siRNA沉默hTERT基因表达对人乳腺癌细胞增殖和凋亡的影响

Effect of hTERT gene silencing by RNA interference on proliferation and apoptosis of human breast cancer cells

目的应用小干扰RNA（small interferingRNA，siRNA）抑制乳腺癌MCF-7细胞hTERT的表达，探讨其对乳腺癌细胞增殖和凋亡的效应。方法体外化学合成针对hTERT基因的siRNA序列，在脂质体介导下转染MCF-7细胞，实时定量PCR检测hTERTmRNA表达水平，Westernblot检测hTERT蛋白表达水平，流式细胞仪检测细胞凋亡状况，MTT法检测细胞增殖活性，平板克隆形成实验检测克隆形成率。结果所设计的3对针对不同靶点的siRNA与对照组相比均可有效抑制hTERT的表达，hTERTsiRNA转染MCF-7细胞48h后，siRNAl-siRNA3组hTERTmRNA表达分别为（35．3±4．2）％、（30．7±2．8）％、（31．3±3．6）％，与阴性对照组（96．4±2．8％）相比，差异有统计学意义。MTT结果显示MCF-7细胞增殖能力显著降低，转染48h后，siRNAl～siRNA4细胞抑制率分别为（57．6±3．6）％、（61．3±4．3）％、（65．6±6．3）％和（3．1±4．5）％，细胞克隆形成能力下降，凋亡率明显增加。结论体外化学合成的hTERTsiRNA可以有效地抑制MCF-7细胞hTERT的表达，从而抑制细胞增殖，促进细胞凋亡。

Objective To evaluate the effect of targeting hTERT gene on the proliferation and apoptosis of MCF-7eells. Methods Chemically synthesized small interfering RNA （siRNA） targeting hTERT was transfected into human breast cancer cell line MCF-7 by LipofeetamineTM 2000. The expression levels of hTERT mRNA and protein were detected respectively by real time PCR and Western blot. Cell proliferation was determined by MTT, and the cell colony formation rate was measured by plate colony formation assay, and cell apoptosis was observed by flow cytometry. Results The expressions of hTERT in both mRNA and protein of MCF-7 cells were effectively decreased by the siRNA targeting human hTERT gene; the hTERT mRNA levels in the groups of siRNA1, siRNA2 and siRNA3 were respectively reduced to （ 35.3 ± 4.2 ） % , （ 30.7 ± 2.8 ） % and （ 31.3 ± 3.6） % at 48 hours after transfection, and were significantly lower than that of the negative control group （96.4 ±2.8% ）. MTT results showed that the growth rate of MCF-7 cells was d,.creased markedly. In the groups of siRNA1, siRNA2, siRNA3 and siRNA4, the inhibition ratios after 48-hour-transfeetion were （57.6 ± 3, 6） %, （61.3 ± 4.3 ） %, （ 65.6± 6.3 ） % and （3.1 ± 4.5 ） %, respectively. Moreover, the cell colony formation rates were statistically decreased in hTERT siRNA groups, and the apoptosis was increased significantly. Conclusion The synthesized siRNA can effectively decrease the hTERT gene expression, inhibit cell proliferation and induce cell apoptosis, hTERT siRNA can be used as a new approach in human breast cancer gene therapy.