siRNA下调叉头框M1基因表达对大肠癌细胞化疗敏感性的影响

Effects of FoxM1 down-regulation by small interfering RNA on chemosensitivity of human colon cancer cell

目的:观察叉头转录因子M1(forkhead box protein M1,FoxM1)基因小干扰RNA(small interfering RNA,siRNA)对大肠癌细胞化疗药物敏感性的影响,并探讨其可能机制。方法:设计合成3个FoxM1 siRNA,转染人大肠癌SW620细胞后,采用定量PCR方法筛选下调FoxM1 mRNA效果最好的siRNA,然后以不同浓度(3.125,6.25,12.5nmol/L)的该siRNA转染处理大肠癌细胞,用RPMI 1640代替FoxM1 siRNA作为空白对照组(Con-A);48 h后,各组细胞分别用伊立替康(Irinotecan,CPT-11,7.5μmol/L)处理,以MTT法检测siRNA转染对各组细胞增殖及对伊立替康敏感性的影响;以蛋白质印迹方法检测Akt磷酸化情况。结果:MTT结果显示,空白对照组、siRNA 3.125 nmol/L组、siRNA 6.25 nmol/L组和siRNA 12.5 nmol/L组SW620细胞生长抑制率分别为18.36%,39.78%,58.18%,78.28%。统计学分析显示,抑制率呈浓度依赖性(r=0.775,P<0.05)。蛋白质印迹结果表明,siRNA转染组Akt磷酸化水平明显下调。结论:FoxM1基因siRNA可提高大肠癌细胞化疗敏感性;下调Akt磷酸化水平是其重要机制之一。

Objective:To study the effects of Forkhead box protein M1(FoxM1) small interfering RNA(siRNA) on chemosensitivity and mechanism of human colon cancer cell.Methods:Three FoxM1 siRNAs were designed and constructed.The best effect of siRNA down-regulation was evaluated by Real time PCR.All cancer cells were divided into different dose of siRNA groups,and RPMI 1640 instead of FoxM1 siRNA as the blank control group(Con-A);after transfected for 48 h,all groups cells were treated with irinotecan(Irinotecan,CPT-11,7.5 μmol/L),respectively.The proliferation and chemosensitivity of cancer cells were evaluated by MTT assay,and the phosphorylation of Akt protein was exmined by Western blot.Results:FoxM1 siRNA could down-regulate the FoxM1 expression in a dose-and time-dependent manner.The MTT results showed that the inhibit rates were 18.36%,39.78%,58.18%,78.28% in different groups[Con-A,siRNA 3.125 nmol/L,siRNA 6.25 nmol/L and siRNA 12.5 nmol/L,respectively.Statistical analysis showed that inhibition rates were in a dose-dependent(r = 0.775,P < 0.05).The phosphorylation of Akt protein was inhibited compared with Con-A group.Conclusion:FoxM1 siRNA could sensitize human colon cancer cells to chemotherapy through inducing phosphorylation of Akt protein.