VHL依赖性的Dicer失调在肾透明细胞癌中的作用及其机制研究

发布时间：2018-08-22 18:56

【摘要】：目的;肾细胞癌是泌尿系统最常见的恶性疾病之一,其中肾透明细胞癌是最主要的病理类型。以往研究表明VHL/HIF通路和rniRNA在肾透明细胞癌的发生发展中起着重要作用,而本课题旨在探寻这两条经典通路之间的关联及其在肾透明细胞癌中的作用和机制。方法：1.通过VHL外显子测序和VHL启动子区域甲基化状态分析检测182例肾透明细胞癌组织中基因组DNA的VHL状态,并选取8例VHL野生型肾透明细胞癌组织与8例VHL缺陷型肾透明细胞癌组织配对,通过微阵列分析两组之间miRNA的表达差异,并通过实时定量PCR进行验证;通过实时定量PCR和Western blot检测miRNA转录后生成过程中的关键酶Dicer、Drosha、Exportin-5和DGCR8在36例VHL野生型肾透明细胞癌组织和36例VHL缺陷型肾透明细胞癌组织中的表达情况,并通过实时定量PCR检测相应癌旁肾组织的Dicer表达情况;通过在肾透明细胞癌细胞系中分别过表达或敲低VHL后检测Dicer的表达情况。2.通过线性回归分析检测肾透明细胞癌组织中Dicer和HIF mRNA表达水平的相关性,并通过在肾透明细胞癌细胞系中过表达和敲低Dicer后检测HIF及其下游基因的表达情况;基于微阵列数据和实时定量PCR的结果,通过生物信息学软件的分析找出与HIF-2a有潜在结合位点的由Dicer介导的miRNA,并通过实时定量PCR、Western blot和荧光素酶实验验证该miRNA能否直接靶向作用于HIF-2α。3.通过克隆形成实验、血管形成实验和裸鼠体内成瘤实验探讨Dicer在VHL缺陷型肾透明细胞癌中的生物学功能以及是否通过HIF-2α发挥作用;对146例VHL缺陷型肾透明细胞癌患者进行随访,通过Kaplan-Meier法和COX回归模型分析Dicer mRNA表达水平高低和临床预后的关系。结果：1.微阵列数据分析和实时定量PCR结果显示VHL缺陷型较VHL野生型肾透明细胞癌组织中的总体miRNA表达是下调的,而下调miRNA的前体表达却是上升的;miRNA转录后生成过程中的关键酶只有Dicer在VHL缺陷型中的表达较VHL野生型中的表达明显下降,同时Dicer的表达水平在VHL缺陷型肾透明细胞癌组织中较癌旁组织明显下调,而Dicer在VHL野生型肾透明细胞癌组织和相应癌旁组织中的表达水平却无明显差异;在肾透明细胞癌细胞系中VHL可以正向调控Dicer的表达水平。2.线性回归分析结果显示Dicer与HIF-2a的mRNA表达水平成负相关,与HIF-1a的mRNA表达水平无相关性;在VHL缺陷型肾透明细胞癌细胞系中Dicer可以反向调控HIF-2a及其下游基因的表达,同时HIF-2a是由Dicer介导的niR-182-5p的直接靶点。3.克隆形成实验和血管形成实验证实了Dicer能在体外抑制VHL缺陷型肾透明细胞癌的增殖和血管生成,裸鼠成瘤实验证实了Dicer也能在体内抑制VHL缺陷型肾透明细胞癌的生长,而且Dicer的这一抑癌功能在某种程度上是通过下调HIF-2a实现的;Kaplan-Meier生存分析显示在VHL缺陷型肾透明细胞癌患者中,Dicer mRNA低表达水平组的术后生存率较Dicer mRNA高表达水平组更差,COX回归分析显示Dicer mRNA低表达水平是术后生存率较低的一个独立危险因素。结论：肾透明细胞癌中Dicer被VHL正向调控并能通过下调HIF-2a及其下游基因的表达来抑制VHL缺陷型肾透明细胞癌的肿瘤表型;Dicer的低表达是VHL缺陷型肾透明细胞癌患者临床预后差的独立危险因素。因此,Dicer在未来有可能成为肾透明细胞癌风险分级的肿瘤标志物和新的治疗靶点,为肾透明细胞癌的诊断和治疗提供新的思路。
[Abstract]:Objective: Renal cell carcinoma (RCC) is one of the most common malignant diseases in the urinary system, and renal clear cell carcinoma (RCCC) is the most important pathological type.Previous studies have shown that the VHL/HIF pathway and rniRNA play an important role in the development of RCC. Methods: 1. The VHL status of genomic DNA in 182 cases of renal clear cell carcinoma was detected by VHL exon sequencing and VHL promoter region methylation analysis, and 8 cases of VHL wild type renal clear cell carcinoma and 8 cases of VHL deficient renal clear cell carcinoma were selected to pair with each other. Differential expression of iRNA was detected by real-time quantitative PCR and Western blot, and the expression of key enzymes Dicer, Drosha, Exportin-5 and DGCR8 in 36 VHL wild renal clear cell carcinoma tissues and 36 VHL deficient renal clear cell carcinoma tissues was detected by real-time quantitative PCR and Western blot. PCR was used to detect the expression of Dicer in the adjacent renal tissues. The expression of Dicer was detected by overexpressing or knocking down VHL in renal clear cell carcinoma cell lines. Dicer-mediated microRNAs with potential binding sites to HIF-2a were identified by bioinformatics software analysis based on microarray data and real-time quantitative PCR results. Real-time quantitative PCR, Western blot and luciferase assay were used to verify the direct targeting of the microRNAs. To investigate the biological function of Dicer in VHL-deficient renal clear cell carcinoma and whether it plays a role through HIF-2alpha through clone formation test, angiogenesis test and tumor formation test in nude mice; to follow up 146 patients with VHL-deficient renal clear cell carcinoma and analyze Dice by Kaplan-Meier method and COX regression model. Results: 1. Microarray data analysis and real-time quantitative PCR showed that the expression of total microRNAs in VHL deficient renal clear cell carcinoma was down-regulated compared with that in VHL wild type renal clear cell carcinoma, but the expression of precursors of down-regulated microRNAs was up-regulated. Dicer was the only key enzyme in the post-transcriptional production of microRNAs. The expression level of Dicer in VHL-deficient renal clear cell carcinoma was significantly lower than that in VHL-deficient renal clear cell carcinoma, but the expression level of Dicer in VHL-deficient renal clear cell carcinoma and its adjacent tissues was not significantly different. Linear regression analysis showed that Dicer was negatively correlated with the mRNA expression of HIF-2a, but not with the mRNA expression of HIF-1a; Dicer could reverse the expression of HIF-2a and its downstream genes in VHL-deficient renal clear cell carcinoma cell lines, and HIF-2a was Dicer-mediated niR-18. Direct target of 2-5p. 3. Cloning and angiogenesis assays confirmed that Dicer could inhibit the proliferation and angiogenesis of VHL-deficient clear cell renal cell carcinoma in vitro. Tumor formation assay in nude mice confirmed that Dicer could also inhibit the growth of VHL-deficient clear cell renal cell carcinoma in vivo, and Dicer's anti-tumor function to a certain extent is universal. Kaplan-Meier survival analysis showed that the survival rate of patients with VHL-deficient renal clear cell carcinoma was worse in the group with low expression of Dicer mRNA than in the group with high expression of Dicer mRNA. COX regression analysis showed that low expression of Dicer mRNA was an independent risk factor for lower survival rate. Dicer is positively regulated by VHL and can inhibit the tumor phenotype of VHL-deficient renal clear cell carcinoma by down-regulating the expression of HIV-2a and its downstream genes. Low expression of Dicer is an independent risk factor for poor clinical prognosis in patients with VHL-deficient renal clear cell carcinoma. Grade I tumor markers and new therapeutic targets provide new ideas for the diagnosis and treatment of renal clear cell carcinoma.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.11

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