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  本文關(guān)鍵詞： 肝細胞肝癌 miRNA-186 增殖 凋亡 遷移與侵襲　出處：《重慶醫(yī)科大學》2017年碩士論文　論文類型：學位論文

【摘要】：目的:檢測miR-186在肝細胞肝癌組織和細胞中的表達及探討其對肝細胞肝癌細胞生物學特性的影響。方法:通過q RT-PCR技術(shù)檢測34對肝細胞肝癌組織和癌旁組織的miR-186表達情況,并同時檢測4種肝細胞肝癌細胞株(hep G2、Hep3B、SMMC-7721、BEL-7402)以及1種正常肝細胞株(HL-7702)中miR-186的表達情況。利用Endo Fection TM-Max轉(zhuǎn)染試劑將已構(gòu)建成功的miR-186真核表達載體轉(zhuǎn)染至肝細胞肝癌細胞系SMMC-7721、BEL-7402中,CCK-8法、流式細胞術(shù)和Transwell遷移和侵襲實驗檢測細胞細的增殖、凋亡、遷移和侵襲能力。并使用q RT-PCR和Western blot技術(shù)做進一步檢測miR-186對靶基因的作用。結(jié)果:HCC組織中miR-186的表達量0.0342±0.0333顯著低于癌旁組織的表達量0.0769±0.0559(p0.001);同時通過對臨床病理學特征分析發(fā)現(xiàn),miR-186的表達量與腫瘤大小和TNM分期有關(guān)。miR-186在HCC細胞的表達量顯著低于正常肝細胞中的表達量(p0.01或p0.001),特別是SMMC-7721、BEL-7402尤為顯著。CCK-8結(jié)果顯示過表達miR-186組在各時間點與對照組相比增殖率下降,而抑制miR-186表達組在各時間點與對照組相比增殖率上升(P0.05或P0.01)。流式細胞術(shù)結(jié)果顯示過表達miR-186組細胞的凋亡率與對照組相比明顯增加,而抑制miR-186表達組細胞凋亡率與對照組相比是明顯減少(P0.05或P0.01)。Traswell遷移與侵襲實驗結(jié)果顯示過表達miR-186使穿過小室的細胞數(shù)增多,而抑制miR-186表達則會減少穿過小室的細胞數(shù)(P0.05或P0.01)。q RT-PCR和Western blot結(jié)果顯示過表達miR-186能夠降低ROCK-1的m RNA及蛋白質(zhì)的表達(P0.05),抑制miR-186則會增加ROCK-1的m RNA及蛋白質(zhì)的表達。結(jié)論:miR-186在肝細胞肝癌組織和肝細胞肝癌細胞中低表達,并能夠抑制肝細胞肝癌細胞的增殖,遷移與侵襲并促進凋亡。說明miR-186在肝細胞肝癌中,是作為抑癌因子存在的,并可能通過抑制ROCK-1的表達來實現(xiàn)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 鹵0.0333, 0.0769 鹵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.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R735.7

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本文編號：1548007