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  

Regulatory effect of microRNA-3175 on homeobox B1 gene expression in human glioma cell line

作　　者：马可[1] 周海霞[2] 田男[3] 田宇[4] 韩亮[4] 

机构地区：[1]吉林大学第一医院儿科急诊,长春130021 [2]吉林大学中日联谊医院VIP病房 [3]浙江中医药大学生命科学学院 [4]吉林大学中日联谊医院神经外科

出　　处：《中华实验外科杂志》2015年第9期2064-2067,共4页Chinese Journal of Experimental Surgery

基　　金：教育部“新世纪优秀人才支持计划”资助项目（NCET-06-03D6）;吉林省科技发展计划国际科技合作项目（20130413028GH）

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.

关 键 词：胶质瘤 同源异形盒基因家族成员B1 微小RNA-3175 

分 类 号：R780.2[医药卫生—口腔医学]