【摘要】 目的从目前已知的人类miRNA中筛选出能特定靶向结合P2X3 3′端非编码区(3′UTR),并能引起P2X3表达下调的miRNA。方法利用生物信息学软件预测P2X3 3′UTR的靶向miRNA,构建P2X3 3′UTR荧光素酶报告基因系统质粒,该质粒同预测的miRNA共转染293TN细胞,并检测相对荧光素酶活性;将筛选出的miRNA转染293TN细胞,选择出能下调P2X3表达的miRNA;构建P2X3 3′UTR突变体荧光素酶报告基因系统质粒,与相应的miRNA共转染后检测相对荧光素酶活性,得到通过结合P2X3 3′UTR下调P2X3表达的miRNA。结果应用TargetScan等软件预测出有21种潜在的miRNAs可能与人P2X3靶向结合,在此基础上构建了包含P2X3 3′UTR全长的双荧光素酶报告系统psiCHECK2-P2X3,该载体质粒分别与上述21种miRNAs共转染293TN细胞后,通过检测报告基因的表达及转录后水平的变化进行筛选,结果初步证实共有3种miRNAs(hsa-miR-491-5p、hsa-miR-1178和hsa-miR-1291)能够靶向结合P2X3的3′UTR,并显著下调其表达。结论本研究通过对人P2X3 miRNAs的筛选和鉴定,初步发现有3种miRNAs(hsa-miR-491-5p、hsa-miR-1178和hsa-miR-1291)可调控P2X3的表达。更多还原

【Abstract】 Objective To systematically characterize the microRNAs targeting P2 X3 by binding the 3′UTR from all the known miRNAs of human. Methods Targeted miRNAs of the P2 X33′UTR were predicted using bioinformatics software.The relative luciferase activity was then detected by constructing a P2 X3 3′UTR luciferase reporter gene plasmid and co-transfection with the predicted miRNA.The selected miRNAs were subsequently transfected into renal cancer cell lines,and miRNAs capable of down-regulating P2 X3 expression were selected.Finally,a P2 X3 3′UTR mutant luciferase reporter gene plasmid was constructed and co-transfected with the corresponding miRNA to detect relative luciferase activity.A miRNA that down-regulates P2 X3 expression by binding to the P2 X3 3′UTR was obtained. Results TargetScan and other softwares were used to predict that there were 21 potential miRNAs that may bind human P2 X3,and a dual luciferase reporter system pSicheck2-P2 X3 containing the full length of P2 X3 3′UTR was constructed.The above 21 miRNAs were co-transfected into 293 TN cells,and then screened by detecting the expression of the reporter gene and the post-transcriptional level.The results confirmed that there were three miRNAs(hsamiR-491-5 p,hsa-miR-1178 and hsa-miR-1291)which were capable of targeting the 3′UTR region of P2 X3 and downregulating its expression significantly. Conclusions In this experiment,preliminary screening and identification of human P2 X3 miRNAs revealed that three miRNAs(hsa-miR-491-5 p,hsa-miR-1178 and hsa-miR-1291)can regulate the expression of P2 X3.更多还原