【作者】 李力；

【导师】 熊建萍；

【作者基本信息】 南昌大学 ， 肿瘤学（专业学位）， 2018， 硕士

【摘要】 目的:探讨miR-93-5p在胃癌中的临床意义,生物学功能以及miR-93-5p与Hippo通路之间的调控关系,同时寻找新型的胃癌治疗靶点,为胃癌诊治提供新思路。方法:1、运用荧光实时定量PCR(qRT-PCR)检测30例胃癌患者新鲜胃癌组织及30例癌旁组织中miR-93-5p的表达;同时分析miR-93-5p表达水平与胃癌组织病理特征之间的关系。2、运用siRNA干扰技术,分别干扰miR-93-5p在SGC-7901及HGC-27胃癌细胞系中的表达,实验分组:对照组为Negative control组,实验组为miR-93-5p mimics组及miR-93-5p inhibitor组,同时运用qRT-PCR检测干扰后胃癌细胞系中miR-93-5p的表达情况。3、运用Western blot实验检测干扰miR-93-5p表达后Hippo通路中关键分子的表达情况,YAP蛋白在胃癌细胞中的分布及EMT蛋白的表达情况。4、运用CCK-8实验及平板克隆实验检测干扰miR-93-5p表达后胃癌细胞增殖能力的变化。5、运用Transwell实验检测干扰miR-93-5p表达后胃癌细胞迁移、侵袭能力的变化。6、运用荧光素酶报告实验检测miR-93-5p的靶基因。结果:1、MiR-93-5p与胃癌的分化及其淋巴结转移相关。2、MiR-93-5p在胃癌中过表达,并且促进胃癌细胞的增殖。3、MiR-93-5p促进胃癌细胞的迁移,侵袭及EMT过程。4、MiR-93-5p调节Hippo通路中多个关键因子。5、FAT4及LATS2是miR-93-5p的靶基因。结论:MiR-93-5p在胃癌中过表达,并通过靶向FAT4及LATS2调节Hippo通路,与胃癌的发生与发展密切相关,可能为胃癌早期诊断标记物或者治疗靶标。更多还原

【Abstract】 Objective:To investigate the biological function and clinical significance of miR-93-5p in gastric cancer(GC),discovery the relationship between miR-93-5p and Hippo pathway,and find a new target for the treatment of GC.Method:1.Quantitative Real-time PCR(qRT-PCR)was used to detect the mRNA expression of miR-93-5p,FAT4 and LATS2 in 30 cases of fresh GC tissue and 30 cases of noncancerous tissues,and analysed the relation between the expression of miR-93-5p and the expression of FAT4 and LATS2;then analyzed the relationship between the expression of miR-93-5p and clinical pathological features in gastric cancer.2.The miR-93-5p mimics and NC were transfected into SGC-7901,and the miR-93-5p inhibitor and inhibitor NC into HGC-27 cells when they reached 50–60% confluence,according to the manufacturer’s instructions.3.Western blot was used to detect the expression of essential components of the Hippo pathway,the position of YAP in GC cells and the expression of EMT protein in GC cells after transfection4.A CCK-8 assay and colony formation assay was conducted to evaluate cell proliferation.The SGC-7901 and HGC-27 cells were transfected with miR-93-5p mimic or inhibitor,and seeded in 96-well plates(2000 cells/well).5.Transwell assay was conducted to detect the ability of migration and invasion in GC cells after transfection.6.Dual-luciferase reporter assay was conducted to detect the target of miR-93-5p.Results:1.The expression of mi R-93-5p significantly associated with tumor differenti-ation and lymph node metastasis status.2.MiR-93-5p is overexpressed in GC tissues and promotes GC-cell proliferation and chemoresistance in vitro.3.MiR-93-5p promotes GC-cell migration,invasion,and EMT in vitro.4.MiR-93-5p regulates multiple components of the Hippo-YAP signaling pathway.5.FAT4 and LATS2 are direct miR-93-5p targets.Conclusion:The results of the present study indicate that miR-93-5p exerts an oncogenic function during GC development,via direct binding to the 3’-UTR of FAT4 and LATS2,thereby inactivating the Hippo pathway.Understanding the mechanism underlying miR-93-5p dysregulation during GC progression will be essential to enable the development of new GC therapeutic strategies in the future.更多还原