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异位表达FOXO4对胃癌细胞凋亡的影响

, PP. 1830-1836

Keywords: FOXO基因,过表达,RNA干扰,细胞凋亡,胃癌细胞

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Abstract:

目的应用pCMV5-Flag和pLL3.7真核表达载体构建ForkheadboxO4(FOXO4)基因的过表达质粒和干扰RNA表达质粒,研究其对胃癌细胞凋亡的影响。方法将FOXO4基因的开放阅读框克隆入pCMV5-Flag载体,将靶向FOXO4基因的siRNA克隆入pLL3.7载体,获得FOXO4的表达质粒Flag-FOXO4和干扰质粒FOXO4-shRNA。将获得的质粒及对照质粒通过脂质体转染MKN45和SGC7901胃癌细胞,Westernblot检测其对细胞中FOXO4蛋白的表达和沉默效率。FCM检测其对细胞凋亡的影响,Westernblot检测CleavedCaspase-3的表达以及应用Real-timePCR检测其对促凋亡转录因子BCL-6的表达的变化。结果成功构建FOXO4的表达质粒Flag-FOXO4和干扰质粒FOXO4-shRNA。Westernblot结果表明,Flag-FOXO4可以明显增加细胞FOXO4的表达,FOXO4-shRNA明显抑制细胞FOXO4的表达。FCM结果显示,Flag-FOXO4组与Control组相比较,MKN45细胞和SGC7901细胞凋亡显著增加了16.54%(P<0.001)和26.23%(P<0.001)。Westernblot结果显示CleavedCaspase-3表达与FCM结果一致。Real-timePCR结果表明Flag-FOXO4组细胞FOXO4下游促凋亡基因BCL-6的表达显著增加(P<0.01),FOXO4-shRNA明显抑制了细胞BCL-6的表达(P<0.01)。结论FOXO4的过表达可能通过增加FOXO4下游促凋亡基因BCL-6的表达促进了胃癌细胞的凋亡。FOXO4的沉默降低了BCL-6的表达。

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