siRNA沉默EZH2表达对人膀胱癌细胞迁移和凋亡的影响

The effect of cell activation and apoptosis by siRNA interference EZH2 expression in human bladder cancer cells

目的探讨小干扰RNA(siRNA)沉默EZH2表达对人膀胱癌细胞增殖、侵袭和迁移能力,以及对凋亡的影响。方法构建靶向EZH2基因的siRNA质粒并转染至人膀胱癌细胞中(干扰组),采用RT-PCR法检测EZH2mRNA的表达情况,利用噻唑蓝(MTT)、Transwell细胞小室及划痕实验检测转染后细胞增殖、侵袭和转移能力的变化,通过流式细胞术实验观察转染后细胞的凋亡情况。结果转染EZH2基因的siRNA质粒后(分干扰1组,干扰2组,干扰3组),干扰各组EZH2mRNA表达较阴性对照组有明显抑制(P<0.05),且干扰2组效果最好,因此选干扰2组作后期实验;在转染48h后,干扰2组生长抑制率达37.9%,较阴性对照组受到明显抑制(P<0.05);划痕实验处理24h后,干扰2组细胞的迁移距离较阴性对照组明显降低(P<0.01);干扰2组细胞侵袭能力较阴性对照组下降了67%,差异有统计学意义(P<0.01);转染48h后,干扰2组早、晚期凋亡率分别为22.80%和3.60%,较阴性对照组增加,且以早期明显,差异有统计学意义(P<0.01)。结论 EZH2基因沉默能有效抑制人膀胱癌细胞的增殖、侵袭和迁移能力,并促进其凋亡,为深入研究膀胱癌的基因治疗提供理论依据。

Objective To investigate the effect of EZH2 knockdown on cell proliferation ,invasion ,migration and apoptosis in hu-man bladder cancer cell line by small interfering RNAs（siRNA） .Methods The siRNA-expressing plasmid targeting EZH2 gene was constructed and transfected into T24 cells .RT-PCR was used to detect the EZH2 gene′s expression at the level of mRNA ;pro-liferation ,invasion and migration of T24 cells were examed in vivo by MTT ,wound healing assay and Transwell chamber migration assay .Finally ,Annexin V-FITC/PI flow cytometric analysis was performed for cell apoptosis .Results The siRNA-expressing plas-mid targeting EZH2 gene successfully inhibited EZH2 gene’s expression in T24 cells .The expression of mRNA was significantly inhibited compared with negative control groups （P〈0 .05） .After the transfection of the plasmid 48 hours ,the growth inhibition rate was 37 .9% ,which was higher than the negative control group（P〈0 .05） .24 hours after wound healing ,the migration distance of transfected group cell was（1 .37 ± 0 .12） ,which was lower than the negative control group（P〈0 .01） .Compared with the nega-tive control group ,invasion capability of EZH2-siRNA group was dropped by 67% （P〈0 .01） .48 hours after transfection ,the early and secondary apoptosis rate of T 24 cells were 22 .80% and 3 .60% respectively ,which were higher than the negative contral group （P〈0 .01） .Conclusion The siRNA interference EZH2 can significantly inhibit cell proliferation ,invasion and migration of T24 cells ,meanwhile promote its apoptosis .It provides a theoretical basis for further study of bladder cancer gene therapy .