FOXQ1介导SHH信号通路诱导胃癌细胞凋亡

Effects of FOXQ1 mediated SHH signaling pathway in inducing apoptosis of gastric cancer cells in vitro

目的探讨Sonic Hedgehog(SHH)信号通路是否参与转录因子叉头框Q1(FOXQ1)基因诱导的胃癌细胞凋亡。方法培养人永生化胃黏膜上皮细胞GES-1及人胃癌细胞SGC-7901、MKN-28、GC-9811和AGS,qRT-PCR检测细胞中FOXQ1 mRNA表达水平。分别转染si-FOXQ1(si-FOXQ1组)、阴性对照si-Control(si-Control组)至SGC-7901细胞,并设置正常对照组(Normal组),转染时间为48 h;用1μmol/L的SHH特异性激活剂purmorphamine(PM)诱导si-FOXQ1组细胞24 h,记为si-FOXQ1+PM组,以si-FOXQ1组细胞为对照。采用qRT-PCR和Western blot法分别检测各组SGC-7901细胞中FOXQ1 mRNA和蛋白表达水平,流式细胞仪检测SGC-7901细胞凋亡率,Western blot检测SGC-7901细胞中Bax、Cleaved Caspase3、SHH信号通路相关蛋白SHH、Smo及下游分子Gli1和Gli3蛋白表达水平。结果与GES-1细胞比较,SGC-7901、MKN-28、GC-9811和AGS细胞中FOXQ1 mRNA表达水平显著降低(P<0.05),且SGC-7901细胞中FOXQ1的表达最高(P<0.05)。与si-Control组比较,si-FOXQ1组SGC-7901细胞mRNA和蛋白表达水平均显著降低(P<0.05),细胞凋亡率显著升高(P<0.05),Bax和Cleaved Caspase3蛋白表达水平显著升高(P<0.05),SHH、Smo、Gli1和Gli3蛋白表达水平显著降低(P<0.05)。Normal组和si-Control组间以上各指标差异均无统计学意义(P>0.05)。与si-FOXQ1组比较,si-FOXQ1+PM组SGC-7901细胞凋亡率显著降低(P<0.05),Bax和Cleaved Caspase3蛋白表达水平显著降低(P<0.05),SHH、Smo、Gli1和Gli3蛋白表达水平显著升高(P<0.05)。结论FOXQ1在胃癌细胞中高表达,沉默FOXQ1可能通过介导SHH信号通路诱导胃癌细胞凋亡,是治疗胃癌的潜在靶点。

Objective To investigate whether SHH signaling pathway is involved in the apoptosis of gastric cancer cells induced by the transcription factor FOXQ1 gene(FOXQ1).Methods Human immortalized gastric mucosal epithelial cells GES-1 and human gastric cancer cells SGC-7901,MKN-28,GC-9811 and AGS were cultured in vitro,and the expression levels of FOXQ1 mRNA in the cells were detected by qRT-PCR.The Si-FOXQ1(si-FOXQ1 group)and negative control si-control(si-control group)were transfected into SGC-7901 cells,respectively,and the transfection time was 48h,and the normal control group(normal control group)was set up.The cells in si-FOXQ1 group were induced by 1μmol/L SHH-specific activator purmorphamine(PM)for 24h,which was regarded as si-FOXQ1+PM group,and the si-FOXQ1 group was used as control group.The expression levels of FOXQ1 mRNA and protein in SGC-7901 cells were detected by qRT-PCR and Western blot,respectively.The apoptosis rate of SGC-7901 cells was detected by flow cytometry.The levels of Bax,Cleaved Caspase3,SHH signaling pathway-associated protein SHH,Smo and downstream molecules Gli1 and Gli3 protein were detected by Western Blot.Results As compared with those in GES-1 cells,the expression levels of FOXQ1 mRNA in SGC-7901,MKN-28,GC-9811 and AGS cells were significantly decreased(P<0.05),and the expression levels of FOXQ1 were the highest in SGC-7901 cells.As compared with those in si-control group,the mRNA and protein expression levels of SGC-7901 cells in si-FOXQ1 group were significantly decreased(P<0.05),however,the apoptosis rate was significantly increased(P<0.05).And the levels of Bax and Cleaved Caspase3 proteins were significantly increased,but the levels of SHH,Smo,Gli1 and Gli3 were significantly decreased(P<0.05).There were no significant differences in the indexes mentioned above between normal control group and si-control group(P>0.05).As compared with thosein si-FOXQ1 group,the apoptosis rate of SGC-7901 cells in si-FOXQ1+PM group was significantly decreased,and the expression levels of Bax and Cleaved Caspase3 proteins were significantly decreased,however,the expression levels of SHH,Smo,Gli1 and Gli3 proteins were significantly increased(P<0.05).Conclusion FOXQ1 is highly expressed in gastric cancer cells.Silencing FOXQ1 may induce apoptosis of gastric cancer cells by mediating SHH signaling pathway,which is a potential target for the treatment of gastric cancer.