miR-186在肝细胞肝癌中的表达及作用

发布时间：2018-02-28 16:12

本文关键词： 肝细胞肝癌 miRNA-186 增殖凋亡迁移与侵袭　出处：《重庆医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:检测miR-186在肝细胞肝癌组织和细胞中的表达及探讨其对肝细胞肝癌细胞生物学特性的影响。方法:通过q RT-PCR技术检测34对肝细胞肝癌组织和癌旁组织的miR-186表达情况,并同时检测4种肝细胞肝癌细胞株(hep G2、Hep3B、SMMC-7721、BEL-7402)以及1种正常肝细胞株(HL-7702)中miR-186的表达情况。利用Endo Fection TM-Max转染试剂将已构建成功的miR-186真核表达载体转染至肝细胞肝癌细胞系SMMC-7721、BEL-7402中,CCK-8法、流式细胞术和Transwell迁移和侵袭实验检测细胞细的增殖、凋亡、迁移和侵袭能力。并使用q RT-PCR和Western blot技术做进一步检测miR-186对靶基因的作用。结果:HCC组织中miR-186的表达量0.0342±0.0333显著低于癌旁组织的表达量0.0769±0.0559(p0.001);同时通过对临床病理学特征分析发现,miR-186的表达量与肿瘤大小和TNM分期有关。miR-186在HCC细胞的表达量显著低于正常肝细胞中的表达量(p0.01或p0.001),特别是SMMC-7721、BEL-7402尤为显著。CCK-8结果显示过表达miR-186组在各时间点与对照组相比增殖率下降,而抑制miR-186表达组在各时间点与对照组相比增殖率上升(P0.05或P0.01)。流式细胞术结果显示过表达miR-186组细胞的凋亡率与对照组相比明显增加,而抑制miR-186表达组细胞凋亡率与对照组相比是明显减少(P0.05或P0.01)。Traswell迁移与侵袭实验结果显示过表达miR-186使穿过小室的细胞数增多,而抑制miR-186表达则会减少穿过小室的细胞数(P0.05或P0.01)。q RT-PCR和Western blot结果显示过表达miR-186能够降低ROCK-1的m RNA及蛋白质的表达(P0.05),抑制miR-186则会增加ROCK-1的m RNA及蛋白质的表达。结论:miR-186在肝细胞肝癌组织和肝细胞肝癌细胞中低表达,并能够抑制肝细胞肝癌细胞的增殖,迁移与侵袭并促进凋亡。说明miR-186在肝细胞肝癌中,是作为抑癌因子存在的,并可能通过抑制ROCK-1的表达来实现其作用。
[Abstract]:Objective: to investigate the expression of miR-186 in hepatocellular carcinoma (HCC) and its effect on the biological characteristics of hepatocellular carcinoma (HCC) cells. Methods: the expression of miR-186 in hepatocellular carcinoma (HCC) tissues and adjacent tissues was detected by Q RT-PCR technique. At the same time, the expression of miR-186 was detected in four kinds of hepatocarcinoma cell lines (HL-7702) and HL-7702 (HL-7402). The constructed miR-186 eukaryotic expression vector was transfected into the hepatocellular carcinoma cell line SMMC-7721 (BEL-7402) by CCK-8 method using Endo Fection TM-Max transfection reagent. Flow cytometry and Transwell migration and invasion assay were used to detect cell proliferation and apoptosis. Q RT-PCR and Western blot techniques were used to further examine the effect of miR-186 on target gene. Results the expression of miR-186 was significantly lower than that in paracancerous tissues (0.0342 卤0.0333, 0.0769 卤0.0559, p0.001). It was found that the expression of miR-186 in HCC cells was significantly lower than that in normal hepatocytes, especially in SMMC-7721 BEL-7402. The results of CCK-8 showed that the overexpression of miR-186 was significantly lower than that of normal hepatocytes at all time points. Compared with the control group, the proliferation rate decreased, However, the proliferative rate of the inhibited miR-186 expression group was significantly higher than that of the control group at different time points (P0.05 or P0.01). Flow cytometry showed that the apoptosis rate of the overexpression miR-186 group was significantly higher than that of the control group. However, compared with the control group, the apoptosis rate of the inhibited miR-186 expression group was significantly lower than that of the control group. The results of migration and invasion experiments showed that overexpression of miR-186 increased the number of cells passing through the chambers. Inhibiting the expression of miR-186 decreased the number of cells passing through the cell (P0.05 or P0.01G. Q RT-PCR and Western blot). The results showed that the overexpression of miR-186 could decrease the expression of m RNA and protein of ROCK-1 (P 0.05), and the inhibition of miR-186 could increase the expression of m RNA and protein of ROCK-1. The expression of miR-186 is low in hepatocellular carcinoma and hepatocellular carcinoma cells. It can inhibit the proliferation, migration and invasion of hepatocellular carcinoma cells and promote apoptosis, which indicates that miR-186 exists as a tumor suppressor in hepatocellular carcinoma, and it may play a role by inhibiting the expression of ROCK-1.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7

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