miR-15a和miR-16通过调控bcl-2蛋白的表达逆转结肠癌细胞的耐药性  

miR-15a and miR-16 modulate drug resistance by targeting bcl-2 in human colon cancer cells

作　　者：徐岷[1] 周冰洁[1] 王国英[1] 王国乙 翁星月 蔡菁[1] 李萍[1] 陈晖[1] 蒋小猛[1] 张尤历[1] 

机构地区：[1]江苏大学附属医院消化内科,镇江212001

出　　处：《中华肿瘤杂志》2014年第12期897-902,共6页Chinese Journal of Oncology

摘　　要：目的研究miR-15a和miR-16靶向调控bcl-2的表达对结肠癌细胞耐药性的逆转作用。方法将miR-15a和miR-16模拟物或抑制物转染结肠癌长春新碱（VCR）敏感细胞株HCT8或耐药细胞株HCT8／VCR，采用实时荧光定量PCR法检测miR-15a和miR-16的表达，Westernblot法检测bcl-2和P糖蛋白（P—gP）蛋白的表达，细胞计数盒8（CCK8）法检测不同浓度VCR对HCT8和HCTS／VCR细胞生长的抑制作用。结果实时荧光定量PCR法检测结果显示，空白对照组、阴性对照组和转染miR-15a模拟物组HCT8／VCR细胞中miR-15a的相对表达量分别为1．00、0．87±0．24和223．44±59．07，转染模拟物后，miR-15a的相对表达量明显升高（P〈0．001）；空白对照组、阴性对照组和转染miR-16模拟物组HCT8／VCR细胞中miR-16的相对表达量分别为1．00、0．66±0．19和107．32±22．58，转染模拟物后，miR-16的相对表达量明显升高（P〈0．001）。Western blot法检测结果显示，空白对照组、阴性对照组、转染miR-15a模拟物组和转染miR-16模拟物组HCT8／VCR细胞中bcl-2蛋白的相对表达量分别为1．00、0．97±0．02、0．51±0．06和0．65±0．03，转染模拟物后，bcl-2蛋白的表达下调（P〈0．05）。而P-gp蛋白的表达在转染模拟物后没有明显变化。CCK8法检测结果显示，经1、5、25和125斗g／mlVCR处理后，空白对照组、阴性对照组、转染miR-15a模拟物组和转染miR-16模拟物组HCTS／VCR细胞存活率的变化趋势基本-致，转染模拟物后，HCTS／VCR细胞的存活率明显降低（P〈0．05）。实时荧光定量PCR法检测结果显示，转染抑制物后，HCT8细胞中miR．15a和miR．16的表达明显降低（P〈0．001）。Western blot法检测结果显示，转染抑制物后，HCT8细胞bcl-2蛋白的表达明显上调（P〈0．05），而P-gP蛋白的表达没有明显变化。CCK8法检测结果显示，转染抑制物后，HCT8细胞的存活率明显高于空白对照�Objective To investigate the reversal effect of targeted modulation of bcl-2 expression by miR-15a and miR-16 on drug resistance of human colon cancer cells. Methods Mimics or inhibitors of miR-15a and miR-16 were transfected into HCT8 or HCTS/VCR cells with the help of Lipofectamine 2000. The expressions of miR-15a and miR-16 mRNA were detected by RT-qPCR. The levels of bcl-2 and P-gp proteins were measured by Western blot. The inhibitory effects of VCR on growth of HCT8 and HCTS/VCR cells were detected by CCKS. Results After transfeetion of the mimics, the expression of miR-15a in the blank control group, negative control group and miR-15a mimic group was 1.00, 0.87 ±0.24, and 223.44 ± 59.07, respectively, and miR-15a was increased significantly （P 〈0. 001 ）. The expression of miR-16 in the blank control group, negative control group and miR-16 mimic group was 1.00, 0. 66 ±0. 19, and 107.32 ± 22. 58, respectively, and miR-16 expression was increased significantly （ P 〈 0. 001 ）. The Western blot assay showed that the relative expressions of bcl-2 protein in the blank control group, negative control group, miR-15a mimic group and miR-16 mimic group were 1.00, 0.97 ±0.02, 0.51±0.06, and 0.65 ±0.03, respectively, and the expression of bcl-2 protein was decreased significantly （ P 〈 0.05 ） , however, the expressions of P-gp protein showed no significant difference. The CCK8 test showed that at 1, 5, 25 and 125 μg/ml concentration of VCR, the survival rates of HCT8/VCR cells were basically the same in the blank control group, negative control group, miR-15a mimic group and miR-16 mimic group, but the survival rate of HCT8/VCR ceils was significantly decreased after transfection of mimics （ P 〈 0.05 ）. After transfection of the inhibitors, the expressions of both miR-15a and miR-16 were decreased significantly （P 〈 0.001 ）. The Western blot showed that the expression of bcl-2 protein was increased （P 〈 0.05）, while the expression of P-gp protein showed no significant differ

关 键 词：结肠肿瘤 肿瘤耐药性 miR-15a miR-16 BCL-2 

分 类 号：R735.35[医药卫生—肿瘤]