水通道蛋白1对MCF-7人乳腺癌细胞增殖能力的影响

Effect of AQP1 on proliferation activity of MCF-7 breast cancer cells

目的采用RNA干扰(RNAi)技术沉默水通道蛋白1(aquaporin 1,AQP1)基因的表达,观察其对人乳腺癌细胞MCF-7增殖和凋亡的影响。方法构建针对AQP1的shRNA重组质粒,脂质体法转染MCF-7细胞,实验分为正常对照组、阴性对照组和4个AQP1-shRNA转染组。通过实时荧光定量PCR(qRT-PCR)和Western blot法检测转染前后MCF-7细胞中AQP1mRNA和蛋白表达水平。CCK-8法检测AQP1对MCF-7细胞增殖活性的影响,流式细胞术检测MCF-7细胞凋亡,Western blot法检测Caspase-3的变化。结果四组特异性shRNA转染MCF-7细胞后48 h,AQP1基因的mRNA和蛋白水平均不同程度降低,其中以AQP1-shRNA4对AQP1的沉默效果最明显(mRNA和蛋白表达抑制率分别为70.54%和72.42%)。AQP1基因沉默48 h后,与正常对照组相比,MCF-7细胞的增殖活性明显受抑(P<0.05);细胞凋亡率显著增加为(27.77±2.82)%(P<0.05);细胞中的Caspase-3蛋白表达水平显著上调(P<0.05)。结论 AQP1表达沉默可有效抑制MCF-7细胞的增殖,促进细胞凋亡,提示AQP1有望成为乳腺癌基因治疗的一个新靶点。

Objective To explore the effects of AQP1 on cell proliferation and apoptosis of MCF-7 breast cancer cells after AQP1 silenced by RNAi technology. Methods The shRNA expression vectors targeting human AQP1 gene were constructed,and then transfected into human breast cancer MCF-7 cells. The cultured MCF-7 cells were divided into normal control group,negative control group and four pairs of AQP1-shRNA transfection group. The expression levels of AQP1 mRNA and protein were detected by qRT-PCR and Western blot to evaluate the interfering efficiency. Cell proliferation activity and apoptosis of MCF-7 cells were detected by CCK-8 assay and FCM,respectively. The expression level of Caspase-3 was detected by Western blot. Results After transfection with the four pairs of AQP1-shRNA for 48 h,AQP1 mRNA and protein expression levels in MCF-7 cells were both reduced. The most effective expression inhibition rate of mRNA and protein were 70. 54% and 72. 42% respectively,which caused by the AQP1-shRNA4 plasmid vector. Cell proliferation activity was blocked in MCF-7 cells after AQP1 silencing for 48 h（ P 0. 05）. The apoptotic rate in the interference group was（ 27. 77 ± 2. 82） %,which was significantly higher than that in normal control group（ P 0. 05）,and the expression level of Caspase-3 in MCF-7 cells was also significantly increased（ P 0. 05）. Conclusion AQP1-shRNA transfection could efficiently inhibit the proliferation of MCF-7 breast cancer and increase the cell apoptosis,which provides a potential target in gene therapy for breast cancer.