PLCε特异性shRNA对人膀胱癌生物学行为的影响

Effect of PLCε shRNA on biological behavior of bladder cancer

目的探讨人源磷脂酶Cε(PLCε)特异性短发夹RNA(shRNA)对人膀胱癌增殖、凋亡、侵袭、转移能力的影响。方法用携带PLCεshRNA基因的真核表达质粒pGenesil-PLCε转染BIU-87细胞,采用逆转录-聚合酶链反应(RT-PCR)和蛋白质印迹法检测各组细胞PLCεmRNA和蛋白的变化。分别用四甲基偶氮唑盐比色法(MTT)、流式细胞术和免疫组化检测BIU-87细胞及裸鼠移植瘤增殖情况和细胞周期的变化;采用RT-PCR检测各组细胞Bcl-2、Bax mRNA的变化;采用Transwell小室体外侵袭法及明胶酶谱分析细胞的侵袭能力。结果转染pGenesil-PLCε质粒的细胞可明显抑制PLCε基因和蛋白表达水平(P<0.05);MTT、流式细胞术与免疫组化检测结果表明转染pGenesil-PLCε质粒的细胞生长明显受抑制(P<0.05),且细胞周期阻滞于G0/G1期;转染pGenesil-PLCε质粒的细胞Bcl-2/Bax mRNA明显降低(P<0.05);明胶酶谱及侵袭实验显示转染pGenesil-PLCε质粒的细胞侵袭、转移能力明显下降(P<0.05)。结论特异性shRNA干扰PLCε基因可抑制膀胱癌的生长,其作用机制可能与抑制肿瘤增殖、诱导细胞凋亡及抑制侵袭转移有关,PLCε为膀胱癌基因治疗的潜在靶点。

Objective To investigate the proliferation,apoptosis and invasion effect by silencing PLCεin bladder cancer. Methods The shRNA recombinant plasmids targeting PLCε gene was transfected into bladder carcinoma cell line BIU-87;RT-PCR and Western-blot detected the expression level of PLCε mRNA and protein. The proliferation and cell cycle were respectively measured by methyl thiazolyl tetrazolium（MTT） ,FCM and IHC. The expressions level of Bcl-2 ,Bax mRNA were detected by RT PCR. Inva sive power of BIU-87 was measured by membrane invasion culture system（Transwell chamber） and gelatin enzymography. Results The expression of PLCε mRNA and protein were effectively reduced in transfected cell（P〈0.05）. The cellular proliferation was obviously inhibited in transfected BIU-87 cell（P〈0.05） and the G0/G1 cell cycle was increased. Bcl-2/Bax mRNA expression was down-regulated in transfected cell（P〈0.05）. The invasion power of cell transfected with positive plasmid was lower than the control group（P〈0.05）. Conclusion Transfection of PLCε shRNA can inhibit the growth of bladder cancer,and the mechanism may relate to the inhibition of proliferation and invasion, and the induction of apoptosis. PLCε shRNA may become to a potential molecular target for bladder cancer in gene therapy.