干扰ANXA3基因表达对乳腺癌细胞增殖和凋亡的影响
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摘要
[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.
[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.
引文
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[2]Suojalehto H,Lindstrom I,Wolff H,et al.Nasal protein profiles in work-related asthma caused by different exposures[J].Allergy,2018,73(3):653-663.
[3]Wang K,Li J.Overexpression of ANXA3 is an independent prognostic indicator in gastric cancer and its depletion suppresses cell proliferation and tumor growth[J].Oncotarget,2016,7(52):86972-86984.
[4]Yu J,Li X,Zhong C,et al.High-throughput proteomics integrated with gene microarray for discovery of colorectal cancer potential biomarkers[J].Oncotarget,2016,7(46):75279-75292.
[5]Mege D,Crescence L,Ouaissi M,et al.Fibrin-bearing microparticles:marker of thrombo-embolic events in pancreatic and colorectal cancers[J].Oncotarget,2017,8(57):97394-97406.
[6]Hamelin-Peyron C,Vlaeminck-Guillem V,Haidous H,et al.Prostate cancer biomarker annexin A3 detected in urines obtained following digital rectal examination presents antigenic variability[J].Clinical biochemistry,2014,47(10):901-908.
[7]Li Y,Wu ZR.The relationship between annexin A3 and the evolution of malignant tumor[J].Medical Review,2014,20(14):2537-2539.[李亚,吴正蓉.膜联蛋白A3与恶性肿瘤演进的关系[J].医学综述,2014,20(14):2537-2539.]
[8]Zhou J,Zhang Z,Song L,et al.Effects of resveratrol on the apoptosis factor Bcl-2/Bax and mitochondrial transmembrane potential after A beta 1-42 stimulation HU-VECs[J].Basic Medicine and Clinic,2016,36(8):1108-1112.[周洁,章卓,宋丽,等.白藜芦醇对Aβ1-42刺激HUVECs后凋亡因子Bcl-2/Bax及线粒体跨膜电位的影响[J].基础医学与临床,2016,36(8):1108-1112.]
[9]Gahl RF,Dwivedi P,Tjandra N.Bcl-2 proteins bid and bax form a network to permeabilize the mitochondria at the onset of apoptosis[J].Cell Death Dis,2016,7(10):e2424.
[10]Gao M,Li Y,Ji X,et al.Disturbance of Bcl-2,Bax,Caspase-3,Ki-67 and C-myc expression in acute and subchronic exposure to benzo(a)pyrene in cervix[J].Acta histochemica,2016,118(2):63-73.
[11]Zeng J,Chen S,Li N,et al.Sasanquasaponin from Camellia oleifera Abel.induces apoptosis via Bcl-2,Bax and caspase-3 activation in HepG2 cells[J].Mol Med Rep,2015,12(2):1997-2002.
[12]Liu X,He Y,Li F,et al.Caspase-3 promotes genetic instability and carcinogenesis[J].Mol Cell,2015,58(2):284-296.