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'UTR-mut，pmiR-RB-REPORT vector与let-7a mimics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-7a mimics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-7a mimics和pmiR-RB-UHRF1-3'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-7a mimics的293T细胞UHRF1表达降低，而转染let-7a inhibitor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.
ObjectiveTo explore the negative regulatory effect of let-7a on the expression of UHRF1 in human renal epithelial cells 293T. MethodsBioinformatics methods were used for let-7a target prediction and analysis. The full length wild type of UHRF1-3' (pmiR-RB-UHRF1-3'UTR-wt) and the mutant type (pmiR-RB-UHRF1-3'UTR-mut plasmid) were constructed, and the 293T cells were co-transfected with pmiR-RB-UHRF1-3'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 293T cells 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 293T cells which had been transfected with let-7a mimics or inhibitor. ResultsBioinformatics analysis indicated that let-7a contained 48 potential target genes including UHRF1、HMGA2 and 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 293T transfected with let-7a mimics 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-7a over-expressed 293T cells, while the expression of UHRF1 was enhanced in let-7a down-expressed 293T cells. ConclusionLet-7a negatively regulates the expression of UHRF1 in human renal epithelial cells 293T through