干扰ANXA3基因表达对乳腺癌细胞增殖和凋亡的影响

Effect of Interfering ANXA3 Gene Expression on Proliferation and Apoptosis of Breast Cancer Cells

[目的]探究干扰膜联蛋白A3(ANXA3)基因表达对乳腺癌细胞增殖、凋亡的影响以及可能作用机制。[方法]以乳腺癌MCF-7细胞为研究对象,RNA干扰技术沉默ANXA3基因表达,荧光定量RT-PCR和Western Blot检测转染后细胞中ANXA3 m RNA及蛋白表达;四甲基偶氮唑(MTT)法检测细胞的增殖能力变化,膜联蛋白V-FITC(Annexin V-FITC)/碘化丙啶(PI)双染法检测细胞的凋亡率,蛋白质印迹法(Western Blot)检测细胞中B细胞淋巴瘤/白血病-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、活化的含半胱氨酸的天冬氨酸蛋白水解酶3(Cleaved Caspase-3)蛋白的水平。[结果]空白对照组、阴性对照组和ANXA3干扰组细胞中ANXA3 m RNA的表达量分别为0.078±0.006、0.072±0.007和0.008±0.001;ANXA3蛋白表达量分别为0.792±0.085、0.764±0.078和0.353±0.039;细胞的凋亡率分别为(3.425±0.643)%、(3.537±0.740)%、(23.455±3.686)%;Bcl-2蛋白水平分别为1.256±0.247、1.239±0.265、0.624±0.065;Bax蛋白水平分别为0.856±0.086、0.842±0.079、1.526±0.344;Cleaved Caspase-3蛋白水平分别为0.522±0.057、0.535±0.062、1.230±0.367。与空白对照组相比,ANXA3干扰组细胞中ANXA3 mRNA和蛋白水平显著性降低(P<0.05),细胞的增殖能力显著性降低(P<0.05),其凋亡率显著性增加(P<0.05);细胞中Bcl-2蛋白水平显著性下调(P<0.05),Bax、Cleaved Caspase-3蛋白表达量显著性上调(P<0.05)。[结论]干扰ANXA3表达抑制乳腺癌细胞增殖,诱导其凋亡,其作用与Bcl-2、Bax、Cleaved Caspase-3蛋白水平变化引起的细胞凋亡通路的变化有关。

[Objective]To investigate the effect of interfering Annexin A3(ANXA3)gene expression on proliferation and apoptosis of breast cancer cells and its mechanism.[Methods]The ANXA3 gene expression in breast cancer MCF-7 cells was silenced by RNA interference technique.The expressions of ANXA3 mRNA and protein were detected by RT-PCR and Western Blot,respectively.Tetramethylzole(MTT)method was used to examine the cell proliferation and the apoptotic rate was determined by Annexin V-FITC/PI staining method.The expression of B cell lymphoma/lewkmia-2(Bcl-2),Bcl-2-related protein X(Bax),cleaved cysteinyl aspartate specific proteinase 3(cleaved Caspase-3)in MCF-7 cells were detected by Western blot.[Results]The expression levels of ANXA3 m RNA were 0.078±0.006,0.072±0.007,0.008±0.001 in blank control group,negative control group and ANXA3 interference group,respectively;and the protein expression levels were 0.792±0.085,0.764±0.078 and 0.353±0.039.The apoptosis rates were(3.425±0.643)%,(3.537±0.740)%,(23.455±3.686)%,respectively,the protein expression levels of Bcl-2 were 1.256±0.247,1.239±0.265,0.624±0.065,respectively;those of Bax were0.856±0.086,0.842±0.079,1.526±0.344,respectively;and those of cleaved Caspase-3 were 0.522±0.057,0.535±0.062,1.230±0.367 in three groups,respectively.Compared with the blank control group,the expression levels of ANXA3 mRNA and protein were significantly decreased(P<0.05),cell proliferation was significantly decreased(P<0.05),and the apoptosis rate was significantly increased(P<0.05).The protein expression levels of Bcl-2 were significantly down-regulated(P<0.05),and the protein expression levels of Bax and cleaved Caspase-3 were significantly up-regulated(P<0.05).[Conclusion]Interference of ANXA3 expression inhibits the proliferation and induces apoptosis of breast cancer MCF-7 cells,which is related to the change in the expression levels of Bcl-2,Bax and cleaved Caspase-3.