小干扰 RNA 表达质粒沉默△Np73 对人乳腺癌细胞 MDA-MB-231增殖和凋亡的影响

Effects of small interfering RNA expression plasmids silencing △Np73 on human breast cancer cell stain MDA-MB-231 proliferation and apoptosis

目的:探讨靶向△Np73的小干扰RNA表达质粒转染人乳腺癌细胞株MDA-MB-231对其增殖和凋亡活性的影响。方法:构建△Np73小干扰表达质粒,脂质体法转染人乳腺癌细胞株MDA-MB-231;荧光显微镜下观察转染细胞绿色荧光蛋白表达,计算转染效率;实时荧光定量PCR检测细胞转染质粒后△Np73和TAp73 mRNA的表达;共转染pcDNA3-HA/DNp73α表达质粒与△Np73小干扰质粒,Western blot检测细胞转染后△Np73和TAp73表达;CCK-8法检测转染质粒后细胞生长抑制率;caspase 3活性分光光度法检测转染质粒并应用阿霉素处理后细胞的凋亡水平。结果:成功构建并转染△Np73小干扰表达质粒,转染效率达60%。△Np73小干扰质粒转染细胞能有效抑制内源性△Np73 mRNA和外源性△Np73蛋白表达,对TAp73无显著抑制作用。经△Np73质粒干扰的细胞增殖活性显著下降,与空白对照组相比差异显著,阿霉素诱导△Np73质粒转染细胞凋亡水平增加,与阴性对照组相比有显著差异。结论:△Np73小干扰RNA表达质粒能显著降低MDA-MB-231细胞中△Np73 mRNA及蛋白的表达,抑制细胞生长增殖,增强其被阿霉素诱导的凋亡水平,表明靶向沉默△Np73可能为未来人乳腺癌的治疗提供了一个新的策略。

Objective：To investigate the effectiveness of human breast cancer cell line MDA-MB-231 proliferation and apopto- sis activity to △Np73 small interfering RNA expression plasmid transfection. Methods： △Np73 small interfering expression plasmids were constructed and transfected into human breast cancer cell line MDA-MB-231. Transfection efficiency was calculated by counting green fluorescent protein expressing cells under fluorescence microscope. △Np73 and TAp73 mRNA expression was measured by real- time PCR. After pcDNA3-HA/DNp73cr expression plasmids were co-transfected with △Np73 small interfering plasmid, Western blot test was used to detect △Np73 and TAp73 expression level. Cell growth inhibition rate was detected by CCK-8 assay. Caspase-3 activi- ty spectrophotometry of A Np73 transfeeted and doxorubiein treated cells was examined to determine its apoptosis level. Results： △Np73 small interfering expression plasmids were successfully constructed and transfeetion efficiency reached 60%. △Np73 siRNA plasmid transfeetion effectively inhibited endogeneous △Np73 mRNA expression and exogenous △Np73 protein expression, showing no significant inhibitory effects on TAp73 mRNA and protein expression. △Np73 siRNA plasmid transfection significantly decreased pro- liferative activity of MDA-MB-231 compared with blank control group. △Np73 siRNA plasmid transfeetion apparently increased doxo- rubicin-induced apoptosis level of MDA-MB-231, with significantly difference from negative control group. Conclusion： △Np73 siRNA expression plasmids can significantly down-regulate △Np73 mRNA and protein expression in MDA-MB-231 cells, inhibit cell growth and proliferation, enhance its level of apoptosis induced by doxorubicin, indicating that targeted silencing △Np73 is likely a hopeful strategy for treatment of breast cancer in the future.