RNA干扰沉默EZH2基因对人膀胱癌EJ细胞增殖的影响

Effect of silencing EZH2 gene expression by RNA interference on proliferation of human bladder carcinoma EJ cell line

目的:研究RNA干扰沉默EZH2基因对人膀胱癌EJ细胞周期和增殖的影响.方法:体外构建EZH2基因的shRNA的表达载体,Lipofectamin 2000介导转染膀胱癌EJ细胞,采用RT-PCR和Western Blot方法检测特异性shRNA对EZH2基因沉默效果,转染后采用MTT法检测shRNA对细胞增殖的作用;应用PI单染流式细胞术分析对细胞周期的改变.结果:EZH2基因的shRNA表达载体有效下调EZH2基因的表达(P<0.05).与对照组比较,细胞增殖明显抑制(P<0.05);同时引起细胞G1期阻滞,RNA干扰后,G1期细胞增加[(84.0±8.7)%vs(52.0±6.8)%,P<0.05],S期细胞减少[(11.0±1.1)%vs(43.0±4.9)%,P<0.05].结论:EZH2基因有望成为应用RNAi技术探索膀胱癌基因治疗的潜在靶基因.

AIM： To investigate whether EZH2 gene downregulation by RNA interference（RNAi） leads to inhibition of proliferation and arrest of cell cycle in human bladder carcinoma EJ cell hne. METHODS： The shRNA expression plasmid targeting to EZH2 gene was constructed and transfected into bladder cancer EJ cell line with Lipofectamin2000. RT-PCR and Western Blot was used to monitor the validity of specific shRNA in downregulation of EZH2 expression. Then the MTT assay was performed for detecting cell proliferation and Annexin V-FITC/ PI flow cytometric analysis for cell cycle. RESULTS： The specific EZH2 shRNA was confirmed to be efficient in silencing EZH2 expression. EZH2 gene downregulation by RNAi inhibited cell proliferation remarkably（P 〈 0.05 ） and arrested cell cycle at G1 phase significantly （P 〈 0.05 ）. There was an increase of cell number at G1 phase [ （84.0 ±8.7）% vs （52.0±6.8）% , P〈0.05] and a decrease ofSphase[（11.0±1.1）% vs （43.0±4.9）%, P〈0.05] in EJ cells treated with EZH2 shRNA compared with untreated EJ cells. CONCLUSION： EZH2 is likely to be potential molecular target for bladder carcinoma in gene therapy by RNAi.