MIF基因沉默对肝癌细胞系增殖凋亡及ERK/RSK2信号通路的影响

Effect of silencing MIF gene by RNAi on proliferation, apoptosis and ERK/RSK2 signal pathway in hepatic carcinoma cell line

摘要:

目的探讨RNA 干扰介导的巨噬细胞移动抑制因子（MIF）基因沉默对肝癌细胞系SMMC-7721与HepG2 凋亡的影响及可能的作用机制。方法将MIF-siRNA 干扰序列转染SMMC-7721 与HepG2 细胞，以Con-siRNA序列转染的细胞作为对照。采用实时荧光定量聚合酶链反应（qRT-PCR）及Western blot检测MIF沉默效果，CCK-8 法测定细胞增殖情况，流式细胞术检测细胞凋亡率。采用Western blot检测MIF 沉默后凋亡相关基因BCL-2、BAX、P53 的蛋白水平及细胞外信号调节激酶（ERK）、P90 核糖体s6 激酶2（RSK2）、糖原合成激酶3β（GSK3β）、Bad 蛋白水平及其磷酸化水平。结果沉默组细胞中MIF mRNA 及蛋白表达水平与对照组比较，差异有统计学意义（p <0.05），沉默组低于对照组；两组细胞增殖能力比较，差异有统计学意义（p < 0.05），MIF沉默组细胞增殖能力低于对照组；两组凋亡率比较，差异有统计学意义（p <0.05），MIF沉默组细胞凋亡率高于对照组；两组细胞BAX、P53、BCL-2 蛋白水平比较，差异有统计学意义（p <0.05），MIF 沉默组细胞BAX、P53 的蛋白表达水平高于对照组，而BCL-2 蛋白水平低于对照组；沉默组与对照组细胞中ERK、RSK2 及Bad 蛋白水平比较，差异均无统计学意义（p >0.05），而沉默组与对照组细胞中GSK3β、p-GSK3β、p-ERK、p-RSK2 及p-Bad 蛋白水平比较，差异均具有统计学意义（p <0.05）。结论MIF 基因沉默抑制SMMC-7721与HepG2细胞的增殖能力并促进细胞凋亡可能是通过调节ERK/RSK2信号通路实现的。

Abstract:

Objective To investigate the effect of silencing MIF gene by RNA interference on apoptosis of hepatic carcinoma cell lines SMMC-7721 and HepG2 and the potential mechanism. Methods The MIF-siRNA interference sequence was transfected into SMMC-7721 and HepG2 cells, Con-siRNA transfected cells were used as the control group. The silencing effect of MIF gene was detected by qRT-PCR and Western blot. The ability of cell proliferation was evaluated by CCK-8 method. The cell apoptosis rate was detected by flow cytometry. The protein levels of Bcl-2, Bax, p53, extracellular signal-regulated kinase (ERK), p-ERK, p90 ribosomal s6 kinase 2 (RSK2), p-RSK2, glycogen synthase kinase 3β(GSK3β), p-GSK3β, Bad and p-Bad were tested by Western blot. Results The expression levels of MIF gene in the cells of the silent groups were significantly lower than that in the control group (p < 0.05). The ability of cell proliferation in the silent groups was obviously lower than that in the control group (p < 0.05). The cell apoptosis rates in the silent groups were significantly higher than that in the control group (p < 0.05). Bax and p53 protein levels in the cells of the silent groups were higher than those of the control group, while the Bcl-2 protein level was lower than that of the control group (p < 0.05). Meanwhile, the protein levels of ERK, RSK2 and Bad were not significantly different between the two silent groups and the control group (p > 0.05). However, the protein levels of GSK3β, p-GSK3β, p-ERK, p-RSK2 and p-Bad were significantly different between the two silent groups and the control group (p < 0.05). Conclusions Silence of gene inhibits the proliferation and promotes the apoptosis of both SMMC -7721 and HepG2 cells maybe via regulation of the ERK/RSK2 signaling pathway.