MIF基因沉默对肝癌细胞系增殖、凋亡的影响及分子机制研究

发布时间：2018-07-29 10:35

【摘要】：目的:观察巨噬细胞移动抑制因子(macrophage migration inhibitory factor,MIF)在肝癌细胞系中的表达情况,探讨siRNA介导的MIF基因沉默后对肝癌细胞系生物学特性的影响及潜在的分子机制。方法:采用实时荧光定量RT-PCR(RT-qPCR)和Western blot技术检测MIF在正常肝细胞系L-O2与肝癌细胞系Huh7、Hep3B、BEL7402、PLC、HepG2、SMMC-7721中的mRNA及蛋白表达情况。采用脂质体法将化学合成的MIF-siRNA干扰序列转染肝癌细胞系SMMC-7721和HepG2,以MIF-siRNA转染的细胞系为实验组,以Con-siRNA转染的细胞系为对照组,采用CCK-8及EdU荧光法检测肝癌细胞系增殖情况;流式细胞术检测细胞周期和细胞凋亡情况;Transwell小室法检测肝癌细胞系的迁移能力;采用Western blot法检测MIF及凋亡相关蛋白Bcl-2、Bax、p53以及ERK/RSK2信号通路相关蛋白的水平。结果:RT-qPCR及Western blot结果显示,MIF在正常肝细胞系与肝癌细胞系中均表达,尤其在HepG2和SMMC-7721细胞中的相对表达量最高。CCK-8及EdU实验结果显示,与对照组相比,MIF基因沉默后可明显抑制肝癌细胞系的增殖;流式细胞术结果显示,实验组G0/G1期细胞所占百分比明显增加且凋亡率明显高于对照组;Transwell实验结果显示,MIF基因沉默后减弱了肝癌细胞系的迁移能力;Western blot结果显示,实验组细胞中Bcl-2表达水平下降,Bax及p53表达水平升高,实验组GSK-3β表达水平升高,p-ERK、p-RSK2、p-Bad和p-GSK-3β水平显著下调,而ERK、RSK2、Bad表达水平无显著变化。结论:MIF在肝癌细胞系中均呈高表达。siRNA介导的MIF基因沉默后,肝癌细胞系的增殖和迁移能力减弱,细胞周期被阻滞在G0/G1期,细胞凋亡率增加,抗凋亡蛋白Bcl-2减少,促凋亡蛋白p53和Bax增加,ERK/RSK2信号通路的相关蛋白水平发生明显变化。初步推测MIF基因沉默抑制肝癌细胞系增殖并促进凋亡可能通过调节ERK/RSK2信号通路实现的。
[Abstract]:Aim: to investigate the expression of macrophage migration inhibitory factor (macrophage migration inhibitory factor-MIF) in hepatocellular carcinoma (HCC) cell lines, and to investigate the effect of MIF gene silencing mediated by siRNA on the biological characteristics of HCC cell lines and its possible molecular mechanism. Methods: real-time fluorescence quantitative RT-PCR (RT-qPCR) and Western blot techniques were used to detect the expression of mRNA and protein in the normal liver cell line L-O2 and the hepatoma cell line Huh7BUE BEL7402PLC+ HepG2SMMC-7721. The chemically synthesized MIF-siRNA interference sequences were transfected into hepatoma cell lines SMMC-7721 and HepG2 by liposome method. MIF-siRNA transfected cell lines were used as experimental group and Con-siRNA transfected cell lines as control group. The proliferation of HCC cell lines was detected by CCK-8 and EdU fluorescence methods. Flow cytometry was used to detect cell cycle and apoptosis. Transwell chamber assay was used to detect the migration ability of hepatoma cell lines, and Western blot assay was used to detect the levels of MIF, Bcl-2Bax-p53 and ERK/RSK2 signaling pathway related proteins. Results the results of 1: RT-qPCR and Western blot showed that both of them were expressed in normal liver cell line and hepatoma cell line, especially in HepG2 and SMMC-7721 cells. The results of CCK-8 and EdU experiments showed that the expression was the highest in the normal liver cell line and the hepatoma cell line, especially in the HepG2 and SMMC-7721 cells. Compared with the control group, the silencing of MIF gene could significantly inhibit the proliferation of hepatoma cell line. The percentage of G0/G1 phase cells in the experimental group was significantly increased and the apoptosis rate was significantly higher than that in the control group. The results showed that the silencing of the G0/G1 gene decreased the migration ability of the hepatoma cell line. Western blot showed that the apoptosis rate was significantly higher in the experimental group than in the control group. In the experimental group, the expression of Bcl-2 decreased and the expression of Bax and p53 increased. The expression of GSK-3 尾 in the experimental group increased and the levels of p-ERKP- RSK2, p-Bad and p-GSK-3 尾 were down-regulated, but the expression of ERK-RSK2Bad did not change significantly. Conclusion after the silencing of MIF gene mediated by .siRNA, the proliferation and migration ability of HCC cell line was decreased, the cell cycle was blocked in G0/G1 phase, the apoptosis rate was increased, and the anti-apoptotic protein Bcl-2 was decreased. Apoptotic protein p53 and Bax increased the level of ERK / RSK2 signaling pathway. It is suggested that MIF gene silencing inhibits the proliferation of HCC cell lines and promotes apoptosis by regulating the ERK/RSK2 signaling pathway.
【学位授予单位】：贵州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7

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