特异性小干扰RNA靶向沉默Oct4基因表达对人膀胱癌EJ细胞生物学的影响

Effect of silencing Oct4 by specific siRNA on the biological behavior of human bladder cancer cell line EJ

目的 观察RNA干扰沉默Oct4基因表达对人膀胱癌EJ细胞生物学的影响.方法 将人膀胱癌EJ细胞,常规分为空白对照组、阴性对照组和实验转染组进行实验.体外化学合成Oct4基因序列特异性小干扰RNA（siRNA）,脂质体Lipofectamine 2000介导转染人膀胱癌细胞株EJ细胞;采用逆转录-聚合酶链反应（RT-PCR）和Western blot方法分别检测特异性siRNA对Oct4基因在mRNA和蛋白水平上沉默效果;转染后采用噻唑蓝（MTT）比色法检测siRNA对细胞增殖的作用;AO/EB荧光染色法检测其凋亡;用细胞划痕和Transwell试验体外检测细胞迁移和侵袭能力.结果 成功转染Oct4-siRNA的膀胱癌EJ细胞,RT-PCR及Western blot分别显示Oct4 mRNA和蛋白表达在相应的癌细胞中明显下降;与阴性对照组比较,细胞增殖明显抑制（P＜0.05）;同时细胞凋亡率增加（52.73%比11.70%）;RNA干扰明显抑制EJ细胞迁移力和侵袭力（15.34±1.85比63.07±1.73）.结论 Oct4-siRNA可有效抑制膀胱癌EJ细胞Oct4基因的表达,从而抑制肿瘤细胞增殖,促进细胞凋亡,抑制细胞迁移和侵袭,Oct4-siRNA有效地调控EJ细胞恶性生物学行为.

Objective To investigate the effect of small interference RNA （siRNA） silencing Oct4 on the biological behavior of human bladder cancer cell line EJ. Methods EJ cells were divided into three groups： the blank control group, the negative control group, the Oct4-siRNA group. Chemically synthesized siRNA targeting Oct4 （Oct4-siRNA） was transfected into EJ cells with high metastatic potential by lipofectamin 2000. Reverse transcription-polymerase chain reaction （RT-PCR） and Western blotting were used to semi-quantify the Oct4 mRNA and protein levels. Then proliferation of EJ cells was determined by methyl thiazol tetrazolium （MTT） assay. Acridine orange-ethidium bromide （AO/EB） fluorescent staining was performed to detect apoptosis. The activities of motility and invasion of EJ cells were assessed by cell wound model and transwell chamber invasion assay in vitro, respectively. Results In the Oct4-siRNA group, the Oct4 mRNA and protein levels were down-regulated remarkably （P 〈 0. 05）, the abilities of proliferation were inhibited, the motility and invation of EJ cells were inhibited significantly （ 15.34 ± 1.85vs 63.07 ± 1.73） in vitro, and the apoptosis rate was increased notablely （52. 73% vs 11.70% ） as compared with the negative control group （ P 〈 0. 05 ）. Conclusion Silencing Oct4 could regulate the maligrant biological behaviors of bladder cancer cell line EJ effectively.