CUL4A通过调控Hippo通路促胃癌细胞增殖和侵袭的分子机制

发布时间：2018-08-04 14:10

【摘要】：目的:探讨CUL4A在胃癌中的作用机制和临床意义方法:免疫组织化学检测124例胃癌组织中CUL4A的表达,分析其表达水平与胃癌患者临床病理特征和生存时间的关系;观察过表达和干扰CUL4A对胃癌细胞增殖、侵袭和EMT的影响;裸鼠动物模型验证干扰CUL4A对胃癌细胞增殖的影响;实时定量PCR、免疫荧光和蛋白质免疫共沉淀等方法探讨CUL4A失活LATS1-Hippo通路的分子机制;荧光素酶报告基因等方法探讨mi R-9和mi R-137对CUL4A的靶向调控作用;最后在体外细胞水平和新鲜胃癌组织中验证miRNAs-CUL4A-LATS1-Hippo调控轴在胃癌细胞中的作用和临床意义。结果:免疫组织化学结果显示CUL4A蛋白表达水平与胃癌患者TNM分期(P=0.025)和淋巴结转移(P=0.003)密切相关,高表达CUL4A蛋白预示胃癌患者预后不良(P=0.026)。过表达CUL4A促进胃癌细胞增殖、侵袭和EMT,干扰CUL4A则抑制胃癌细胞增殖、侵袭和EMT。过表达CUL4A能降低LATS1和p-YAP蛋白的表达,而不影响MST1/2蛋白的表达,促进YAP蛋白入核,干扰CUL4A表达则相反,并且显著抑制YAP相关靶基因的转录。免疫共沉淀结果显示CUL4A与LATS1蛋白结合发挥作用。分别上调miR-9和miR-137的表达抑制胃癌细胞增殖和侵袭,下调CUL4A蛋白表达,而不影响CUL4A mRNA表达,荧光素酶报告基因结果表明miR-9和miR-137靶向调控CUL4A的表达。不仅如此,miR-9和miR-137通过靶向CUL4A调控Hippo通路,过表达CUL4A能部分逆转miR-9和miR-137抑制胃癌细胞增殖和侵袭的作用。在胃癌组织中,miR-9和miR-137的表达水平均与CUL4A和YAP蛋白表达量呈负相关。结论:miR-9/137-CUL4A-LATS1-Hippo调控轴失衡参与了胃癌细胞恶性进展,CUL4A可作为治疗胃癌的一个潜在分子靶点。
[Abstract]:Objective: to investigate the mechanism and clinical significance of CUL4A in gastric cancer. Methods: the expression of CUL4A was detected by immunohistochemistry in 124 cases of gastric cancer, and the relationship between the expression level and clinicopathological features and survival time of gastric cancer was analyzed. The effects of overexpression and interference of CUL4A on the proliferation, invasion and EMT of gastric cancer cells were observed, and the effects of interfering CUL4A on the proliferation of gastric cancer cells were verified in nude mice. Real-time quantitative PCR, immunofluorescence and protein immunoprecipitation were used to investigate the molecular mechanism of inactivated LATS1-Hippo pathway of CUL4A, luciferase reporter gene and other methods were used to investigate the targeting regulation of CUL4A by miR-9 and miR-137. Finally, the role and clinical significance of miRNAs-CUL4A-LATS1-Hippo regulatory axis in gastric cancer cells were verified in vitro and fresh gastric cancer tissues. Results: immunohistochemical results showed that the expression of CUL4A protein was closely related to the TNM stage (P0.025) and lymph node metastasis (P0. 003) of gastric cancer patients. The high expression of CUL4A protein could predict the poor prognosis of gastric cancer patients (P0. 026). Overexpression of CUL4A promoted the proliferation, invasion and EMTof gastric cancer cells, while interfering with CUL4A inhibited the proliferation, invasion and EMTof gastric cancer cells. Overexpression of CUL4A decreased the expression of LATS1 and p-YAP, but did not affect the expression of MST1/2, promoted the entry of YAP into the nucleus, interfered with the expression of CUL4A, and significantly inhibited the transcription of YAP related target genes. The results of immunoprecipitation showed that CUL4A combined with LATS1 protein. The up-regulated expression of miR-9 and miR-137 inhibited the proliferation and invasion of gastric cancer cells, down-regulated the expression of CUL4A protein, but did not affect the expression of CUL4A mRNA. The results of luciferase reporter gene showed that miR-9 and miR-137 regulated CUL4A expression. Moreover, miR-137 and miR-9 regulated the Hippo pathway through targeting CUL4A, and overexpression of CUL4A partially reversed the inhibitory effect of miR-9 and miR-137 on proliferation and invasion of gastric cancer cells. The expression levels of miR-9 and miR-137 were negatively correlated with the expression of CUL4A and YAP in gastric carcinoma. Conclusion the imbalance of the axis regulated by 1: miR-9 / 137-CUL4A-LATS1-Hippo is involved in the malignant progression of gastric cancer cells. CUL4A may be a potential molecular target for the treatment of gastric cancer.
【学位授予单位】：南昌大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.2

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