miRNA-370对肾癌细胞株ACHN和786-O中抑癌基因p21表达的调控作用及其对细胞生长的影响  

Regulatory effect of miRNA-370 on expression of tumor suppressor gene p21 in renal cell carcinoma cell lines ACHN and 786-O and its effect on cell growth

作　　者：黄耿[1] 姜卫东[1] 毛青[1] 桂定文[1] 

机构地区：[1]鄂东医疗集团黄石市中心医院湖北理工学院附属医院泌尿外科肾脏疾病发生与干预湖北省重点实验室,435000

出　　处：《肿瘤研究与临床》2017年第9期589-592,共4页Cancer Research and Clinic

摘　　要：目的 探讨miRNA-370（miR-370）对肾癌细胞株ACHN和786-O中抑癌基因p21表达的调控作用以及对细胞生长的影响.方法 采用脂质体Lipofectamine 3000将已知的缺少人类基因同源性的dsRNA（对照组）和miR-370（实验组）分别转染ACHN和786-O细胞.采用实时荧光定量聚合酶链反应（RT-qPCR）和蛋白印迹法分别检测p21 mRNA和蛋白的表达变化.流式细胞术鉴定细胞周期分布,采用MTS法和集落培养实验检测细胞活力和增殖能力.结果 在ACHN细胞中,对照组和实验组中p21 mRNA的表达量分别为1.04±0.33、3.68±0.62,两者比较差异有统计学意义（t=7.535,P〈0.001）.在786-O细胞中,对照组和实验组中p21 mRNA的表达量分别为1.04±0.31、3.15±0.29,两者比较差异有统计学意义（t=9.975,P〈0.001）.在两种细胞中,实验组p21 mRNA表达与对照组相比均上调.蛋白印迹法检测结果显示,p21蛋白表达水平增加,与p21 mRNA水平上调一致.ACHN和786-O细胞转染miR-370后,细胞周期被阻滞在G0-G1期.MTS结果显示,在ACHN细胞中,对照组和实验组中细胞形成的集落数分别为（113±30）、（53±17）个,两者比较差异有统计学意义（t=2.982,P=0.041）.在786-O细胞中,对照组和实验组中细胞形成的集落数分别为（106±27）、（50±16）个,两者比较差异有统计学意义（t=3.089,P=0.037）.在两种细胞中,实验组形成的集落数与对照组相比均减少.结论 miR-370可上调肾癌细胞中抑癌基因p21的表达,并抑制肾癌细胞的生长.Objective To investigate the regulatory effect of miRNA-370（miR-370）on the expression of tumor suppressor gene p21 in renal cell carcinoma cell lines ACHN and 786-O and its effect on cell growth. Methods RCC cells were transfected with dsRNA known lack homology to human genes （control group） and miR-370 （experimental group） by Lipofectamine 3000 respectively. Real-time fluorescence quantitative polynucleotide chain reaction （RT-qPCR） and Western blot were used to detect the expression of p21 mRNA and protein. The cell cycle distribution was identified by flow cytometry （FCM）. Cell viability and proliferation ability were measured by cell viability assay （MTS） and colony culture assay. Results The expression of p21 mRNA in ACTN and 786-O cells in control group was 1.04±0.33, 1.04±0.31, respectively. The expression of p21 mRNA in experimental group was significantly increased by 3.68±0.62 （t=7.535, P〈0.001）, 3.15±0.29 （t=9.975, P〈0.001）. Western blot further demonstrated that the increased expression of p21 protein in both renal cell lines was consistent with the upregulation of p21 mRNA level. FCM results showed that the cell cycle of more cells was blocked in G0-G1phase after transfection of miR-370.MTS results showed that after transfection of miR-370,the number of colonies formed by ACHN and 786-O cells in the control group was 113±30 and 106±27 respectively. The number of colonies formed by experimental group was significantly reduced by 53±17 （t=2.982, P=0.041） and 50±16 （t=3.089, P=0.037）. Conclusion miR-370 can significantly up-regulate the expression of tumor suppressor gene p21 in renal cell carcinoma and inhibit the growth of renal cell carcinoma.

关 键 词：肾肿瘤 微RNAS 癌基因蛋白质p21(ras) 

分 类 号：R737.11[医药卫生—肿瘤]