脑型糖原磷酸化酶对胃癌细胞凋亡的影响及机制

Effect of brain glycogen phosphorylase induced apoptosis in gastric cancer cells and its mechanism

目的探讨脑型糖原磷酸化酶(PYGB)对胃癌(GC)细胞凋亡的影响及其机制。方法收集29例初诊GC患者的一般临床资料和GC组织及距肿瘤边缘>5 cm的癌旁组织作为组织标本,同时选取GC细胞系HGC-27、MGC-803、MKN45、AGS及人胃黏膜上皮细胞GES-1作为细胞标本,采用实时荧光定量PCR(RT-qPCR)检测2种组织和上述5种细胞中PYGB mRNA的表达,采用蛋白印迹法(Western blot)检测各种细胞PYGB蛋白的表达,采用χ^(2)检验分析PYGB mRNA表达与患者临床病理特征的关系;选择PYGB mRNA表达量最高的AGS细胞作后续研究,采用RNA干扰技术根据PYGB基因合成3对siRNA(si-PYGB 1~3)序列,以Lipofectamine^(TM) 3000为转染试剂转染胃癌细胞AGS,分为Control组(空白对照)、si-NC组(阴性对照)、si-PYGB 1组(si-PYGB 1)、si-PYGB 2组(si-PYGB 2)和si-PYGB 3组(si-PYGB 3),分别采用RT-qPCR和Western blot检测各组AGS细胞PYGB在mRNA和蛋白水平上的表达;保留干扰效果最好的si-PYGB 2组,采用流式细胞术检测si-NC组和si-PYGB 2组细胞周期、凋亡及细胞内活性氧(ROS)水平。结果与癌旁组织比较,GC组织中PYGB mRNA水平表达上调(P<0.05);与GES-1比较,GC细胞AGS和MKN45中PYGB mRNA和蛋白水平上表达上调、GC细胞HGC-27和MGC-803中表达下调(P<0.05);与si-NC组比较,si-PYGB 2组AGS细胞S期比例下降、G2/M期比例上升(P<0.05),同时细胞凋亡率上升、细胞内ROS水平增加(P<0.05)。结论沉默PYGB可诱导AGS细胞凋亡,其机制可能与细胞内ROS水平增加有关。

Objective To investigate the effect and mechanism of brain glycogen phosphorylase(PYGB)on the apoptosis of gastric cancer(GC)cells.Methods The cancer tissues(GC tissues)of 29 newly diagnosed GC patients and the adjacent tissues more than 5 cm from the tumor edge were selected as tissue specimens,and general clinical data of the patients were collected.At the same time,GC cell strains HGC-27,MGC-803,MKN45,AGS,and human gastric mucosal epithelial cells GES-1 were used as cell specimens.Real time fluorescent-quantitative PCR(RT-qPCR)was used to detect the expression of PYGB mRNA in these two tissues and the above mentioned 5 cells;Western blot was used to detect expression of PYGB protein in various cells.The χ^(2) test was used to analyze the relationship between the expression of PYGB mRNA and the clinical pathological characteristics of the patients;selecting the AGS cells with the highest expression of PYGB mRNA for follow-up research.Using RNA interference technology to synthesize 3 pairs of siRNA(si-PYGB 1-3)sequences based on the PYGB gene,and transfecting the GC AGS cell with Lipofectamine^(TM) 3000 as the transfection reagent[dividing them into control group(blank control),si-NC group(negative control),si-PYGB 1 group(si-PYGB 1),si-PYGB 2 group(si-PYGB 2),and si-PYGB 3 group(si-PYGB 3)].Using RT-qPCR and Western blot to detect the expression of PYGB mRNA and protein levels in AGS cell of each group respectively.Keeping 2 groups of si-PYGB with the best interference effect.Flow cytometry was adopted to detect cell cycle,apoptosis and intracellular reactive oxygen species(ROS)levels in si-NC group and si-PYGB 2 group.Results Compared with adjacent tissues,the expression of PYGB mRNA in GC tissues was up-regulated(P<0.05).Compared with GES-1,the expression of PYGB mRNA and protein level in GC cells AGS and MKN45 was up-regulated,and the expression in GC cells HGC-27 and MGC-803 was down-regulated(P<0.05).Compared with the si-NC group,the ratio of AGS cells in the S phase of the si-PYGB 2 group decreased,and the ratio of G2/M phase increased(P<0.05).At the same time,the apoptosis rate increased and the intracellular ROS level increased(P<0.05).Conclusion Silencing PYGB can induce apoptosis of AGS cells,and the mechanism may be related to the increase of intracellular ROS levels.