RNA干扰hTERT基因对人乳腺癌细胞株MCF-7周期及凋亡影响的研究

Effect of human breast cancer line MCF-7 cell cycle and apoptosis using RNAi hTERT mRNA

目的观察载体介导的RNA干扰技术能否有效抑制人乳腺癌细胞株MCF-7的hTERT的表达及其对细胞周期及凋亡的影响。方法构建2个针对hTERT基因表达短发夹状siRNA的真核表达载体(pshRNA-hTERT)并转染人乳腺癌细胞株MCF-7,通过RT-PCR检测该基因mRNA,Western blot蛋白质检测,端粒酶活性检测,流式细胞仪检测细胞周期及凋亡等方法检测RNAi效果,从中筛选出基因沉默效果好的靶位点。结果转染乳腺癌细胞后,pshRNA-hTERT1,2质粒均能抑制hTERT基因表达,mRNA表达分别下调了54.6%,55.2%;蛋白表达分别下调46.6%,47.8%。端粒酶活性均明显下降。对乳腺癌细胞周期中S期细胞明显减少,G1/G0期细胞显著增加。结论 RNAi在体外明显抑制乳腺癌细胞中hTERT基因的表达和瘤细胞增殖。

Objective To investigate whether RNA interference（RNAi） induced by plasmid vector could suppress hTERT.expression in bureau breast carcinoma cells MCF - 7 and the effect on cell cycle and apoptosis. Methods The small hairpin RNA （shRNA） eukaryotic expression vector targeting hTERT gene, named pshRNA - hTERT - 1/ pshRNA - hTERT - 2, were constructed and transfected into cultured human breast carcinoma cell of line MCF - 7 via GenEseortTM U ; The inhibited effects on hTERT gene were determined by RT - PCR detecting hTERT - mRNA, western blot detecting its protein expression, TRAP - Hyb Kit detecing telomerase activation and flow cytometry analysis Cell cycle and apoptosis. Then screening gene silencing effective site. Results After the shRNA expression vector was success- fully transfected into MCF -7 cells via GenEscortTM n , pshRNA - hTERT1,2 reduced hTERT gene expression by compared with the control group, hTERT - rnRNA expression decrease 54. 6% , 55. 2% , protein expression decrease 46.6% ,47. 8%. Telomerase activation decrease significantly. Cell cycle analysis showed accumulation of cells in S phase significant decrease,G0 - G1 phase significant increase. Conclusion RNAi obviously inhabit hTERT gene expression of the breast cancer cell line MCF - 7 and cell proliferation.