) 将CD513B-cas9-sgFBXO7质粒转染AN3 CA细胞，48小时后将细胞消化成均匀的细胞悬液，计数细胞。取1块六孔板，将细胞悬液按每孔1000个细胞种植到六孔板；(1) Transfect AN3 CA cells with CD513B-cas9-sgFBXO7 plasmid, digest the cells into a homogeneous cell suspension after 48 hours, and count the cells. Take a six-well plate, and plant the cell suspension into the six-well plate with 1000 cells per well;

(2) 将六孔板放入恒温培养箱中培养10天，待肉眼可见细胞集落形成时，将单个细胞集落用胰酶消化后转移至24孔板中，置于恒温培养箱中继续培养，每孔单克隆细胞作为一组； (2) Put the six-well plate in a constant temperature incubator and cultivate it for 10 days. When cell colonies are visible to the naked eye, digest a single cell colony with trypsin and transfer it to a 24-well plate, and place it in a constant temperature incubator to continue culturing. Monoclonal cells per well as a group;

(3) 待细胞均匀铺满24孔板后，按上述方法将每组细胞转移至12孔板，待细胞均匀铺满孔板后同法转移至6孔板，待细胞密度至80%，将六孔板中每组细胞按1传3进行传代至新的6孔板，置于恒温培养箱中继续培养； (3) After the cells evenly cover the 24-well plate, transfer the cells of each group to the 12-well plate according to the above method, and transfer the cells to the 6-well plate in the same way after the cells evenly cover the well plate. When the cell density reaches 80%, transfer the cells to the 12-well plate Each group of cells in the six-well plate was subcultured to a new 6-well plate according to 1 passage and 3, and placed in a constant temperature incubator to continue culturing;

(4) 待细胞密度达80%时，每组细胞取一孔细胞进行Western Blotting鉴定；(4) When the cell density reaches 80%, take a well of cells from each group of cells for Western Blotting identification;

(5) 在FBXO7蛋白敲除的子宫内膜癌细胞株中瞬时转染回补野生型FBXO7蛋白和子宫内膜癌来源的FBXO7突变体蛋白，用Western Blotting鉴定。(5) The wild-type FBXO7 protein and FBXO7 mutant protein derived from endometrial cancer were transiently transfected into the FBXO7 protein knockout endometrial cancer cell line, and identified by Western Blotting.

3、克隆形成实验3. Colony formation experiment

(1) 取生长良好的AN3 CA野生型细胞株，FBXO7蛋白敲除AN3 CA细胞稳定株，将贴壁细胞消化成均匀细胞悬液，进行细胞计数；(1) Take the well-growing AN3 CA wild-type cell line and the FBXO7 protein knockout AN3 CA cell stable line, digest the adherent cells into a uniform cell suspension, and perform cell counting;

(2) 取数个6孔细胞板，每孔加入2×10³个细胞，每个处理组设置3个复孔，置于恒温培养箱，每3天换液一次，共培养10−15天；(2) Take several 6-well cell plates, add 2×¹⁰³ cells to each well, set up 3 duplicate wells for each treatment group, place them in a constant temperature incubator, change the medium every 3 days, and culture for 10−15 days ;

(3) 当六孔板内肉眼可见细胞集落时，弃去孔内培养基，用PBS缓冲液润洗2次，每孔加入适量4%多聚甲醛固定细胞，放置30 min；(3) When cell colonies are visible to the naked eye in the six-well plate, discard the culture medium in the well, rinse with PBS buffer twice, add an appropriate amount of 4% paraformaldehyde to each well to fix the cells, and place for 30 min;

(4) 弃去固定液，用PBS缓冲液润洗2次，向每孔加入800 µL 0.1%结晶紫染料，放于摇床上染色10 min，回收染料，用PBS缓冲液润洗3次，置于37℃烘箱过夜，待晾干后拍照并统计数据。 (4) Discard the fixative, rinse twice with PBS buffer, add 800 µL of 0.1% crystal violet dye to each well, place on a shaker for staining for 10 min, recover the dye, rinse three times with PBS buffer, and place Oven overnight at 37°C, take pictures and count data after drying.

4、FBXO7蛋白对INF2蛋白的泛素化降解4. Ubiquitination and degradation of INF2 protein by FBXO7 protein

(1) 在HEK293T细胞中瞬时转染FLAG-INF2重组蛋白质粒，在此基础上瞬时转染梯度递增的Myc-FBXO7重组蛋白质粒，最后用Western blotting检测到外源FBXO7蛋白水平与外源INF2蛋白水平呈现明显负相关；(1) Transiently transfect the FLAG-INF2 recombinant protein plasmid in HEK293T cells, and then transiently transfect the gradient increasing Myc-FBXO7 recombinant protein plasmid on this basis, and finally detect the level of exogenous FBXO7 protein and exogenous INF2 protein by Western blotting The levels showed a significant negative correlation;

(2) 在HEK293T细胞中瞬时转染FLAG-INF2、Myc-FBXO7和HA-Ub质粒，在收集细胞裂解液前8小时，使用MG132抑制INF2的泛素-蛋白酶体途径降解，而后使用富含 FLAG 抗体的商品化 M2 珠子免疫共沉淀INF2，最后Western blotting 使用HA抗体检测到INF2被FBXO7泛素化修饰；(2) Transiently transfect FLAG-INF2, Myc-FBXO7 and HA-Ub plasmids in HEK293T cells, 8 hours before collecting the cell lysate, use MG132 to inhibit the degradation of the ubiquitin-proteasome pathway of INF2, and then use enriched FLAG The commercialized M2 beads of the antibody co-immunoprecipitate INF2, and finally Western blotting uses HA antibody to detect that INF2 is modified by FBXO7 ubiquitination;

(3) 在HEK293T细胞中瞬时转染FLAG-INF2重组蛋白质粒，在此基础上瞬时转染梯度递增的Myc-FBXO7野生型和子宫内膜癌来源突变体重组蛋白质粒，最后用Westernblotting检测到子宫内膜癌来源的FBXO7突变体无法降解INF2。 (3) Transiently transfect the FLAG-INF2 recombinant protein plasmid in HEK293T cells, and then transiently transfect the Myc-FBXO7 wild-type and endometrial cancer-derived mutant recombinant protein plasmids on this basis, and finally detect the uterus by Western blotting Endometrial cancer-derived FBXO7 mutants fail to degrade INF2.

5、Mdivi-1药物治疗5. Mdivi-1 drug treatment

取生长良好的AN3 CA野生型细胞株，FBXO7蛋白敲除AN3 CA细胞稳定株，行克隆形成实验。Mdivi-1溶解于DMEM高糖完全培养基中，最终浓度为1μM。待克隆形成铺板的细胞贴壁，将培养基更换为含有单纯DMSO（Mdivi-1的初始溶剂，排除溶剂毒性干扰）的DMEM高糖完全培养基和含有Mdivi-1的DMEM高糖完全培养基，每3天换液一次，共培养10−15天。观察Mdivi-1对敲除FBXO7蛋白的子宫内膜癌细胞稳定株增殖能力的影响。Take the well-growing AN3 CA wild-type cell line and the FBXO7 protein knockout AN3 CA cell stable line for colony formation experiments. Mdivi-1 was dissolved in DMEM high glucose complete medium to a final concentration of 1 μM. When the clones form the plated cells to adhere to the wall, replace the medium with DMEM high-glucose complete medium containing pure DMSO (the initial solvent of Mdivi-1, to exclude solvent toxicity interference) and DMEM high-glucose complete medium containing Mdivi-1, The medium was changed every 3 days and co-cultured for 10−15 days. To observe the effect of Mdivi-1 on the proliferation ability of stable endometrial cancer cell lines knocked out of FBXO7 protein.

二、实验结果分析2. Analysis of Experimental Results

本发明确保实验数据的完整、准确、无误。免疫组织化学图片经由2位专业病理科专业人员在不知情实验分组的情况下对图片进行打分，打分的结果经由GraphPad Prism软件进行作图并分析，所有数据统计检验均取双侧概率，按照α=0.05的检验水准进行统计检验，若P<0.05，即认为两组间的差异有统计学意义，反之则认为差异无统计学意义。克隆形成实验结果经由Image J软件进行分析，分析结果经由GraphPad Prism软件进行作图并分析，所有数据统计检验均取双侧概率，按照α=0.05的检验水准进行统计检验，若P<0.05，即认为两组间的差异有统计学意义，反之则认为差异无统计学意义。The invention ensures that the experimental data is complete, accurate and error-free. Immunohistochemical pictures were scored by 2 professional pathologists without knowing the experimental grouping. The scoring results were drawn and analyzed by GraphPad Prism software. =0.05 for statistical testing, if P<0.05, the difference between the two groups was considered statistically significant, otherwise, the difference was considered not statistically significant. The results of clone formation experiments were analyzed by Image J software, and the analysis results were drawn and analyzed by GraphPad Prism software. All data statistical tests were carried out with two-sided probability, and the statistical test was carried out according to the test level of α=0.05. If P<0.05, that is The difference between the two groups was considered to be statistically significant, otherwise the difference was considered not to be statistically significant.

我们发现在子宫内膜癌组织和正常子宫内膜组织中，FBXO7蛋白在子宫内膜癌组织中的表达显著低于正常子宫内膜组织，FBXO7蛋白在正常子宫内膜组织中的弱阳性率为80% (4/5)，阳性率为20%(1/5)；在子宫内膜癌组织中，阴性率为62.8% (61/97)，弱阳性率为37.2% (36/97)（图1 A, B；*P<0.05）。We found that in endometrial cancer tissue and normal endometrial tissue, the expression of FBXO7 protein in endometrial cancer tissue was significantly lower than that in normal endometrial tissue, and the weak positive rate of FBXO7 protein in normal endometrial tissue was 80% (4/5), the positive rate was 20% (1/5); in endometrial cancer tissue, the negative rate was 62.8% (61/97), and the weak positive rate was 37.2% (36/97) ( Figure 1 A, B; *P<0.05).

克隆形成实验显示，与野生型AN3 CA细胞相比，敲除FBXO7蛋白的细胞克隆集落的数量明显增加。这些结果提示均提示检测FBXO7蛋白质的相对表达对子宫内膜癌的诊断具有较高价值（图2A, B, C；*P<0.05）。Colony formation experiments showed that the number of clonal colonies of cells knocked out of FBXO7 protein was significantly increased compared with wild-type AN3 CA cells. These results suggest that detecting the relative expression of FBXO7 protein has a high value in the diagnosis of endometrial cancer (Fig. 2A, B, C; *P<0.05).

同时我们发现野生型FBXO7蛋白对INF2蛋白进行泛素化降解，而子宫内膜癌来源的FBXO7蛋白则无法降解INF2蛋白(图3A, B, C)，INF2蛋白在线粒体分裂起始阶段招募Drp1，随后引发线粒体分裂，因此我们猜测FBXO7可能通过抑制线粒体分裂来抑制子宫内膜癌的发生，而子宫内膜癌患者体内异常降低或者突变的FBXO7蛋白可能导致细胞内线粒体分裂的亢进。At the same time, we found that wild-type FBXO7 protein can ubiquitinate and degrade INF2 protein, while endometrial cancer-derived FBXO7 protein cannot degrade INF2 protein (Fig. 3A, B, C). INF2 protein recruits Drp1 at the initiation stage of mitochondrial division, Mitochondrial fission is subsequently triggered, so we speculate that FBXO7 may inhibit the occurrence of endometrial cancer by inhibiting mitochondrial fission, and the abnormally reduced or mutated FBXO7 protein in endometrial cancer patients may lead to the hyperactivity of mitochondrial fission in cells.

为此本发明应用线粒体分裂抑制剂Mdivi-1治疗FBXO7蛋白低表达或者突变的子宫内膜癌，发现Mdivi-1显著抑制了敲除FBXO7蛋白的AN3 CA细胞稳定株的增殖能力（图4A, B, C; *P<0.05），提示Mdivi-1在精准靶向治疗FBXO7蛋白低表达或者突变的子宫内膜癌的可行性。For this reason, the present invention uses the mitochondrial fission inhibitor Mdivi-1 to treat endometrial cancer with low expression or mutation of FBXO7 protein, and finds that Mdivi-1 significantly inhibits the proliferation ability of the stable strain of AN3 CA cells knocked out of FBXO7 protein (Fig. 4A, B , C; *P<0.05), suggesting the feasibility of Mdivi-1 in precise targeted therapy of endometrial cancer with low expression or mutation of FBXO7 protein.

该发明通过检测FBXO7蛋白在子宫内膜癌中的差异表达，和敲除FBXO7蛋白的AN3CA细胞株的克隆形成实验，证明FBXO7蛋白作为子宫内膜癌诊断标志物的可行性。并通过发现FBXO7的下游信号分子通路，将FBXO7蛋白应用于子宫内膜癌的精准靶向治疗。The invention proves the feasibility of FBXO7 protein as a diagnostic marker for endometrial cancer by detecting the differential expression of FBXO7 protein in endometrial cancer and the clone formation experiment of AN3CA cell line with FBXO7 protein knocked out. And by discovering the downstream signaling molecular pathway of FBXO7, the FBXO7 protein is applied to the precise targeted therapy of endometrial cancer.

上述说明并非对本发明的限制，本发明也并不限于上述举例。本技术领域的普通技术人员在本发明的实质范围内，做出的变化、改型、添加或替换，也应属于本发明的保护范围。The above description does not limit the present invention, and the present invention is not limited to the above examples. Changes, modifications, additions or substitutions made by those skilled in the art within the essential scope of the present invention shall also belong to the protection scope of the present invention.

Claims (4)

Translated fromChinese

1.一种蛋白质FBXO7在制备子宫内膜癌诊断标志物和/或治疗药物中的应用。1. The application of a protein FBXO7 in the preparation of endometrial cancer diagnostic markers and/or therapeutic drugs.2.根据权利要求1所述的一种蛋白质的应用，其特征在于：所述的蛋白质FBXO7突变体在制备子宫内膜癌诊断试剂中的应用。2. The application of a protein according to claim 1, characterized in that: the application of the protein FBXO7 mutant in the preparation of endometrial cancer diagnostic reagents.3.根据权利要求1所述的一种蛋白质的应用，其特征在于：所述的蛋白质FBXO7在制备子宫内膜癌精准靶向治疗药物中的应用。3. The application of a protein according to claim 1, characterized in that: the application of the protein FBXO7 in the preparation of drugs for precise targeted therapy of endometrial cancer.4.一种线粒体分裂抑制剂Mdivi-1在制备FBXO7蛋白低表达和/或突变的子宫内膜癌中的应用。4. The application of a mitochondrial division inhibitor Mdivi-1 in the preparation of endometrial cancer with low expression and/or mutation of FBXO7 protein.

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