HIF下游基因DBC1、STC1对肾细胞癌形成的作用机制研究

发布时间：2018-05-31 10:09

本文选题：肾细胞癌 + VHL　；参考：《天津医科大学》2017年硕士论文

【摘要】：研究目的:绝大多数肾脏恶性肿瘤的组织学类型是肾透明细胞癌。约75%-80%的肾透明细胞癌患者中存在染色体缺失、基因突变及启动子甲基化导致的VHL等位基因失活表达缺失。很多研究也发现了VHL基因缺失、表达失活在肾透明细胞癌的发生及进展过程中发挥着非常重要的作用。氧存在时VHL蛋白(p VHL)呈递缺氧诱导因子HIF-α并与之结合使其通过泛素连接酶-蛋白酶体途径降解,使HIF-α分子在细胞中处于合成与降解的动态平衡。肾细胞癌细胞系786-O细胞中VHL基因表达缺失,细胞内HIF-2α不能被识别并降解呈现持续高表达的状态,进一步诱导下游百余种靶基因(HRGs)的表达。课题组前期实验中通过裸鼠体内成瘤的实验证实了HIF-2α对肾癌细胞系786-O成瘤的重要作用,但HIF下游靶基因对肾癌细胞的生长则表现为促进、抑制或没影响等不同情况。通过基因序列表达谱分析发现乳腺癌缺失基因-1(DBC1)及斯钙素蛋白-1(STC1)属于被HIF-2α诱导表达升高的下游靶基因,而且检索文献发现DBC1、STC1在多种恶性肿瘤均发挥着重要的作用,但它们对肾细胞癌肿瘤形成的作用及机制并没有系统深入的研究。本研究的目的在于验证DBC1、STC1会被HIF-2α诱导表达,属于HIF-2α下游基因,并探讨它们对肾细胞癌肿瘤形成的作用及可能机制,旨在寻找潜在的肾细胞癌靶向治疗的新靶点及诊断或预后新的生物学标志物,优化肾癌治疗的个体化方案,改善晚期肾癌患者的预后。研究方法:1.培养本室构建并保存的四种人类肾透明细胞癌细胞786-O(VHL-/-、VHL+/+、VHL-/-HIF-2αsh566、VHL+/+HIF-2α-d PA),使用实时荧光定量PCR技术检测DBC1、STC1在四种细胞系中m RNA水平的表达,验证其是否属于HIF-2α诱导表达升高的基因;2.通过使用慢病毒携带sh RNA DBC1、sh RNA STC1及SCR感染786-O(VHL-/-)肾癌细胞分别沉默DBC1、STC1的表达及对照组细胞,嘌呤霉素筛选得到稳定转染的细胞系,使用Western Blot检测相应细胞中DBC1、STC1的表达情况;3.MTT分别检测阴性对照的786-O(VHL-/-)细胞和沉默DBC1表达及沉默STC1表达的786-O(VHL-/-)细胞的体外增殖情况;4.分别使用沉默DBC1表达及沉默STC1表达的786-O(VHL-/-)细胞注入裸鼠一侧皮下作为实验组,对侧皮下注入携带对应阴性对照的786-O(VHL-/-)细胞作为对照组来进行体内成瘤实验,8周后处死裸鼠取出皮下肿瘤组织,进行称重后统计并比较两组实验组及对照组裸鼠移植瘤生长情况。结果:1.DBC1及STC1在786-O(VHL+/+、VHL-/-HIF-2αsh566)细胞的表达情况低于786-O(VHL-/-、VHL+/+HIF-2α-d PA)细胞中的表达情况,表明DBC1和STC1属于HIF-2α的下游靶基因,能够被HIF-2α诱导而表达升高。2.MTT实验结果表明沉默DBC1在786-O(VHL-/-)细胞中的表达没有对肾癌细胞的体外增殖产生影响,沉默STC1在786-O(VHL-/-)细胞中的表达则抑制肾癌细胞的体外增殖。3.沉默DBC1和STC1在786-O VHL-/-细胞中的表达后裸鼠移植瘤成瘤能力明显小于对照,DBC1组和STC1组中实验组与对照组的差异均具有统计学意义(P0.01)。结论:1.DBC1、STC1在人类肾透明细胞癌细胞786-O中被HIF-2α诱导表达升高,是HIF-2α的下游靶基因。2.DBC1的表达对肾癌细胞体外增殖没有影响,STC1的表达促进肾癌细胞的体外增殖;DBC1和STC1都明显促进裸鼠移植瘤成瘤,提示它们在肾癌中是促癌因素。沉默DBC1及STC1表达抑制了786-O(VHL-/-)肾癌细胞系裸鼠移植瘤的形成,表明DBC1、STC1是可能的肾癌分子靶向治疗的新靶点。3.VHL-HIF-HRGs调节通路是促进肾细胞癌发生发展的关键,但仍有一大批被HIF-2α诱导表达升高的靶基因在肾细胞癌肿瘤形成的过程中发挥的作用还没有被系统得证实,它们可能通过参与不同的信号通路,发挥着对肾细胞癌发生发展相关的作用。对这些基因进行进一步的筛选和验证有助于深入全面地了解肾细胞癌肿瘤发生发展的机制,并可以探索更多肾癌分子靶向治疗的新靶点。
[Abstract]:Objective: the histologic type of the vast majority of renal malignant tumors is clear cell carcinoma of the kidney. There are chromosomal deletion, gene mutation and promoter methylation of VHL allele inactivation in about 75%-80% of renal clear cell carcinoma. Many studies have also found VHL gene deletion and inactivation in renal clear cell carcinoma. When oxygen exists, the VHL protein (P VHL) presents hypoxia inducible factor HIF- alpha and combines with the ubiquitin ligase proteasome pathway to make the HIF- alpha molecule in the cell in the dynamic equilibrium of synthesis and degradation. The expression of VHL gene in the cell line of renal cell carcinoma cell line 786-O is deficient. The HIF-2 alpha in the cell can not be identified and degraded to present a continuous high expression state, which further induces the expression of more than 100 target genes (HRGs) in the lower reaches of the group. In the early experiments of the group, the tumor formation in nude mice confirmed the important role of HIF-2 alpha on the tumor cell line 786-O in the renal cell line, but the target gene of the downstream HIF on the growth of renal cell carcinoma cells -1 (DBC1) and -1 (STC1) are the downstream target genes which are elevated by HIF-2 alpha induced expression by gene sequence expression spectrum analysis, and the retrieval literature found that DBC1, STC1 plays an important role in various malignant tumors, but they are to renal cells. The role and mechanism of cancer formation have not been systematically studied. The purpose of this study is to verify that DBC1, STC1 can be induced by HIF-2 alpha, belongs to the HIF-2 alpha downstream gene, and to explore the role and possible mechanism of them on the formation of renal cell carcinoma, aiming to find new targets and diagnosis or prognosis of potential treatment of renal cell carcinoma. A new biological marker to optimize the individualized regimen for the treatment of renal carcinoma to improve the prognosis of patients with advanced renal cancer. 1. to cultivate four kinds of human renal cell carcinoma cell 786-O (VHL-/-, VHL+/+, VHL-/-HIF-2 a sh566, VHL+/+HIF-2 alpha -d PA) constructed and preserved in this room, and detect DBC1 with real-time fluorescent quantitative PCR technique, STC1 in four kinds of cells The expression of M RNA level in the system is to verify whether it belongs to the gene of elevated HIF-2 alpha induced expression; 2. by using lentivirus to carry sh RNA DBC1, sh RNA STC1 and SCR infected 786-O (VHL-/-) kidney cancer cells are silent, and the cells of the control group and purinamycin are screened for the stable transfected cell lines. The expression of DBC1 and STC1 in the cells; 3.MTT detection of negative control 786-O (VHL-/-) cells and silent DBC1 expression and the proliferation of 786-O (VHL-/-) cells expressing STC1 expression, respectively. 4. cells were injected subcutaneously into the nude mice by silencing DBC1 expression and silent STC1 expression as experimental group, subcutaneous injection of the contralateral side 786-O (VHL-/-) cells with negative control were used as the control group to carry out the tumor formation experiment in the body. After 8 weeks, the nude mice were killed and the subcutaneous tumor tissues were taken out. After weighing, the growth of the transplanted tumor in the two groups and the control group was compared. Results: the expression of 1.DBC1 and STC1 in 786-O (VHL+/+, VHL-/-HIF-2 alpha sh566) cells was low. The expression in 786-O (VHL-/-, VHL+/+HIF-2 alpha -d PA) cells indicates that DBC1 and STC1 belong to the downstream target gene of HIF-2 a, which can be induced by HIF-2 alpha and expressed in.2.MTT. The expression of.2.MTT in.2.MTT shows that the expression of silent DBC1 in 786-O cells does not affect the proliferation of renal cell carcinoma cells. The expression of.3. inhibited the proliferation of renal cell carcinoma cells in vitro and the expression of DBC1 and STC1 in 786-O VHL-/- cells was significantly less than that of the control. The difference between the experimental group and the control group in the DBC1 and STC1 groups was statistically significant (P0.01). Conclusion: 1.DBC1, STC1 is HIF-2 alpha in the 786-O of human renal cell carcinoma cells. The expression of.2.DBC1, the downstream target gene of HIF-2 alpha, has no effect on the proliferation of renal cancer cells in vitro. The expression of STC1 promotes the proliferation of renal cancer cells in vitro, and both DBC1 and STC1 obviously promote the tumor formation in nude mice, suggesting that they are cancer promoting factors in renal carcinoma. The silence of DBC1 and STC1 inhibits the 786-O (VHL-/-) renal cell line. The formation of xenografts in nude mice shows that DBC1, STC1 is a possible new target of molecular targeting therapy for renal cancer,.3.VHL-HIF-HRGs regulation pathway is the key to promote the development of renal cell carcinoma, but a large number of target genes which are induced by HIF-2 alpha induced expression have not been systematically confirmed by the role of the target gene in the process of renal cell carcinoma. They may play a role in the development of renal cell carcinoma by participating in different signaling pathways. Further screening and verification of these genes will help to understand the mechanism of the development of renal cell carcinoma and explore more new targets for molecular targeting therapy of renal cell carcinoma.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.11

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