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

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Hep G2凋亡的影响及可能的作用机制。方法将MIF-si RNA干扰序列转染SMMC-7721与Hep G2细胞,以Con-si RNA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(q RT-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 m RNA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Hep G2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。

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.