【摘要】 目的:通过过表达手段上调细胞周期调节蛋白依赖性激酶2-关联蛋白1(CDK2-AP1)基因在乳腺癌细胞MCF-7中的表达,并观察其对MCF-7细胞生长和细胞周期调控的作用。方法:将CDK2-AP1基因的编码框构建于慢病毒表达载体,导入MCF-7细胞,应用实时定量PCR和Western印迹验证CDK2-AP1基因mRNA和蛋白的表达效率。利用MTT法绘制生长曲线、克隆形成实验观察CDK2-AP1基因过表达后MCF-7细胞生长的变化,PI染色流式细胞仪检测MCF-7细胞周期的改变。通过Western印迹检测CDK2-AP1过表达后,细胞周期相关蛋白(CDK2,CDK4,P16Ink4A,P21Cip1/Waf1)的表达。结果:过表达CDK2-AP1基因的慢病毒感染MCF-7细胞可上调其mRNA表达6.94倍,蛋白表达也十分显著地增高,两者相一致。生长曲线显示MCF-7细胞过表达CDK2-AP1基因后,增殖能力显著降低(P<0.05);克隆形成实验表明,其形成的克隆数目同样显著减少(P<0.05);流式细胞仪检测证实MCF-7细胞过表达CDK2-AP1能够使细胞周期出现G1期阻滞,并且出现凋亡峰;CDK2-AP1基因表达上调导致P21Cip1/Waf1和P16Ink4A蛋白表达上调,CDK2和CDK4蛋白表达下调。结论:CDK2-AP1基因具有抑癌基因的功能,在乳腺癌MCF-7细胞过表达该基因能够抑制细胞的生长和克隆形成能力,并且使细胞阻滞于G1期。更多还原

【Abstract】 Objective: To over-express cyclin-dependent kinase 2-associated protein 1(CDK2-AP1) gene,and investigate its effect on the proliferation and cell cycle regulation in breast cancer cell line MCF-7. Methods: CDK2-AP1 gene coding region was cloned into lentivirus vector.Lentivirus particles were infected into MCF-7 cells to upregulate the expression of CDK2-AP1 gene.The expression level of CDK2-AP1 was detected at both mRNA and protein levels by real-time PCR and Western blot.MTT assay,colony formatting assay,and flow cytometry were performed to detect the change of proliferation and cell cycle in MCF-7 cells.We examined the expression of cell cycle associated genes(CDK2,CDK4,P16Ink4A,and P21Cip1/Waf1) followed by CDK2-AP1 over-expression by Western blot. Results: CDK2-AP1 gene was up-regulated significantly at both mRNA(6.94 folds) and protein level.MTT based growth curve,colony formatting assay and flow cytometry showed that CDK2-AP1 over-expression lentivirus inhibited the proliferation of MCF-7 cells with statistical difference(P<0.05).In addition,with CDK2-AP1 over-expression,MCF-7 cells were arrested in G1 phase accompanied by apoptosis.Western blot showed that the expression level of P21Cip1/Waf1 and P16 Ink4A was upregulated,while the expression level of CDK2 and CDK4,members of the CDK family,was downregulated. Conclusion: CDK2-AP1 gene plays a cancer suppressor role in breast cancer.Its function includes inhibiting the proliferation of MCF-7 cells and arresting the cell cycle in G1 phase.更多还原