COMMD7基因活化PI3K-AKT信号通路促进HepG2细胞增殖转移的机制研究

发布时间：2018-05-01 18:17

本文选题：肝细胞癌 + COMMD7　；参考：《第三军医大学》2016年硕士论文

【摘要】：目的和意义:在世界范围内,肝细胞癌的发病率在常见的恶性肿瘤中已经排名第五,且每年至少增加100万新发病例,且中绝大部分发生在我国。作为一个乙肝大国,肝癌在我国有着广泛的疾病基础。肝脏移植和手术治疗是最为理想的治疗方式,但是手术切除率低、术后复发率高,病人生存率及生存质量偏低,总体治疗效较差。随着基因研究水平的不断提高,我们对肝癌的认识越加深入,意识到肝癌是一个受到多基因调控的肿瘤。分析鉴定肝癌相关基因及研究不同调控基因间的相互作用机制,寻找调控网络中的关键基因及调控因素,研发抗肿瘤的靶向治疗药物已经成为肝细胞癌研究的热点。方法:设计针对COMMD7基因的特异性siRNA(smallinterference RNA)转染人类肝癌细胞株HepG2,作为干扰组(Si-HepG2);并设置PTEN抑制剂处理组(Si+Bpv-HepG2);空白对照组(HepG2);空载体对照组(N-HepG2)。q RT-PCR检测转染后各组COMMD7、PTEN基因m RNA的表达变化;Western blot检测各组COMMD7、PTEN、p-AKT和AKT蛋白的表达变化;MSP法检测PTEN基因甲基化水平;CCK8法检测细胞增殖能力;Transwell实验检测细胞在转染前后转移及侵袭能力的改变。结果:siRNA-Commd7转染HepG2细胞后,qRT-PCR检测结果显示与空白对照组及空载体对照组相比较干扰组COMMD7基因的表达量降低了90%,PTEN基因的表达量升高了184%,均有统计学差异(P0.05);Western blot检测结果显示:siRNA干扰组COMMD7表达明显降低,PTEN表达升高;下游p-AKT表达受到显著抑制;PTEN抑制剂处理组PTEN表达受到显著抑制,下游p-AKT的表达明显增强;MSP法检测PTEN基因的甲基化水平:干扰组与空白对照组及空载体对照组相比较发生明显的去甲基化,表明抑制COMMD7表达可诱导PTEN去甲基化。CCK8实验结果:干扰组细胞较空白对照组及空载体对照组和PTEN抑制剂处理组增殖速度明显降低,均有统计学差异(P0.05);Transwell实验实验结果显示:干扰组穿膜细胞数(17.4±2.7)较空载体对照组(36.2±3.2)、空白对照组(41.6±4.5)及PTEN抑制剂处理组(47.6±1.8)明显减少,均有统计学差异P0.05。结论:siRNA干扰下调COMMD7基因的表达,可诱导HepG2细胞内抑癌基因PTEN的去甲基化,增加PTEN蛋白的表达而抑制PI3K/AKT信号通路,以降低HepG2细胞的增殖、迁移及侵袭能力。
[Abstract]:Objective and significance: in the world, the incidence of hepatocellular carcinoma has ranked fifth among the common malignant tumors, and at least 1 million new cases are increasing every year, most of which occur in China. As a large country of hepatitis B, liver cancer has a broad disease base in China. Liver transplantation and surgical treatment are the most ideal treatment methods, but the resection rate is low, the recurrence rate is high, the survival rate and quality of life are low, the overall curative effect is poor. With the development of gene research, our understanding of liver cancer is more and more profound, and we realize that liver cancer is a tumor regulated by many genes. Analysis and identification of hepatoma related genes and study of the interaction mechanism between different regulatory genes, search for the key genes and regulatory factors in regulatory networks, research and development of anti-tumor targeted therapy drugs have become a hot spot in the research of hepatocellular carcinoma. Methods: the specific siRNA(smallinterference RNAs targeting COMMD7 gene were designed and transfected into human hepatoma cell line HepG2 as interference group, and PTEN inhibitor treatment group was set up with SiBpv-HepG2; blank control group with HepG2; empty vector control group with N-HepG2TEN. Q RT-PCR was used to detect the m RNA of COMMD7PTEN gene after transfection. The expression of PTEN gene methylation level was detected by PTEN gene methylation level and the cell proliferation ability was detected by CCK8 method. Transwell assay was used to detect the change of cell metastasis and invasion ability before and after transfection. Results after transfection of HepG2 cells with 10% siRNA-Commd7, the results of qRT-PCR showed that the expression of COMMD7 gene in interference group decreased compared with blank control group and blank vector control group, and the expression of COMMD7 gene in interference group was increased by 1844%. The results showed that the expression of COMMD7 decreased significantly in the group of small siRNA interference, and the expression of PTEN increased significantly. The downstream p-AKT expression was significantly inhibited in the PTEN inhibitor treated group, and the PTEN expression was significantly inhibited in the PTEN inhibitor treatment group. The expression of downstream p-AKT was significantly enhanced to detect the methylation level of PTEN gene. There was significant demethylation in the interference group compared with the blank control group and the blank vector control group. The results showed that inhibition of COMMD7 expression could induce PTEN demethylation. The results showed that the proliferation rate of the cells in the interference group was significantly lower than that in the blank control group, empty vector control group and PTEN inhibitor treatment group. The results of Transwell experiment showed that the number of perforating cells in the interference group was significantly lower than that in the blank vector control group (36.2 卤3.2), the blank control group (41.6 卤4.5) and the PTEN inhibitor treatment group (47.6 卤1.8). Conclusion the down-regulation of COMMD7 gene expression by COMMD7 interference can induce the demethylation of PTEN, increase the expression of PTEN protein, and inhibit the PI3K/AKT signaling pathway in HepG2 cells, so as to reduce the proliferation, migration and invasion of HepG2 cells.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.7

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