摘要
目的:探讨miR-17-5p在HCC细胞系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细胞系HepG2,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检测细胞或组织中miR-17-5p及MICB mRNA表达情况,利用蛋白免疫印迹法检测MICB蛋白表达情况。对比转染质粒后各组细胞中miR-17-5p和MICB mRNA及蛋白的表达情况,分析Target Scan预测和荧光素酶活性测定结果、miR-17-5p与MICB蛋白在HCC组织中的表达及与临床病理特征的关系、miR-17-5p与MICB蛋白在HCC组织中表达的相关性,并对比各组MICB蛋白表达和NK细胞介导的细胞毒性、丙戊酸钠各组NK细胞毒性。结果:①与miR-17-5p-NC组相比,miR-17-5p-mimic组HepG2细胞中miR-17-5p表达水平较高,MICB mRNA和蛋白表达水平均较低(P <0. 05);与anti-miR-17-5p-NC组相比,anti-miR-17-5p组HepG2细胞中miR-17-5p表达水平均较低,MICB mRNA和蛋白表达水平均较高(P <0. 05);与miR-17-5p-NC+MICB 3'UTR-Wt组相比,miR-17-5p-mimic+MICB 3'-UTR-Wt组荧光素酶活性较低(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组织中的表达水平较高,而MICB蛋白表达水平较低(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NK细胞毒性、MICB蛋白表达水平均较低(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蛋白表达水平均较高(P <0. 05);与anti-miR-17-5p-NC组相比,anti-miR-17-5p组NK细胞毒性和MIBC蛋白表达均较高(P <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表达水平较低(P <0. 05)。在不同效靶比时,与Ctrl组相比,丙戊酸钠组NK细胞毒性较高(P <0. 05)。与丙戊酸钠+miR-17-5p-NC组相比,丙戊酸钠+miR-17-5p-mimic组NK细胞毒性较低(P <0. 05)。结论:miR-17-5p通过靶向MICB降低Hep G2细胞对NK细胞介导的细胞毒性敏感性,从而抑制NK细胞对肝癌细胞的毒性。
Objective: To investigate the regulatory effect of miR-17-5 p on NK cell-mediated cytotoxicity in HCC cell line HepG2 cells. Methods: A plasmid vector containing the wild type MICB gene 3' UTR sequence,the mutant MICB gene 3' UTR sequence,the miR-17-5p mimic sequence,the miR-17-5p-NC sequence,the anti-miR-17-5 p sequence and the anti-miR-17-5 p-NC sequence respectively,or co-transfected HCC cell line HepG2. Treatment of transfected or untransfected cells with sodium valproate. Real time fluorescence quantitative PCR was used to detect the expressions of miR-17-5p and MICB mRNA in cells or tissues. The expression of MICB protein was detected by Western blot. The expressions of miR-17-5p,MICB mRNA and protein in each group after transfection were compared. The results of Target Scan prediction and luciferase activity assay were analyzed,as well as the expressions of miR-17-5p and MICB protein in HCC tissues and their relationships with clinicopathological characteristics,the correlation between the expression of miR-17-5 p and MICB protein in HCC tissues. The MICB protein expression and NK cell mediated cytotoxicity in each group were compared,as well as the toxicity of NK cells in each group of valproate. Results: ① Compared with miR-17-5p-NC group,miR-17-5 pmimic group miR-17-5p expression level was higher,MICB mRNA and protein expression levels were lower( P < 0. 05). Compared with anti-miR-17-5p-NC group,the expression of miR-17-5p in anti-miR-17-5 p group was lower,and the expression of MICB mRNA and protein was higher( P < 0. 05). The luciferase activity of miR-17-5 p-mimic + MICB 3'-UTR-Wt group was significantly lower than that of miR-17-5 p-NC + MICB 3'UTR-Wt group( P < 0. 05). There was no significant difference in the luciferase activity between miR-17-5p-NC + MICB 3'UTR-Mut group and miR-17-mimic + MICB 3'-UTR-Mut group( P > 0. 05). ②The expression level of miR-17-5p in HCC tissues was significantly higher than that in the adjacent non-tumor tissues,and the expression level of MICB protein was significantly lower( P < 0. 05). The expression of miR-17-5 p 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-17-5 p and MICB protein expression in HCC tumor tissues( P < 0. 05). ③Compared with group miR-17-5 p-NC,the NK cytotoxicity and the level of MICB protein expression in group miR-17-5 p-mimic were lower( P < 0. 05). Compared with the miR-17-5 p-mimic + MICB 3 'UTR-Mut group,the toxicity of NK cells and the expression of MICB protein in the miR-17-5 p-mimic + MICB-3'UTR-Wt co transfection group were higher( P < 0. 05). Compared with group anti-miR-17-5 p-NC,the NK cytotoxicity and MIBC protein expression in anti-miR-17-5 p group were higher( P < 0. 05). ④Compared with sodium valproate + miR-17-5 p-NC group,the expression level of MICB in valproate + miR-17-5 p-mimic group was lower( P < 0. 05). Compared with the control group,the NK cytotoxicity of sodium valproate was higher at different target ratios( P < 0. 05). Compared with the sodium valproate + miR-17-5 p-NC group,the NK cytotoxicity of sodium valproate + miR-17-5 p-mimic group was significantly lower( P < 0. 05). Conclusion: miR-17-5 p reduces the cytotoxic sensitivity of Hep G2 cells to NK cells by targeting MICB,and thus inhibits the cytotoxicity of NK cells to hepatoma cells.
引文
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