hTERT短发夹状RNA抑制前列腺癌细胞生长的研究

Study on Inhibition of Growth and Proliferation in Prostate Cancer Cell by the shRNA of hTERT

目的研究靶向端粒酶逆转录酶(hTERT)的短发夹状RNA(shRNA)基因转染对前列腺癌细胞体外生长的抑制效应及其促细胞凋亡作用。方法在脂质体介导下将针对hTERT基因的shRNA表达载体psilencer-TRT转染前列腺癌细胞PC-3m,得到稳定细胞株PC-3m/shRNA-TRT。采用RT-PCR检测hTERT基因表达情况,western blot分析各组细胞hTERT及c-myc蛋白表达变化,细胞计数并绘制细胞生长曲线,Hoechst33258染色、透射电镜、流式细胞仪检测细胞凋亡情况。结果重组质粒psi-lencer-TRT转录生成的shRNA使实验组细胞的hTERT基因表达显著下调,抑制率约为89.02%;同时实验组细胞hTERT及c-myc蛋白水平较对照组有明显下降,细胞的生长增殖能力也显著降低(P<0.05),生长速率明显变慢,部分细胞呈现凋亡形态学改变,凋亡率为(19.69±4.75)%。结论hTERT短发夹状RNA能有效抑制前列腺癌细胞中hTERT表达及癌细胞生长,诱导PC-3m细胞凋亡,可望为前列腺癌的基因治疗提供新方法。

Objeetive To investigate the effect of the short hairpin RNA （shRNA） against human telom- erase reverse transcriptase （hTERT） on the proliferation and apoptosis of prostate cancer cells PC-3m in vitro. Methods The recombinant plasmid psilencer-TRT was transfected into prostate cancer cell line PC-3m via liposome reagent. The level of hTERT mRNA was examined by reverse transcription polymerase chain reaction （RT-PCR）. The expressions of hTERT and c-myc protein were detected by western blot analysis. The effect of hTERT shRNA on the cellular proliferation capacity of PC-3m cells was assayed by the growth curve. The cell apoptosis was detected by Hoechst33258 staining, electron microscope, and flow cytometry analysis. Results The vector-mediated shRNA significantly reduced the level of hTERT mRNA by 89. 02% after psilencer-TRT transduction in PC-3m cells. Meanwhile, the levels of hTERT and c-myc protein were also decreased in transfected cells. The cell proliferation was markedly inhibited compared with the control cells. Partial cancer cells presented morphological changes of apoptosis, and the apoptosis rate was （19. 69 ± 4. 75） %. Conclusion hTERT shRNA can suppress hTERT expression and cell proliferation, in addition to acceleration of apoptosis. This implied the possibility of RNA interfering to hTERT as the potential method for gene therapy of prostate cancer.