人胶质瘤细胞中微小RNA-148b-3p对长链非编码RNA HOX转录反义RNA表达的调控作用

Regulatory effect of microRNA-148b-3p on long non-coding RNA HOX transcript antisense RNA gene expression in human glioma cell line

目的观察在人胶质瘤细胞中微小RNA(miRNA,miR)-148b-3p对长链非编码RNA HOX转录反义RNA(HOTAIR)表达的调控作用。方法通过生物信息学可见miR-148b-3p与HOTAIR基因相互配对。生物合成miR-148b-3p mimics、miR-148b-3p inhibitor和阴性对照(NC),采用Lipofectamine 2000转染至胶质瘤细胞A172,通过实时定量反转录聚合酶链反应(RT-qPCR)分析miR-148b-3p在人胶质瘤细胞A172中对HOTAIR表达的调控作用。构建HOTAIR基因野生型(WT)和变异型(MUT)荧光素酶载体,利用双荧光素酶报告基因系统检测miR-148b-3p对HOTAIR调控的靶向性。结果RT-qPCR结果显示,NC组HOTAIR mRNA的表达量为1.06±0.15,miR-148b-3p mimics转染A172细胞24、48、72 h后,HOTAIR mRNA的表达量分别为0.79±0.12、0.63±0.18和0.39±0.06。miR-148b-3p inhibitor转染A172细胞24、48、72 h后,HOTAIR mRNA的表达量分别为1.24±0.09、2.70±0.24和3.56±0.18。与NC组比较,miR-148b-3p mimics下调HOTAIR基因的表达(t=2.824、3.102、3.648,P<0.05),miR-148b-3p inhibitor上调HOTAIR基因的表达(t=2.742、3.761、3.928,P<0.05)。双荧光素酶报告基因系统显示,胶质瘤A172细胞中,共转染NC+pmirGLO-WT-HOTAIR、NC+pmirGLO-MUT-HOTAIR、miR-148b-3p mimics+pmirGLO-WT-HOTAIR和miR-148b-3p mimics+pmirGLO-MUT-HOTAIR的荧光素酶活性分别为1.18±0.03、1.23±0.09、0.64±0.02和1.12±0.01。共转染miR-148b-3p mimics+pmirGLO-MUT-HOTAIR的A172细胞与共转染NC和重组载体的A172细胞之间荧光素酶活性差异无统计学意义(t=0.459、0.427,P>0.05)。而共转染miR-148b-3p mimics+pmirGLO-WT-HOTAIR的A172细胞与共转染miR-148b-3p mimics+pmirGLO-MUT-HOTAIR的A172细胞比较,荧光素酶活性明显受到抑制(t=3.362,P<0.05)。结论在胶质瘤细胞中,miR-148b-3p对HOTAIR基因具有直接靶向调控作用,HOTAIR为miR-148b-3p的靶基因。

Objective To investigate the regulation of HOX transcript antisense RNA(HOTAIR)gene by microRNA(miRNA,miR)in human glioma cell line.Methods Bioinformatics analysis was applied to predict the HOTAIR targeted miRNA.MiR-148b-3p mimics,miR-148b-3p inhibitor and their negative controls were bio-synthetized.Real-time quantitative reverse transcriptase-polymerase chain reaction(RT-qPCR)was performed to analyze the regulation of HOTAIR gene expression by miR-148b-3p in human glioma cell line A172 after transferred with miR-148b-3p mimics.Lipofectamine 2000 transfecton and the Dual Luciferase Reporter Gene System were applied to detect the effect of miR-148b-3p on the HOTAIR gene in A172 cell.Results Results of RT-qPCR found that the expression level of HOTAIR in negative control group was 1.06±0.15.24,48 and 72 h after miR-148b-3p mimics transfection,the expression level of HOTAIR mRNA was 0.79±0.12,0.63±0.18 and 0.39±0.06 respectively.24,48 and 72 h after miR-148b-3p inhibitor transfection,the expression level of HOTAIR mRNA was 1.24±0.09,2.70±0.24 and 3.56±0.18 respectively.Compared with the negative control group,miR-148b-3p mimics could decrease the expression of HOTAIR gene(t=2.824,3.102,3.648,P<0.05),while miR-148b-3p inhibitor could increase the expression of HOTAIR gene(t=2.742,3.761,3.928,P<0.05).The Dural-Luciferase assay shows that the luciferase activity of A172 cells co-transfected with NC+pmirGLO-WT-HOTAIR,NC+pmirGLO-MUT-HOTAIR,miR-148b-3p mimics+pmirGLO-WT-HOTAIR and miR-148b-3p mimics+pmirGLO-MUT-HOTAIR was 1.18±0.03,1.23±0.09,0.64±0.02and 1.12±0.01 respectively.There were no significant differences between the luciferase activity of A172 cells co-transfected with NC+pmirGLO-WT-HOTAIR,NC+pmirGLO-MUT-HOTAIR,and miR-148b-3p mimics+pmirGLO-MUT-HOTAIR(t=0.459,0.427,P>0.05);however,the luciferase activity of A172 cells co-transfected with mimics+pmirGLO-WT-HOTAIR was significantly decreased compared with A172 cells co-transfected with miR-148b-3p mimics+pmirGLO-MUT-HOTAIR(t=3.362,P<0.05).Conclusion MiR-148b-3p play a regulatory role of HOTAIR expression by directly targeted combination in human glioma cell lines.HOTAIR is a direct target of miR-148b-3p.