上调miR-181b表达对乳腺癌细胞功能的影响  

The effect of increasing the expression of miR-181b on the function of human breast cancer cells

作　　者：李晓宇[1] 罗颀枫[1] 魏传奎 李登峰[1] 房林[1] 

机构地区：[1]同济大学附属第十人民医院甲状腺乳腺外科,上海200072

出　　处：《肿瘤》2014年第3期197-203,共7页Tumor

基　　金：国家自然科学基金资助项目(编号:81272240)

摘　　要：目的 :探讨上调miR-181b的表达对人乳腺癌MDA-MB-231和MCF-7细胞功能的影响及其可能的靶向基因。方法 :应用脂质体介导的方法将miR-181b模拟物(miR-181b mimics)分别转染MDA-MB-231和MCF-7细胞后,通过MTT、Transwell和FCM法分别检测细胞增殖和迁移能力及细胞周期的变化。将含细胞周期蛋白依赖性激酶8(cyclin-dependent kinase 8,CDK8)基因3’端非翻译区(3’-untranslated region,3’-UTR)的重组载体psiCHECK-2/CDK8-3’UTR和miR-181b mimics共转染入人胚肾HEK293T细胞,应用双荧光素酶报告系统验证CDK8基因3’-UTR是否有miR-181b的结合位点。蛋白质印迹法检测miR-181b mimics转染后MDAMB-231和MCF-7细胞中CDK8蛋白的表达情况。结果 :与转染miR-181b模拟物阴性对照(negative control,NC)(miR-181b mimics-NC)组比较,miR-181b mimics转染组MDA-MB-231和MCF-7细胞的增殖和迁移能力受到抑制(P<0.05),细胞周期主要阻滞于G0/G1期(P<0.01);psiCHECK-2/CDK8-3’UTR和miR-181b mimics共转染组HEK293T细胞的荧光素酶活性下降(P<0.01),证实miR-181b与CDK 8基因3’-UTR可特异性靶向结合。与转染miR-181b mimics-NC组比较,miR-181b mimics转染组MDA-MB-231和MCF-7细胞中CDK8蛋白的表达水平明显下调(P<0.05)。结论 :上调miR-181b的表达可显著抑制人乳腺癌细胞的增殖和迁移,这种作用可能与miR-181b靶向调节CDK8的表达有关。Objective： To investigate the effects of increasing the expression of miR-181b on the function of human breast cancer cell lines MDA-MB-231 and MCF-7, and to explore its possible target gene. Methods： miR-181b mimics were transfected into MDA-MB-231 and MCF-7 cells by LipofectAMiNE 2000 and then the capabilities of proliferation and migration as well as the cell cycle distribution of MDA- MB-231 and MCF-7 cells were evaluated by MTT, Transwell and flow cytometry methods, respectively. Recombinant vector psiCHECK-2/cyclin-dependent kinase 8 （CDK8）-3＇-untranslated region （3＇-UTR） and miR-181b mimics were co-transfected into human embryonic kidney HEK293T cells and then the targeting of miR-181b combined with 3＇-UTR of CDK8 gene was detected by dual luciferase report system. The expression of CDK8 in MDA-MB-231 and MCF-7 cells after transfection with miR-181b mimics was determined by Western blotting. Results： As compared with the miR-181b mimics-negative control （NC） tranfection group, the capabilities of proliferation and migration in MDA-MB-231 and MCF-7 cells after transfection with miR-181b mimics were significantly inhibited （P 〈 0.05）, and the cell cycle was arrested at Go/G1 phase （P 〈 0.01）. The luciferase activity of HEK293T cells after co-transfection with psiCHECK-2/CDK8-3＇UTR and miR-181b mimics was reduced （P 〈 0.01）, and it confirmed that miR- 181b specifically targeted with CDK8 3＇-UTR. As compared with the miR-181b mimics-NC transfection group, the expression level of CDK8 in MDA-MB-231 and MCF-7 cells after transfection with miR-181b mimics was down-regulated （P 〈 0.05）. Conclusion： The up-regulation of miR-181 b expression can inhibit the proliferation and migration of human breast cancer cells. This effect may be related to miR-181b targeting of the expression of CDK8.

关 键 词：乳腺肿瘤 微RNAS 细胞增殖 细胞周期蛋白依赖性激酶8 miR-181b 细胞 MDA—MB一231 MCF-7 

分 类 号：R737.9[医药卫生—肿瘤]