Let-7a负性调控人肾上皮细胞293T中UHRF1基因的表达

MicroRNA Let-7a Negatively Regulates the Expression of UHRF1 in Human Renal Epithelial Cells 293T

目的:探讨人肾上皮细胞293T中let-7a对靶基因UHRF1的负性调控作用.方法:运用生物信息学技术对let-7a靶基因进行预测和分析.构建含有UHRF1 3'UTR全长的野生型(pmiR-RB-UHRF1-3'UTR-wt)和突变型荧光素酶报告质粒(pmiR-RB-UHRF1-3'UTR-mut),分别将pmiR-RB-UHRF1-3'UTR-wt,pmiR-RB-UHRF1-3'UTRmut,pmiR-RB-REPORT vector与let-7amimics或mimics control共转染293T细胞,双荧光素酶报告系统检测转染后293T细胞的荧光素酶表达水平.293T细胞分别转染let-7amimics和inhibitor,western blot和real-time RT PCR检测UHRF1基因的表达.结果:生物信息学结果显示let-7a有48个预测靶基因,包括UHRF1,HMGA2,MAPK6等,主要涉及microRNA在肿瘤、细胞周期、PI3K-AKT、p53等信号通路.其中UHRF1 3'UTR含有一个let-7a的结合位点,且在多个物种中呈高度保守.进一步双荧光素酶检测发现,共转染let-7amimics和pmiR-RBUHRF1-3'UTR-wt的293T细胞荧光素酶活性显著降低(p<0.01).Western blot和real-time RT PCR结果显示转染let-7amimics的293T细胞UHRF1表达降低,而转染let-7ainhibitor的293T细胞UHRF1表达增高.结论:人肾上皮细胞293T中let-7a能通过与UHRF13'UTR靶向结合,负性调控UHRF1的表达.

Objective To explore the negative regulatory effect of let-7a on the expression of UHRF1 in human renal epithelial cells 293 T.Methods Bioinformatics methods were used for let-7atarget prediction and analysis.The full length wild type of UHRF1-3＇（pmiR-RB-UHRF1-3＇UTR-wt）and the mutant type（pmiR-RBUHRF1-3＇UTR-mut plasmid）were constructed,and the 293 T cells were co-transfected with pmiR-RBUHRF1-3＇UTR-wt or pmiR-RB-UHRF1-3＇UTR-mut plasmid or pmiR-RB-REPORT vector with let-7a mimics or mimics control intervention.Luciferase expression level of the 293 Tcells was detected by the dual luciferase reporter assay system.Western blot and real-time RT PCR were used to detect the expression of UHRF1 gene in the 293 Tcells which had been transfected with let-7amimics or inhibitor.Results Bioinformatics analysis indicated that let-7acontained 48 potential target genes including UHRF1、HMGA2and MAPK6,which were mainly involved in the signaling pathways of cancer,cell cycle,PI3K-AKT and p53.Among these targets,UHRF1 was found to contain a phylogenetically conserved binding site with let-7a.Furthermore,dual luciferase reporter displayed that luciferase activity in 293 Ttransfected with let-7amimics and pmiR-RB-UHRF1-3＇UTR-wt intervention was significant decreased（p〈0.01）.Moreover,western blot and real-time RT PCR demonstrated that the expression of UHRF1 declined in let-7aover-expressed 293 Tcells,while the expression of UHRF1 was enhanced in let-7adown-expressed 293 Tcells.Conclusion Let-7anegatively regulates the expression of UHRF1 in human renal epithelial cells 293 T through binding UHRF13＇UTR.