RASSF1A甲基化对肝细胞癌变影响的相关研究

发布时间：2018-06-14 13:45

本文选题：肝癌细胞株 + RASSF1A基因甲基化　；参考：《青岛大学》2017年硕士论文

【摘要】：目的探讨RASSF1A基因甲基化在肝癌细胞株中表达情况及5-Aza-Cd R对人肝癌细胞株Hep G2细胞增殖及RASSF1A基因m RNA表达的影响。方法体外培养肝癌细胞株及正常肝细胞株,应用MSP法分别检测3种肝癌细胞株Hep G2、Hep3B、SK-HEP1及正常肝细胞株LO2 RASSF1A甲基化水平,以探讨RASSF1A甲基化在肝癌细胞株的表达情况。以肝癌细胞株Hep G2为研究对象,实验组分别以不同浓度的甲基化抑制剂5-Aza-Cd R(0.5μmol/L、5μmol/L、50μmol/L)对人肝癌细胞株Hep G2去甲基化组处理,对照组5-Aza-Cd R浓度为0μmol/L;采用CCK-8法检测两组肝癌细胞增殖情况的变化,以观察甲基化抑制剂对肝癌细胞增值的影响,并采用RT-PCR检测5-Aza-Cd R处理前、后Hep G2细胞抑癌基因RASSF1A基因m RNA表达水平的变化。结果人肝癌细胞株Hep G2、Hep3B、SK-HEP1均检测到RASSF1A基因甲基化表达,而正常肝细胞株LO2中未检测到RASSF1A基因的甲基化表达,提示肝细胞癌变与RASSF1A基因甲基化有关。5-Aza-Cd R可以抑制肝癌细胞Hep G2的生长增值。处理72小时后,抑制率最明显,且随着药物浓度增加,增殖抑制率越明显,表明去甲基化可以抑制细胞生长增值(F=44.164,P0.01)。5-Aza-Cd R处理后实验组较对照组RASSF1A基因m RNA表达明显上调,呈剂量依赖性。在50μmol/L时去甲基化效果最明显。50μmol/L 5-Aza-Cd R处理24h、48h、72h时RASSF1A基因m RNA相对表达量分别为(0.2798±0.0048、0.3678±0.0161、0.3873±0.00105),与对照组比较差异有统计学意义(P0.05),表明去甲基化后可以增加Hep G2细胞RASSF1A基因m RNA表达。结论1.肝细胞癌变与抑癌基因RASSF1A甲基化有关。2.5-Aza-CdR可以抑制HepG2细胞生长增殖,呈剂量依赖性,其机制可能与其调控RASSF1A基因启动子甲基化水平,从而使RASSF1A基因m RNA表达上调有关。
[Abstract]:Objective to investigate the expression of RASSF1A gene methylation in human hepatoma cell line and the effect of 5-Aza-CD R on proliferation and mRNA expression of RASSF1A gene in human hepatoma cell line Hep G2. Methods the methylation level of RASSF1A methylation in three hepatoma cell lines Hep G2H2H2Bh3BP1and normal hepatocyte line LO2 RASSF1A was detected by MSP method in order to investigate the expression of RASSF1A methylation in hepatoma cells. Human hepatoma cell line HepG2 was treated with methylation inhibitor 5-Aza-CD R 0.5 渭 mol / L 5 渭 mol 路L ~ (-1) / L ~ (50 渭 mol 路L ~ (-1), respectively. In the control group, the concentration of 5-Aza-CD R was 0 渭 mol / L, the proliferation of hepatoma cells was detected by CCK-8 method, and the effect of methylation inhibitor on the proliferation of hepatoma cells was observed, and the effect of 5-Aza-CD R on the proliferation of hepatoma cells was detected by RT-PCR. Changes of mRNA expression of tumor suppressor gene RASSF1A in Hep G2 cells. Results RASSF1A gene methylation was detected in human hepatoma cell line Hep G _ 2H _ 3B _ (3) B _ (+) -HEP1, but no methylation expression of RASSF1A gene was detected in normal hepatoma cell line LO2, and the methylation of RASSF1A gene was not detected in normal hepatoma cell line (LO2). The results suggest that the carcinogenesis of hepatocytes is related to the methylation of RASSF1A gene. 5-Aza-CD R can inhibit the proliferation of HepG2 cells. After 72 hours of treatment, the inhibition rate was the most obvious, and with the increase of drug concentration, the inhibition rate of proliferation was more obvious, which indicated that demethylation could inhibit the growth and proliferation of the cells. The mRNA expression of RASSF1A gene in the experimental group was significantly up-regulated than that in the control group. In a dose-dependent manner. The relative mRNA expression of RASSF1A gene was 0.2798 卤0.0048 卤0.3678 卤0.0161 卤0.3873 卤0.00105 after treatment with 50 渭 mol / L 5-Aza-CD R for 24 h and 48 h / 72 h, respectively, which was significantly different from that of the control group (P 0.05), indicating that the mRNA expression of RASSF1A gene in Hep G2 cells could be increased after demethylation. Conclusion 1. The carcinogenesis of hepatocytes is related to the methylation of tumor suppressor gene RASSF1A. 2.5-Aza-CdR can inhibit the growth and proliferation of HepG2 cells in a dose-dependent manner. The mechanism may be related to its regulation of methylation level of RASSF1A gene promoter and up-regulation of mRNA expression of RASSF1A gene.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7

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