Objective      To construct the expression vector and the small interfering RNA containing human Forkhead box O4 gene (FOXO4) by using the pCMV5-Flag and pLL3.7 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 pCMV5-Flag

and the siRNA sequence targeting FOXO4 gene was cloned into the plasmid pLL3.7 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% (P0.01) and 26.23% (P0.01) 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 (P0.01)

and it was decreased in the cells with FOXO4-shRNA transfection (P0.01).       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.