Let-7a负性调控人肾上皮细胞293T中UHRF1基因的表达

发布时间：2018-07-10 19:00

本文选题：微RNA + UHRF　；参考：《西南大学学报(自然科学版)》2017年07期

【摘要】：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(p0.01).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.
[Abstract]:Aim: to investigate the negative regulation of let-7a on target gene UHRF1 in human renal epithelial cell line 293T. Methods: let-7a target gene was predicted and analyzed by bioinformatics. Wild type (pmiR-RB-UHRF1-3UUTR-wt) and mutant luciferase reporter plasmid (pmiR-RB-UHRF1-3UUTR-mut) were constructed, respectively, and pmiR-RB-UHRF1-3 UTR-wtpmiR-RB-UHRF1-3UTRPORT vector was cotransfected with let-7amimics or mimics control into 293T cells. The expression of luciferase in 293T cells was detected by double luciferase report system. The expression of UHRF1 gene was detected by real-time RT PCR and let-7amimics transfected with 293T cells. Results: bioinformatics showed that let-7a had 48 predictive target genes, including UHRF1, HMGA2mMAPK6, which were mainly involved in the signaling pathways of microRNAs in tumor, cell cycle, PI3K-AKTP-p53 and so on. UHRF1 3 UTR contains a let-7a binding site and is highly conserved in multiple species. Further double luciferase assay showed that luciferase activity of 293T cells co-transfected with let-7amimics and pmiR-RBUHRF1-3UTR-wt decreased significantly (p0.01). Western blot and real-time RT PCR showed that UHRF1 expression in 293T cells transfected with let-7amimics was decreased, but UHRF1 expression in 293T cells transfected with let-7ainhibitor was increased. Conclusion: let-7a can negatively regulate the expression of UHRF1 by targeting with UHRF13 UTR in human renal epithelial cell line 293T.
【作者单位】：重庆医科大学细胞生物学及遗传学教研室;重庆医科大学生物信息学教研室;重庆医科大学实验教学中心;
【基金】：国家自然科学基金面上项目(81270912) 重庆市科委一般项目(cstc2013jcyjA10044)
【分类号】：Q78

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