短发夹状RNA抑制缺氧诱导因子1α在前列腺癌细胞中的表达

目的:构建产生缺氧诱导因子1α(HIF-1α)特异性短发夹状RNA(shRNA)的载体并检测其抑制作用,探讨该技术在前列腺癌治疗中的应用前景。方法:设计、合成针对HIF-1α的特异性shRNA模板序列,将其插入含有U6启动子的psilencerTM2.1-U6载体中,得到shRNA表达载体psilencer-HIF,在脂质体介导下转染前列腺癌细胞株PC-3M,应用绿色荧光蛋白质粒pEGFP共转染评价转染效率,RT-PCR及Western印迹检测其对HIF-1α表达的影响。结果:重组质粒psilencer-HIF经测序分析证实与设计的完全一致,共转染24 h后质粒转染效率为(89.26±4.72)%,其产生的shRNA在PC-3M细胞中能诱导RNA干扰(RNAi),转染后48 h HIF-1αmRNA和蛋白水平分别下降82.09%和81.61%(P<0.01);而阴性对照组HIF-1α表达水平无明显改变(P>0.05)。结论:成功构建了HIF-1α基因shRNA表达质粒,其可有效抑制前列腺癌细胞中的HIF-1α的表达,为前列腺癌的基因治疗提供了新思路。

Vector-Mediated shRNA Inhibits HIF-1α Expression in Prostate Cancer Cells

Objective: To construct a short hairpin RNA(shRNA) vector of the hypoxia inducible factor-1α(HIF-1α),determine its inhibitory effect on the expression of the HIF-1α gene in PC-3M cells,and investigate its application prospects in the treatment of prostate cancer.Methods: We designed and synthesized the shRNA template sequence specific against HIF-1α,inserted it into the vector psilencer2.1-U6 to generate the plasmid psilencer-HIF,transfected the recombinant plasmid into prostate cancer cell line PC-3M cells and detected the transfection efficiency by cotransfection with the pEGFP vector as well as the expression of HIF-1α by RT-PCR and Western blot.Results: The DNA sequencing analysis showed a complete consistency of the recombinant plasmid psilencer-HIF with the design.Twenty-four hours after the transfection,the rate of transfected pasmid was about(89.26±4.72)% and the vector-mediated shRNA induced RNA interference(RNAi),while 48 hours' transfection reduced the HIF-1α mRNA and protein levels by 82.09% and 81.61% respectively(P<0.01) in PC-3M cells.Conclusion: The shRNA vector was successfully constructed,which can effectively suppress the expression of HIF-1α in prostate cancer cells.