水通道蛋白-5对结直肠癌细胞迁移、凋亡的影响及机制研究

Effect of aquaporin-5 on migration and apoptosis of colorectal cancer cells and its mechanism

目的:探讨短发夹RNA(short hairpin RNA,shRNA)沉默水通道蛋白-5(aquaporin-5,AQP5)对人结直肠癌HT-29细胞和HCT116细胞迁移、凋亡的影响及相关机制。方法:构建pRNA-H1.1-AQP5的干扰质粒和pRNA-H1.1-NC的对照质粒转染HT-29和HCT116细胞,利用Real-time PCR(RT-PCR)、Western blot方法验证AQP5基因敲降效率,划痕试验检测AQP5-shRNA对结直肠癌细胞迁移率的影响,流式细胞术检测各组细胞的细胞凋亡率,Western blot检测凋亡相关蛋白Bax和Bcl-2基因在蛋白水平的表达。选用NF-κB抑制剂BAY 11-7082(BAY)处理转染AQP5-shRNA的HT-29和HCT116细胞,划痕试验检测各组细胞的迁移率,Western blot检测凋亡基因Bax和Bcl-2的表达情况。结果:AQP-shRNA转染HT-29和HCT116细胞中,AQP5表达量明显低于NC-shRNA转染的HT-29和HCT116细胞(P<0.05)。划痕试验结果表明,对比NC组,AQP5-shRNA明显抑制HT-29和HCT116细胞的迁移(P<0.05)。流式细胞术结果表明AQP5-shRNA显著提高HT-29和HCT116细胞的凋亡率(P<0.05)。对比NC-shRNA组,AQP5-shRNA组细胞中Bax蛋白表达明显提高,而Bcl-2蛋白表达显著下降。BAY处理后,AQP5-shRNA所抑制的结直肠癌细胞迁移率显著下降,AQP5-shRNA所促进的细胞凋亡率显著升高,且对比AQP5-shRNA组,AQP5-shRNA与BAY共同处理的细胞中Bax蛋白表达明显升高,Bcl-2蛋白表达明显降低。结论:AQP5-shRNA抑制结直肠癌细胞迁移,促进结直肠癌细胞凋亡受NF-κB信号调控。

Objective:To investigate the functions of aquaporin-5(AQP5) on the cell migration and apoptosis of colorectal cancer cells(HT-29 and HCT116),as well as the underlying mechanism by short hairpin RNA(shRNA) silencing AQP5.Methods:The plasmid containing shRNA targeting AQP5 or NC was transfected into HT-29 and HCT116 cells,and Real-time PCR(RT-PCR) and Western blot were used to detect the inhibition efficiency of AQP5-shRNA.Scratch test was utilized to detect the cell migration and flow cytometry was used to detect the apoptosis among groups.Western blot was used to detect the expression of apoptosis-related protein Bax and Bcl-2.The inhibitor of NF-κB,BAY 11-7082(BAY) was treated the HT-29 snd HCT116 which transfected with AQP5-shRNA.Scratch test was used to detect the migration and Western blot was used to detect the expression of Bax and Bcl-2 among groups.Results:The expression of AQP5 was suppressed by AQP5-shRNA in both HT-29 and HCT116 cells(P<0.05).Results showed that AQP5-shRNA inhibited the migration of HT-29 and HCT116 cells compared to NC(P<0.05).The results also indicated that AQP5-shRNA increased the apoptosis rate of HT-29 and HCT116 cells(P<0.05).Compared to NC-shRNA group,Bax expression was increased,and Bcl-2 expression was decreased.After the treatment of BAY,the migration regulated by AQP5-shRNA was further suppressed,and apoptosis induced by AQP5-shRNA was enhanced significantly.Conclusion:AQP5-shRNA inhibited the migration and induced the apoptosis of HT-29 and HCT116 cells regulating by NF-κB signaling pathway.