利用RNA干扰抑制p21^(WAF1/CIP1)基因的表达及周期阻滞效应

Inhibition of the Expression of p21^(WAF1/CIP1) Gene by RNA Interference and its Effect on Cell Cycle Arrest

目的:构建p21WAF1/CIP1基因小干扰RNA(siRNA)的真核表达载体,观察其对p21WAF1/CIP1表达的影响和细胞周期的变化。方法:合成了针对p21WAF1/CIP1基因的siRNA,将其克隆到siRNA表达载体pSliencer2.1-U6neo上,将重组质粒和带FLAG标签的p21WAF1/CIP1共转染293T人胚肾细胞,通过Westernblot检验RNA干扰(RNAi)敲低外源p21WAF1/CIP1的效果;将重组质粒单独转染293T人胚肾细胞,利用p21WAF1/CIP1抗体检测RNAi敲低内源p21WAF1/CIP1的效果;利用流式细胞仪检测敲低后细胞周期的变化。结果:测序证明构建了p21WAF1/CIP1siRNA真核表达载体;Westernblot和流式细胞分析证明,构建的siRNA能有效降低p21WAF1/CIP1基因的表达,并且使G1期细胞数减少14.03%,S期细胞增多13.45%。结论:构建了p21WAF1/CIP1siRNA的真核表达载体,该siRNA能有效抑制p21WAF1/CIP1基因的表达并部分解除了G1期阻滞。

Objective： To construct the eukaryotic expression vector of p21WAF1/CIP1 siRNA, and investigate its effect on the expression of p21WAF1/CIP1 gene and cell cycle. Methods：p21WAF1/CIP1 siRNA was synthesized and inserted into pSliencer 2.1-U6 neo expression vector. Then human embryo kidney 293T cells were transfected with the recombination plasmid and the p21WAF1/CIP1 plasmid tagged with FLAG to detect the effect of the exogenous gene expression; 293T cells were transfected with the recombination alone to detect the effect of the endogenous gene expression by Western blot. Then the effect of p21WAF1/CIP1 siRNA on cell cycles was identified by FCM assays. Results： The expression vector of p21WAF1/CIP1 siRNA was constructed and confirmed by DNA sequencing. Western blot and FCM assays showed that the siRNA could effectively weaken the gene expression. The cell number of G1 phase reduce by 14.03% and the cell number of S phase increase by 13.45%. Conclusion： The eukaryotic expression vector of p21WAF1/CIP1 siRNA was constructed successfully. The siRNA could effectively inhibit the expression of p21WAF1/CIP1 gene and partly abolish the G1 arrest.