目的

应用小干扰RNA(small interfering RNA, 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hTERT mRNA 表达水平,Western blot 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,hTERT siRNA 转染MCF-7 细胞48 h 后,siRNA1 ～siRNA3 组hTERT mRNA 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48 h 后,siRNA1 ～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hTERT siRNA 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-7cells.

Methods

Chemically synthesized small interfering RNA (siRNA) targeting hTERT was transfected into human breast cancer cell line MCF-7 by Lipofectamine^TM 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 decreased markedly. In the groups of siRNA1, siRNA2, siRNA3 and siRNA4, the inhibition ratios after 48-hour-transfection 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.