摘要
目的探讨miR-192对人肝癌细胞株HepG2中RB1基因表达的调节作用。方法运用生物信息学方法对miR-192进行靶基因预测并分析其潜在靶基因RB1;将含有miR-192结合位点的RB1mRNA 3′端非编码区(3′UTR)片段和在miR-192结合位点进行突变的RB1 3′UTR突变片段克隆至报告基因载体pMIR-Report luciferase vector,重组质粒分别命名为pMIR-RB1和pMIR-RB1-mut;将重组质粒、Beta-gal内参质粒和microRNA共转染HepG2细胞,双荧光素酶报告基因系统检测各实验组中细胞荧光素酶的表达;SYBR Green荧光定量PCR和Western blot分别在mRNA和蛋白水平检测miR-192对内源性RB1表达的调节作用。结果生物信息学分析筛选出89个miR-192的潜在靶基因,RB1基因是其中之一;测序验证重组质粒pMIR-RB1和pMIR-RB1-mut构建成功;过表达miR-192时,共转染pMIR-RB1质粒的细胞相对荧光素酶活性(4.80±0.36)较相应过表达miR-NC组(7.90±0.91)明显降低(P<0.05),而共转染pMIR-Luc或pMIR-RB1-mut质粒的细胞相对荧光素酶活性[pMIR-Luc:(7.68±1.04);pMIR-RB1-mut:(7.56±0.99)]较相应过表达miR-NC组[pMIR-Luc:(7.86±0.73);pMIR-RB1-mut:(7.82±1.05)]无显著差异;上调miR-192水平显著降低HepG2细胞中内源性RB1mRNA[(0.56±0.10)vs(1.05±0.13)]和蛋白(47%vs 100%)的表达。结论 RB1基因是miR-192的一个靶基因,在HepG2细胞中,miR-192通过直接结合RB1mRNA 3′UTR而负性调控RB1基因表达。
Objective To explore the regulatory effect of miR-192 on the expression of RB1 in human hepatocellular carcinoma cell line HepG2.Methods Bioformatics analysis was used for miR-192 target prediction.Among all the potential targets,RB1 was chosen for further investigation.The fragments of 3′-untranslated region（3′UTR）of RB1 mRNA encompassing a putative miR-192 binding site and the mutant forms were cloned into pMIR-Report luciferase vector.The recombinant plasmids were named as pMIR-RB1 and pMIR-RB1-mut,respectively.Recombinant plasmids,Beta-gal control plasmid and microRNAs were co-transfected into HepG2 cells.The luciferase and β-galactosidase activities of each group were detected by using dual-light luciferase assay.SYBR Green quantitative PCR and Western blot were performed to examine the regulatory effect of miR-192 on the expression of endogenous RB1 mRNA and protein.Results Eighty-nine genes were isolated as potential targets of miR-192 by using bioformatics analysis and one of them was RB1 gene.The recombinant plasmids pMIR-RB1 and pMIR-RB1-mut were confirmed by sequencing.As compared with miR-NC group（7.90±0.91）,overexpression of miR-192 significantly reduced the relative luciferase activity of cells co-transfected with pMIR-RB1 vector（4.80±0.36）.In contrast,the relative luciferase activity of cells co-transfected with pMIR-Luc or pMIR-RB1-mut vector in miR-192 group[pMIR-Luc：（7.68±1.04）;pMIR-RB1-mut：（7.56±0.99）]had no significant changes as compared with that in miR-NC group[pMIR-Luc：（7.86±0.73）;pMIR-RB1-mut：（7.82±1.05）].Overexpression of miR-192 suppressed dramatically the expression of endogenous RB1 at mRNA[（0.56±0.10） vs（1.05±0.13）]and protein levels（47% vs 100%）in HepG2 cells.Conclusion RB1 gene was a target of miR-192,and miR-192 negatively regulated the expression of RB1 gene by directly binding to RB1 mRNA 3′ UTR in HepG2 cells.
出处
《华中科技大学学报（医学版）》
CAS
CSCD
北大核心
2011年第6期656-661,共6页
Acta Medicinae Universitatis Scientiae et Technologiae Huazhong
基金
国家自然科学基金资助项目(No.30872237
No.81101824)
