Rab6A通过调控AKT/p38信号通路影响胃癌细胞增殖和凋亡

Rab6A affects cell proliferation and cell apoptosis of gastric cancer cells by regulating AKT/p38 signaling pathway

目的观察Rab6A在胃癌组织中的表达及其对胃癌细胞增殖、细胞周期和细胞凋亡的影响,探讨其可能的分子机制。方法在西安交通大学第一附属医院肿瘤外科收集53例胃癌组织和53例癌旁组织样本,采用实时定量PCR和Western blot分别检测组织中Rab6A在mRNA和蛋白质水平的表达情况。利用癌症基因组图谱(the cancer genome altas,TCGA)与基因型-组织表达工程(the genotype-tissue expression project,GTEX)数据库分析Rab6A在胃癌组织和正常胃组织中的表达水平。胃癌细胞系BGC-823和SGC-7901分别分为3组:阴性对照组、siRNA-1组和siRNA-2组,分别转染NC-siRNA、Rab6A siRNA-1和Rab6A siRNA-2。采用实时定量PCR和Western blot法分别检测Rab6A siRNAs在胃癌细胞系BGC-823和SGC-7901中的沉默效率。MTT法分析转染Rab6A siRNAs对胃癌细胞系BGC-823和SGC-7901细胞增殖活力的影响。流式细胞术分析转染Rab6A siRNAs对BGC-823和SGC-7901细胞周期和细胞凋亡的影响。Western blot检测转染Rab6A siRNAs对BGC-823和SGC-7901细胞中AKT/p38信号通路蛋白(p-AKT,AKT,CDK4,Cyclin D1,p-p38,p38)表达的影响。结果与癌旁组织相比,胃癌组织中Rab6A在mRNA和蛋白质表达均显著上调(P<0.01)。TCGA和GTEX数据库分析显示,与正常组织相比,Rab6A在胃癌组织中高表达(P<0.01)。与阴性对照组相比,在BGC-823和SGC-7901细胞中Rab6A siRNA-1组和siRNA-2组Rab6A mRNA和蛋白质表达均显著降低(P<0.01)。与阴性对照组相比,Rab6A siRNA-1组和siRNA-2组BGC-823和SGC-7901细胞的增殖能力显著下降(P<0.01)。与阴性对照组相比,在BGC-823和SGC-7901细胞中Rab6A siRNA-1组和siRNA-2组G1/G0期细胞比例显著增加(P<0.01),S期和G2/M期细胞比例显著减少(P<0.01)。与阴性对照组相比,在BGC-823和SGC-7901中Rab6A siRNA-1组和siRNA-2组早期凋亡和晚期凋亡显著增加(P<0.01)。与阴性对照组相比,Rab6A siRNA-1组和siRNA-2组AKT/p38信号通路相关蛋白p-AKT、CDK4和Cyclin D1表达显著下降(P<0.01),p-p38表达显著增加(P<0.01)。结论Rab6A在胃癌组织中表达上调,并通过影响AKT/p38信号通路促进胃癌细胞增殖、周期转换,抑制细胞凋亡。

Objective To observe the expression of Rab6A in gastric cancer tissue and its effects on the cell proliferation,the cell cycle and the cell apoptosis of gastric cancer cells,and to explore its possible molecular mechanism.Methods A total of 53 gastric cancer tissue and 53 adjacent normal tissue samples were collected from the Department of Oncology of the First Affiliated Hospital of Xi’an Jiaotong University.Real-time quantitative PCR and Western blot were used to detect the expression of Rab6A at mRNA and protein levels in these tissues.The cancer genome altas(TCGA)and the genotype tissue expression project(GTEX)were used to analyze the expression level of Rab6A in gastric cancer tissues and normal gastric tissues.Gastric cancer cell lines BGC-823 and SGC-7901 were respectively divided into three groups:negative control group,siRNA-1 group and siRNA-2 group,and transfected with NC-siRNA,Rab6A siRNA-1 and Rab6A siRNA-2,respectively.The silencing efficiency of siRNAs on Rab6A in BGC-823 and SGC-7901 cells was detected by real-time quantitative PCR and Western blot,respectively.MTT was used to analyze the effects of Rab6A siRNAs on the proliferation activities of BGC-823 and SGC-7901 cells.Flow cytometry was used to analyze the effects of Rab6A siRNAs on cell cycle and cell apoptosis of BGC-823 and SGC-7901 cells.The effects of Rab6A siRNAs on the expression of AKT/p38 signal pathway-related proteins(p-AKT,AKT,CDK4,Cyclin D1,p-p38,p38)in BGC-823 and SGC-7901 cells were detected by Western blot.Results Compared with adjacent gastric tissues,the expression of Rab6A was significantly up-regulated at both mRNA and protein levels in gastric cancer tissues(P<0.01).TCGA and GTEX database analysis showed that Rab6A was highly expressed in gastric cancer compared with normal tissues(P<0.01).The expression levels of Rab6A mRNA and protein were significantly lower in BGC-823 and SGC-7901 cells in Rab6A siRNA-1 group and siRNA-2 group than in negative control group(P<0.01).Compared with negative control group,the cell proliferation ability was decreased significantly in Rab6A siRNA-1 group and siRNA-2 group(P<0.01).Compared with negative control group,the proportion of G1/G0 cells was increased in BGC-823 and SGC-7901 cells in Rab6A siRNA-1 group and siRNA-2 group(P<0.01),while the proportion of S phase and G2/M phase cells was decreased significantly(P<0.01).Compared with negative control group,the number of early apoptosis and late apoptosis of BGC-823 and SGC-7901 cells was increased significantly in Rab6A siRNA-1 group and siRNA-2 group(P<0.01).Compared with negative control group,the expression of AKT/p38 signal pathway-related proteins p-AKT,CDK4 and cyclin D1 was decreased significantly(P<0.01),while the expression of p-p38 was increased significantly in Rab6A siRNA-1 group and siRNA-2 group(P<0.01).Conclusion Rab6a is up-regulated in gastric cancer,and it can promote the proliferation and the cycle transition,and inhibit the apoptosis of gastric cancer cells by affecting AKT/p38 signaling pathway.