人胶质瘤细胞中微小RNA-3175对同源异形盒基因家族成员B1基因表达的调控作用

摘要

目的 观察在人胶质瘤细胞中微小RNA(miRNA,miR)-3175通过3'端非编码区(3'-UTR)对同源异形盒基因家族成员B1(HOXB1)表达的调控作用.方法 通过生物信息学可见miR-3175与HOXB1基因3'-UTR相互配对,构建HOXB1基因3'-UTR野生型和变异型荧光素酶载体,生物合成miR-3175 mimics、miR-3175 inhibitor和各自阴性对照.采用Lipofectamine 2000转染和双荧光素酶报告基因系统检测人胶质瘤细胞株(A172、U87)中miR-3175对HOXB1基因3'-UTR的调控作用.寡核苷酸转染24、48、72 h后,通过实时定量反转录聚合酶链反应(RT-qPCR)和Western blot分析miR-3175在人胶质瘤细胞株(A172、U251、U87)对HOXB1基因的表达调控.结果 双荧光素酶报告基因系统提示共转染HOXB1基因野生型载体和miR-3175 mimics后荧光素酶活性明显下降(P＜0.05),A172细胞株荧光素酶活性下降(37.38±10.98)％,U87细胞株荧光索酶活性下降(24.62±3.22)％.与对照组比较miR-3175 mimics可下调HOXB1基因的表达,转染72h后HOXB1 mRNA表达水平在A172细胞株中下降(42.57±5.52)％,U251细胞株中下降(71.06±0.41)％,U87细胞株中下降(55.54±7.57)％;miR-3175 inhibitor可上调HOXB1基因的表达,转染72 h后HOXB1 mRNA表达水平在A172细胞株中升高(92.39±31.88)％,U251细胞株中升高(250.25±60.37)％,U87细胞株中升高(128.44±25.11)％.转染后HOXB1蛋白水平的变化也得出了相类似的结果.结论 HOXB1基因3'-UTR为miR-3175调控直接靶点,并且miR-3175 mimics为靶向负调控,miR-3175 inhibitor为靶向正调控.%Objective To explore the regulation of homeobox B1 (HOXB1) gene by microRNA (miRNA,miR)-3175 targeting 3' untranslated region (3'-UTR) in the human glioma cell line.Methods Bioinformatics analysis was applied to predict the HOXB1 gene 3'-UTR targeted miRNA.The wild-type and mutation-type of HOXB1 gene 3'-UTR luciferase vectors were constructed.MiR-3175 mimics,miR-3175 inhibitor and their negative controls were bio-synthetized.Lipofectamine 2000 transfection and the Dual Luciferase Reporter Gene System were applied to detect the effect of miR-3175 on the HOXB1 gene 3'-UTR in the human glioma cell lines (A172 and U87).Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) and Western blotting were performed to analyze the regulation of HOXB1 gene expression by miR-3175 in the human glioma cell lines (A172,U251 and U87) after oligonucleotide transfection in 24,48,and 72 h.Results The luciferase assay revealed that miR-3175 mimics could significantly down-regulate the luciferase activity of the wild-type HOXB1 gene vector.The luciferase activity of A172 cell line was declined by (37.38 ± 10.98)％ and that of U87 cell line by (24.62 ± 3.22)％ respectively.MiR-3175 mimics could decrease the expression of HOXB1 gene.After transfection over 72 h,the HOXB1 mRNA expression levels were decreased by (42.57 ± 5.52) ％ in A172 cell line,(71.06 ± 0.41) ％ in U251 cell line,and (55.54 ± 7.57) ％ in U87 cell line.MiR-3175 inhibitor increased the expression of HOXB1 gene.After transfection over 72 h,HOXB1 mRNA expression levels were elevated by (92.39 ±31.88)％ in A172 cell line,(250.25 ±60.37)％ in U251 cell line,and (128.44 ± 25.11)％ in U87 cell line.The similar results were obtained about the change of HOXB1 protein levels.Conclusion HOXB1 gene 3'-UTR is a direct target of rniR-3175.MiR-3175 mimics is negative regulation for target,and miR-3175 inhibitor is positive regulation.