论文部分内容阅读
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。
OBJECTIVE: To construct a vector expressing short hairpin RNA (shRNA) of hypoxia inducible factor 1α (HIF-1α) and to examine its inhibitory effect, and to explore its potential application in the treatment of prostate cancer. Methods: The specific shRNA targeting HIF-1α was designed and synthesized and inserted into psilencerTM2.1-U6 vector containing U6 promoter. The shRNA expression vector psilencer-HIF was obtained and transfected into prostate by liposome The expression of HIF-1α was detected by RT-PCR and Western blotting. The expression of HIF-1α was detected by co-transfection of green fluorescent protein plasmid pEGFP. Results: The recombinant plasmid psilencer-HIF was confirmed by sequencing analysis. The efficiency of plasmid transfection was (89.26 ± 4.72)% 24 h after co-transfection, and the shRNA produced by this method could induce RNA interference in PC-3M cells The mRNA and protein levels of HIF-1α decreased by 82.09% and 81.61%, respectively, 48 h after transfection (P <0.01). However, the expression of HIF-1α did not change significantly in the negative control group (P> 0.05). Conclusion: The shRNA expression plasmid of HIF-1α gene was successfully constructed, which can effectively inhibit the expression of HIF-1α in prostate cancer cells and provided a new idea for the gene therapy of prostate cancer.