慢病毒介导的细胞分裂周期相关蛋白2基因沉默对乳腺癌细胞增殖和凋亡的影响  

Effect of CDCA2 gene inhibition by lentivirus-mediated small interfering RNA on proliferation and apoptosis of breast cancer cells

作　　者：矫璐宇 秦春新 杨小青[1] 于浩[1] 王子良[1] JIAO Luyu;QIN Chunxin;YANG Xiaoqing;YU Hao;WANG Ziliang(Department of Thyroid and Breast Surgery,Weihai State Hospital,Weihai,Shandong 264200,China)

机构地区：[1]威海市立医院甲状腺乳腺外科,山东威海264200

出　　处：《安徽医药》2021年第10期2052-2056,共5页Anhui Medical and Pharmaceutical Journal

摘　　要：目的探讨应用慢病毒介导小干扰核糖核酸(siRNA)沉默细胞分裂周期相关蛋白2(CDCA2)基因对乳腺癌细胞增殖和凋亡的影响。方法采用实时荧光定量逆转录聚合酶链反应(q RT-PCR)与蛋白质印迹法(Western blotting)法分别检测人正常乳腺上皮细胞株MCF-10A与乳腺癌细胞MCF-7、MDA-MB-231、BT549中CDCA2 mRNA及蛋白表达。构建慢病毒表达载体转染乳腺癌MCF-7细胞,实验分为对照组、Lv-NC组、Lv-siRNA-CDCA2组。采用噻唑蓝(MTT)实验及克隆形成实验检测沉默CDCA2表达对细胞增殖能力的影响;采用流式细胞仪检测沉默CDCA2表达对细胞凋亡能力的影响。Western blotting法检测细胞周期素D1(Cyclin D1)、CDK4、P21、B细胞淋巴瘤-2(Bcl-2)、Bcl-2相关X(Bax)蛋白表达。采用裸鼠移植瘤实验观察LvsiRNA-CDCA2对移植瘤体积及体质量的影响。结果乳腺癌细胞MCF-7、MDA-MB-231、BT549中CDCA2 mRNA[(2.18±0.13)、(1.56±0.09)、(1.83±0.10)比(1.02±0.06)]及蛋白表达水平[(1.13±0.15)、(0.56±0.08)、(0.79±0.11)比(0.23±0.04)]与MCF-10A相比显著升高(P<0.05),乳腺癌MCF-7细胞中CDCA2的表达水平升高最为显著;与Lv-NC组相比,Lv-siRNA-CDCA2组MCF-7细胞增殖活性与克隆形成率显著降低(P<0.05),而细胞凋亡率显著升高(P<0.05),可促进P21、Bax表达(P<0.05),而抑制Cyclin D1、CDK4、Bcl-2表达(P<0.05);与Lv-NC组比较,Lv-siRNA-CDCA2组移植瘤体积和体质量均显著降低(P<0.05)。结论慢病毒介导的CDCA2基因沉默可乳腺癌细胞细胞增殖,诱导细胞凋亡,并抑制肿瘤生长。Objective To investigate the effects of lentiviral-mediated small interfering ribonucleic acid(siRNA)silencing of cell division cycle-associated protein 2(CDCA2)gene on proliferation and apoptosis of breast cancer cells.Methods The expressions of CDCA2 mRNA and protein in human normal mammary epithelial cell line MCF-10 A and breast cancer cells MCF-7,MDA-MB-231 and BT549 were detected by qRT-PCR and Western blotting.The lentiviral expression vector was constructed and transfected into breast cancer MCF-7 cells.The experiment was assigned into Control group,Lv-NC group and Lv-siRNA-CDCA2 group.The effects of silencing CDCA2 expression on cell proliferation were detected by Methyl Thiazolyl Tetrazolium(MTT)assay and colony formation assay.The effect of silencing CDCA2 expression on apoptosis was detected by flow cytometry.The expression of Cyclin D1,CDK4,P21,Bcl-2 and Bax protein was detected by Western blotting.The effect of Lv-siRNA-CDCA2 on the volume and weight of transplanted tumor was observed by nude mice xenograft experiments.Results The expression levels of CDCA2 mRNA[(2.18±0.13),(1.56±0.09),(1.83±0.10)vs.(1.02±0.06)]and protein[(1.13±0.15),(0.56±0.08),(0.79±0.11)vs.(0.23±0.04)]in breast cancer cells MC.F-7,MDA-MB-231 and BT549 were significantly higher than those in MCF-10 A(P<0.05),and the highest level of CDCA2 expression was observed in breast cancer MCF-7 cells.Compared with Lv-NC group,the proliferation activity and colony formation rate of MC.F-7 cells in Lv-siRNA-CDCA2 group were significantly decreased(P<0.05),while the apoptosis rate was significantly increased(P<0.05),P21 and Bax expression were promoted(P<0.05),and Cyclin D1,CDK4 and Bcl-2 expression were inhibited(P<0.05).Compared with the Lv-NC group,the volume and weight of the transplanted tumors in the Lv-siRNA-CDCA2 group were significantly lower(P<0.05).Conclusion Lentiviral-mediated CDCA2 gene silencing inhibits breast cancer cells proliferation,induces apoptosis,and inhibits tumor growth.

关 键 词：乳腺肿瘤 细胞分裂周期相关蛋白2 增殖 凋亡 慢病毒属 

分 类 号：R737.9[医药卫生—肿瘤]