shRNA干扰介导PLCε基因表达下调对膀胱癌T24细胞生长的影响

shRNA-mediated interference of gene expression PLCε downturn on T24 bladder cancer cells

目的:探讨shRNA干扰沉默PLCε基因对人膀胱癌T24细胞生长的影响。方法:体外构建PLCε基因的shRNA的重组质粒,Lipofectamine2000介导法转染T24细胞,采用RT-PCR方法检测特异性shRNA的重组质粒对PLCε基因沉默效果,转染后采用MTT法检测shRNA的重组质粒对细胞增殖的作用,免疫细胞化学染色法检测T24细胞PCNA的表达,电镜观察细胞超微结构的改变。结果:shRNA的重组质粒能有效下调PLCε基因的表达,抑制率为78.01%,与对照组比较其差异有统计学意义(P<0.01);细胞增殖活性受到明显抑制,与对照组比较其差异有统计学意义(P<0.01);细胞内PCNA含量明显低于空白对照组和阴性质粒组,其差异有统计学意义(P<0.01);细胞形态改变产生凋亡小体。结论:PLCε基因有望成为应用RNAi技术探索治疗膀胱癌潜在的靶基因。

Objective：To investigate whether PLCε gene down regulation by RNA interference（RNAi） leads to inhibition of proliferation in human bladder carcinoma T24 cell.Methods： The shRNA recombinant plasmids targeting to PLCε gene was constructed and transfected into bladder carcinoma T24 cell with Lipofectamine 2000.RT-PCR was used to monitor the validity of specific s h R N A in down regulation of PLCε.Then MTT assay was performed for detecting cell proliferation,the changes of PCNA were analyzed by immunocytochemical method, Electron microscope was used to observe the morphological changes. Results： The specific PLC shRNA was confirmed to be efficient in silencing PLCε expression. PLCε gene down regulation by the shRNA recombinant plasmids inhibition cell proliferation rate about 78.01%, They were significantly different from that of control group （P〈0.01 ;After transfected the specific recombinant plasmids, PCNA expression was significantly decreased,They were significantly different from that of control group （P〈0.01 ;morphological changes produce apoptotic body. Conclusion： PLCε is likely to be potential molecular target for bladder carcinoma in gene therapy by RNAi.