miR-17-5p靶向MICB减弱NK细胞对肝癌细胞的毒性作用  

miR-17-5p targeting MICB attenuates toxic effects of NK cells on hepatocarcinoma cells

作　　者：贾萌[1] 薛焕洲[1] 申权[1] 余淼[1] 贾江坤[1] 王佳佳[1] 徐建 JIA Meng;XUE Huan-Zhou;SHEN Quan;YU Miao;JIA Jiang-Kun;WANG Jia-Jia;XU Jian(Department ofHepatobiliary Surgery,Henan Province People's Hospital,Zhengzhou 450000,China)

机构地区：[1]河南省人民医院肝胆外科,郑州450000

出　　处：《中国免疫学杂志》2018年第12期1776-1782,共7页Chinese Journal of Immunology

基　　金：2016年度河南省医学科技攻关计划项目(201602275)

摘　　要：目的:探讨miR-17-5p在肝细胞癌(HCC)HepG2细胞对NK细胞介导的细胞毒性敏感性中的调控作用。方法:使用含野生型MICB基因3'UTR序列、突变型MICB基因3'UTR序列、miR-17-5p mimic序列、miR-17-5p-NC序列、anti-miR-17-5p序列的质粒载体分别或共转染HCC HepG2细胞,使用丙戊酸钠处理转染或未转染细胞。应用荧光素酶活性测定检测miR-17-5p对MICB的调控作用。应用实时荧光定量PCR或蛋白免疫印迹检测细胞或组织中miR-17-5p及MICB mRNA和蛋白表达情况。结果:(1)与miR-17-5p-NC组相比,miR-17-5p-mimic组HepG2细胞中miR-17-5p表达水平显著升高,MICB mRNA和蛋白表达水平均显著降低(P<0. 05);与anti-miR-17-5p-NC组相比,anti-miR-17-5p组HepG2细胞中miR-17-5p表达水平均显著降低,MICB mRNA和蛋白表达水平均显著增加(P<0. 05)。与miR-17-5p-NC+MICB 3'UTR-Wt组相比,miR-17-5p-mimic+MICB 3'-UTR-Wt组荧光素酶活性显著降低(P<0. 05)。miR-17-5p-NC+MICB 3'UTR-Mut组与miR-17-mimic+MICB 3'-UTR-Mut组的荧光素酶活性差异无统计学意义(P>0. 05)。(2)与对应癌旁非肿瘤组织相比,miR-17-5p在HCC组织中的表达水平显著增加,而MICB蛋白表达水平显著降低(P<0. 05)。miR-17-5p和MICB蛋白的表达与肿瘤直径、远处转移、TNM分期、分化程度等临床病理特征有关(P<0. 05)。在HCC肿瘤组织中miR-17-5p与MICB蛋白表达水平呈明显负相关(P<0. 05)。(3)与miR-17-5p-NC组相比,miR-17-5p-mimic组MICB蛋白表达水平均显著降低(P<0. 05)。与miR-17-5p-mimic+MICB 3'UTR-Mut组相比,miR-17-5p-mimic+MICB-3'UTR-Wt共转染组中NK细胞毒性和MICB蛋白表达水平均显著增加(P均<0. 05)。与antimiR-17-5p-NC组相比,anti-miR-17组NK细胞毒性和MICB蛋白表达水平均显著增加(P均<0. 05)。(4)与丙戊酸钠+miR-17-5p-NC组相比,丙戊酸钠+miR-17-5p-mimic组细胞中MICB表达水平显著降低(P<0. 05)。在不同效靶比时,与Ctrl组相比,丙戊酸钠组NK细胞毒性显著增加(P<0. 05)。与丙戊酸钠+miR-17-5Objective:To investigate the regulatory effect of miR-17-5p on NK cell-mediated cytotoxicity in HCC cell line HepG2cells.Methods:A plasmid vector containing the wild type MICB gene3'UTR sequence,the mutant MICB gene3'UTR sequence,the miR-17-5p mimic sequence,the miR-17-5p-NC sequence,the anti-miR-17-5p sequence and the anti-miR-17-5p-NC sequence respectively,or co-transfected HCC cell HepG2.Treatment of transfected or untransfected cells with sodium valproate.The inhibitory effect of miR-17-5p on MICB was detected by luciferase activity assay.The mRNA expression of miR-17-5p and MICB was detected by qPT-PCR,meantime,the protein expression of MICB determined by Western blot was performed.Results:①Compared with miR-17-5p-NC group,miR-17-5p-mimic group miR-17-5p expression level was significantly increased,MICB mRNA and protein expression levels were significantly reduced(P<0.05).Compared with anti-miR-17-5p-NC group,the expression of miR-17-5p in anti-miR-17-5p group was significantly decreased,and the expression of MICB mRNA and protein was significantly increased(P<0.05).The luciferase activity of miR-17-5p-mimic+MICB3'-UTR-Wt group was significantly lower than that of miR-17-5p-NC+MICB3'UTR-Wt group(P<0.05).There was no significant difference in the luciferase activity between miR-17-5p-NC+MICB3'UTR-Mut group and miR-17-mimic+MICB3'-UTR-Mut group(P>0.05).②The expression level of miR-17-5p in HCC tissues was significantly higherthan that in the adjacent non-tumor tissues,and the expression level of MICB protein was significantly decreased(P<0.05).The expression of miR-17-5p and MICB protein was correlated with tumor diameter,distant metastasis,TNM stage,differentiation and other clinicopathological features(P<0.05).There was a significant negative correlation between miR-17-5p and MICB protein expression in HCC tumor tissues(P<0.05).③Compared with miR-17-5p-NC group,NK cell toxicity and MICB protein expression in miR-17-5p-mimic group were significantly decreased(P<0.05).Compared with miR-17-5p-mimic+MICB3'

关 键 词：肝细胞癌 HEPG2细胞 自然杀伤细胞 miR-17-5p 主要组织相容性复合物Ⅰ类链相关基因B 

分 类 号：R735.7[医药卫生—肿瘤]