SH2B1沉默对胃癌SGC-7901细胞增殖、凋亡及PI3K/AKT通路的影响

Effects of SH2B1 silencing on proliferation,apoptosis and PI3K/AKT pathway of gastric cancer SGC-7901 cells

目的研究沉默SH2B1基因表达对胃癌SGC-7901细胞增殖、凋亡及3-磷酸肌醇激酶(PI3K)/蛋白质丝氨酸苏氨酸蛋白激酶(AKT)通路的影响。方法体外培养胃癌SGC-7901细胞,采用SH2B1 siRNA转染SGC-7901细胞作为研究组,采用国际通用的与所有基因均无同源序列的non-target siRNA转染作为阴性对照组(NC组),以未经处理的胃癌SGC-7901细胞作为空白对照组(BC组),48h后收集各组转染成功细胞,采用荧光定量聚合酶链反应(qRT-PCR)检测SH2B1 mRNA表达情况,采用蛋白质印迹法(Western Blot)检测SH2B1蛋白表达情况;采用细胞计数试剂盒(CKK-8)检测细胞增殖情况,采用流式细胞仪检测细胞凋亡,同时检测Ki67、增殖细胞核抗原(PCNA)、Caspase-9、PI3K、AKT、p-AKT蛋白表达情况。结果研究组SGC-7901细胞SH2B1蛋白及mRNA表达量较BC组和NC组明显降低,差异有统计学意义(P<0.05);转染后,siRNA组SGC-7901细胞增殖明显受到抑制、平板克隆形成率较BC组和NC组明显降低,凋亡率明显升高,差异均有统计学意义(P<0.05);与BC和NC组比较,研究组SGC-7901细胞PI3K、p-AKT蛋白、Ki67及PCNA蛋白呈低表达,差异有统计学意义(P<0.05),Caspase-9蛋白呈高表达,AKT蛋白表达差异无统计学意义(P>0.05)。结论沉默SH2B1基因表达可能通过抑制PI3K/ATK信号通路激活,抑制SGC-7901细胞增殖,促进凋亡。

Objective To study the effects of silencing SH2B1 gene expression on proliferation,apoptosis and 3-phosphate inositol kinase(PI3K)/protein serine threonine protein kinase(AKT)pathway of gastric cancer SGC-7901 cells.Methods SGC-7901 cells were cultured in vitro and transfected with SH2B1 siRNA as research group,the non-target siRNA without homologous sequences of all genes was transfected into the negative control group(NC),the untreated gastric cancer SGC-7901 cells were used as blank control group(BC),the transfected cells were collected 48 hours later.The expression of SH2B1 mRNA was detected by qRTPCR and the expression of SH2B1 protein was detected by Western blot.Cellcounting kit(CKK-8)was used to detect cell proliferation,cell apoptosis was detected by flow cytometry,the expressions of Ki67,PCNA,Caspase-9,PI3K,AKT and p-AKT were detected by Western blot.Results The expressions of SH2B1 protein and mRNA in SGC-7901 cells in research group were significantly lower than those in BC group and NC group(P<0.05).After transfection,the proliferation of SGC-7901 cells in research group was significantly inhibited,the formation rate of plate clone was significantly lower than that in BC group and NC group,and the apoptosis rate was significantly higher(P<0.05).Compared with BC group and NC group,the expressions of PI3K,p-AKT protein,Ki67 and PCNA protein in SGC-7901 cells of research group were lower(P<0.05),the expression of Caspase-9 protein was higher and the expression of AKT protein was not significantly different(P>0.05).Conclusion Silencing SH2B1 gene expression might inhibit the proliferation of SGC-7901 cells and promote apoptosis by inhibiting the activation of PI3K/ATK signaling pathway.