HSP90、SIRT3基因多态性与肝癌发病风险及其在肝癌中表达与侵袭间的相关性研究

发布时间：2018-02-20 06:48

本文关键词： HSP90 SIRT3 基因多态性肝癌细胞增殖转移侵袭　出处：《安徽医科大学》2016年博士论文　论文类型：学位论文

【摘要】：目的本课题通过研究HSP90和SIRT3在肝癌细胞中的表达与侵袭以及两者的多态性位点与肝癌发病风险之间的相关性,旨在探讨HSP90与SIRT3二者对肝癌发生发展的影响,并通过此项群体研究发现和鉴定新的肝细胞癌易感基因,试图为肝癌易感人群的疾病预防提供新的标志物,同时也为开发和研制新的肝细胞癌临床用药提供新的靶标,以期对提高肝细胞癌的临床防治效果提供帮助。方法(1)选取在2013年1月-2015年10月期间住院的198例肝癌患者,年龄在35-76岁范围;同时采集198例体检中心健康自愿者作为对照,年龄在33-69岁间。所有受试者均基于自愿并签署知情同意书。对HSP90与SIRT3基因多态性与肝癌患病风险进行相关性分析,此外还对具有饮酒史患者与肝癌易感性进行相关性分析。(2)将p GCSIL-GFP-HSP90重组慢病毒载体转染人肝癌AMMC-7721细胞,荧光定量PCR检测转染后细胞中HSP90 m RNA的表达;MTT法检测细胞生长情况;流式细胞术检测转染后对各组细胞凋亡的影响;Transwell小室法检测细胞的侵袭能力;平板克隆形成实验检测细胞的增殖能力;细胞划痕实验检测细胞的迁移能力;Caspase-3活性检测细胞的凋亡情况。(3)采用RNA干扰的方法沉默人肝癌细胞AMMC-7721中HSP90基因的表达获得HSP90基因沉默细胞株si HSP90-AMMC-7721,分别将野生型WT-HSP90和突变型Mut-HSP90转染si HSP90-AMMC-7721细胞;荧光定量PCR检测各组细胞SIRT3 m RNA表达;western blot法检测各组细胞SIRT3蛋白的表达。结果(1)HSP90 rs8005905与rs3742429基因多态性与肝癌的发生风险之间存在显著相关性;HSP90 rs3742429与具有饮酒史的人群的肝癌发生风险呈显著相关性。(2)上调AMMC-7721细胞中HSP90的表达后使细胞的增殖能力明显增高;流式细胞仪检测结果表明,上调细胞中HSP90的表达后对AMMC-7721细胞凋亡情况并未见显著影响;通过Transwell小室法检测结果表明上调HSP90的表达后视野平均侵袭细胞数量显著增多;平板克隆形成实验结果表明增高AMMC-7721细胞中HSP90的表达时,细胞克隆的形成能力明显上升;细胞划痕实验检测表明上调HSP90后AMMC-7721细胞的迁移性明显增强;caspase-3活性检测结果表明,上调HSP90表达后AMMC-7721细胞中caspase-3活性并未受到显著影响。(3)用荧光定量PCR以及western blot法分别对转染后各组细胞中SIRT3蛋白的表达情况进行检测,结果表明野生型HSP90转染后si HSP90-AMMC-772细胞中SIRT3m RNA和相应蛋白的表达明显降低;突变型HSP90转染后si HSP90-AMMC-7721细胞中SIRT3 m RNA和相应蛋白的表达无明显变化。结论HSP90基因多态性以及患者是否有饮酒史与肝癌的发生之间存在密切联系,且HSP90对肝癌细胞增殖、侵袭以及转移过程的促进作用可能是通过抑制SIRT3的表达实现的。
[Abstract]:Objective to investigate the relationship between the expression and invasion of HSP90 and SIRT3 in HCC cells and the relationship between the polymorphic sites of HSP90 and SIRT3 and the risk of HCC in order to explore the effect of HSP90 and SIRT3 on the occurrence and development of HCC. Through this study, a new susceptibility gene of hepatocellular carcinoma was found and identified in order to provide a new marker for the prevention of HCC and a new target for the development and development of new clinical drugs for HCC. Methods A total of 198 patients with liver cancer, aged 35-76 years, were selected from January 2013 to October 2015, and 198 healthy volunteers were collected as control. All subjects were based on voluntary and signed informed consent letters. The association between polymorphism of HSP90 and SIRT3 gene and the risk of liver cancer was analyzed. In addition, the relationship between drinking history and susceptibility to liver cancer was analyzed. (2) the recombinant lentivirus vector of p GCSIL-GFP-HSP90 was transfected into human hepatoma AMMC-7721 cells, and the expression of HSP90 m RNA in transfected cells was detected by fluorescence quantitative PCR. The effect of transfection on cell apoptosis was detected by flow cytometry. RNA interference method was used to silence the expression of HSP90 gene in human hepatoma cell line AMMC-7721. The cell line si HSP90-AMMC-7721 was silenced by HSP90 gene. The wild type WT-HSP90 was isolated from the cell line si HSP90-AMMC-7721. And mutant Mut-HSP90 were transfected into si HSP90-AMMC-7721 cells. The expression of SIRT3 m RNA was detected by fluorescence quantitative PCR and the expression of SIRT3 protein was detected by western blot. Results there was a significant correlation between the polymorphism of HSP90 rs8005905 and rs3742429 gene and the risk of liver cancer. There was a significant correlation between HSP90 rs3742429 and people with drinking history. There was a significant correlation between the risk of HCC development and the proliferation of AMMC-7721 cells after upregulating the expression of HSP90. The results of flow cytometry showed that the up-regulation of HSP90 expression had no significant effect on the apoptosis of AMMC-7721 cells, and the results of Transwell chamber assay showed that the number of invading cells in the visual field was significantly increased after the expression of HSP90 was upregulated. The results of plate clone formation assay showed that the ability of cell clone formation was significantly increased when the expression of HSP90 was increased in AMMC-7721 cells, and the migration of AMMC-7721 cells after HSP 90 was significantly enhanced by cell scratch assay. The activity of caspase-3 in AMMC-7721 cells was not significantly affected by up-regulation of HSP90 expression. The expression of SIRT3 protein was detected by fluorescence quantitative PCR and western blot. The results showed that the expression of SIRT3m RNA and corresponding protein in si HSP90-AMMC-772 cells was significantly decreased after transfection of wild type HSP90. There was no significant change in the expression of SIRT3 m RNA and corresponding proteins in si HSP90-AMMC-7721 cells transfected with mutant HSP90. Conclusion there is a close relationship between the polymorphism of HSP90 gene and the history of drinking alcohol in patients with HCC, and the proliferation of HCC cells induced by HSP90. The promotion of invasion and metastasis may be achieved by inhibiting the expression of SIRT3.
【学位授予单位】：安徽医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.7

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