异位表达FOXO4对胃癌细胞凋亡的影响

Effects of ectopic expression of Forkhead box O4 on cell apoptosis in human gastric cancer cells

目的应用p CMV5-Flag和p LL3.7真核表达载体构建Forkhead box O4(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p CMV5-Flag载体,将靶向FOXO4基因的siRNA克隆入p LL3.7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,Western blot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,Western blot检测Cleaved Caspase-3的表达以及应用Real-time PCR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。Western blot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)。Western blot结果显示Cleaved Caspase-3表达与FCM结果一致。Real-time PCR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。

Objective To construct the expression vector and the small interfering RNA containing human Forkhead box O4 gene（ FOXO4） by using the p CMV5-Flag and p LL3. 7 eukaryon expression vector,and determine the effects on the apoptosis of gastric cancer cells after transfected with the vectors. Methods The open reading frame sequence of FOXO4 gene was cloned into the plasmid p CMV5-Flag,and the siRNA sequence targeting FOXO4 gene was cloned into the plasmid p LL3. 7 in order to generate the expression plasmid Flag-FOXO4 and interference plasmid FOXO4-shRNA. The plasmids and control of the plasmids were transfected into the gastric cancer MKN45 and SGC7901 cells respectively by lipofectamine-mediated transfection. The expression and silencing efficiency were determined by Western blotting. Cell apoptosis were detected by flow cytometry,and the protein levels of cleaved Caspase-3 was detected by Western blotting.Real-time PCR was employed to measure the mRNA expression of apoptosis promoting transcription factor BCL-6（ a downstream gene of FOXO4 gene） in the gastric cancer cells after transfection with the vectors.Results The expression plasmid（ Flag-FOXO4） and interference plasmid（ FOXO4-shRNA） were successfully constructed respectively. The protein level of FOXO4 was significantly increased in the gastric cancer MKN45 and SGC7901 cells after plasmid Flag-FOXO4 transfection,and was obviously decreased in the cells transfected with FOXO4-shRNA vector. Flow cytometry showed that the Flag-FOXO4 transfection enhanced the apoptotic rates of the MKN45 cells and SGC7901 cells by 16. 54%（ P 〈 0. 01） and 26. 23%（ P 〈 0. 01） respectively when compared with the cells without transfection. Western blot result showed the expression level of cleaved Caspase-3 was also increased. Real-time PCR showed that,the mRNA expression level of BCL-6 was increased in the gastric cancer cells with Flag-FOXO4 transfection than the cells without（ P 〈 0. 01）,and it was decreased in the cells with FOXO4-shRNA transfection（ P 〈 0. 01）. Conclusion Over-expression of FOXO4 enhances the apoptosis in gastric cancer cells through up-regulating the expression of its downstream pro-apopotic gene BCL-6,while,silencing of FOXO4 decreases the expression of BCL-6.