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

【摘要】：目的:檢測miR-186在肝細(xì)胞肝癌組織和細(xì)胞中的表達(dá)及探討其對肝細(xì)胞肝癌細(xì)胞生物學(xué)特性的影響。方法:通過q RT-PCR技術(shù)檢測34對肝細(xì)胞肝癌組織和癌旁組織的miR-186表達(dá)情況,并同時檢測4種肝細(xì)胞肝癌細(xì)胞株(hep G2、Hep3B、SMMC-7721、BEL-7402)以及1種正常肝細(xì)胞株(HL-7702)中miR-186的表達(dá)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(dá)載體轉(zhuǎn)染至肝細(xì)胞肝癌細(xì)胞系SMMC-7721、BEL-7402中,CCK-8法、流式細(xì)胞術(shù)和Transwell遷移和侵襲實(shí)驗(yàn)檢測細(xì)胞細(xì)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(jìn)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(dá)量0.0342±0.0333顯著低于癌旁組織的表達(dá)量0.0769±0.0559(p0.001);同時通過對臨床病理學(xué)特征分析發(fā)現(xiàn),miR-186的表達(dá)量與腫瘤大小和TNM分期有關(guān)。miR-186在HCC細(xì)胞的表達(dá)量顯著低于正常肝細(xì)胞中的表達(dá)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(dá)miR-186組在各時間點(diǎn)與對照組相比增殖率下降,而抑制miR-186表達(dá)組在各時間點(diǎn)與對照組相比增殖率上升(P0.05或P0.01)。流式細(xì)胞術(shù)結(jié)果顯示過表達(dá)miR-186組細(xì)胞的凋亡率與對照組相比明顯增加,而抑制miR-186表達(dá)組細(xì)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(shí)驗(yàn)結(jié)果顯示過表達(dá)miR-186使穿過小室的細(xì)胞數(shù)增多,而抑制miR-186表達(dá)則會減少穿過小室的細(xì)胞數(shù)(P0.05或P0.01)。q RT-PCR和Western blot結(jié)果顯示過表達(dá)miR-186能夠降低ROCK-1的m RNA及蛋白質(zhì)的表達(dá)(P0.05),抑制miR-186則會增加ROCK-1的m RNA及蛋白質(zhì)的表達(dá)。結(jié)論:miR-186在肝細(xì)胞肝癌組織和肝細(xì)胞肝癌細(xì)胞中低表達(dá),并能夠抑制肝細(xì)胞肝癌細(xì)胞的增殖,遷移與侵襲并促進(jìn)凋亡。說明miR-186在肝細(xì)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(dá)來實(shí)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(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R735.7

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本文編號：1548007