短发夹状RNA对PC-3M细胞端粒酶逆转录酶基因表达和端粒酶活性的影响

Effects of short hairpin RNA on hTRT expression and telomerase activity in PC-3M cells

目的:研究短发夹状RNA(shRNA)对PC 3M细胞中端粒酶逆转录酶(hTRT)基因表达及端粒酶活 性的影响。方法:构建针对hTRT基因的shRNA表达质粒psilencer TRT,在质脂体介导下转染人前列腺癌PC 3M细胞,应用RT PCR及免疫组织化学检测hTRTmRNA及蛋白表达水平,应用SYBR Green染色法检测端粒 酶活性的变化。结果:重组体psilencer TRT转染细胞24h和48h后显著下调hTRTmRNA及蛋白水平,作用 48h后,其抑制率分别为85.39%和79.17%,较空白对照组差异有统计学意义(P<0.01)。同时,随着时间的延 长,端粒酶活性亦有明显下降(P<0.01)。结论:利用短发夹状RNA干扰技术能有效抑制靶基因的表达,为前 列腺癌的基因治疗提供新思路。

Objective:To study the effect of short hairpin RNA(shRNA) on the expression of human telomerase reverse transcriptase(hTRT) and telomerase activity in PC-3M cells.Methods:Plasmid psilencer-TRT expressing hTRT shRNA was constructed. The recombinant plasmid was transfected into prostate cancer cell line PC-3M by lipofectamine. The cellular hTRT expression and telomerase activities were detected by RT-PCR, immunohistochemical method and SYBR-Green assay respectively.Results:ShRNA could significantly reduce the levels of hTRT mRNA and protein. When transfected with psilencer-TRT for 48 h,the expressions of hTRT mRNA and protein were inhibited by 85.39% and 79.17% respectively(P< 0.01). Telomerase activity of PC-3M cells was also down-regulated significantly with time after psilencer-TRT treatment(P< 0.01).Conclusions:The shRNA express vector was successfully constructed. The RNA interference through shRNA can effectively inhibit the expression of target gene, and can be potentially useful in gene therapy of hTRT related prostate cancers.