CUL53’-UTR双荧光素酶报告载体构建及与MiR-148a-3p靶向验证

Construction of the CUL5 3'-UTR dual luciferase reporter vector and validation of the relationship between CUL5 and miR-148a-3p

目的 :构建CUL5基因3’-UTR双荧光素酶报告载体,并验证miR-148a-3p与CUL5的靶向关系。方法 :利用生物信息学软件预测miR-148a-3p与CUL5结合位点;利用PCR扩增CUL5基因3'-UTR序列,将其克隆到pmiR-RB-Report TM双荧光素酶报告基因载体中,同时构建CUL5 3’UTR突变载体;将miR-148a-3p及阴性对照分别与野生型has-CUL5-wt 3'-UTR及突变型has-CUL5-mut 3'-UTR双荧光素酶报告质粒共转染至293T细胞中,双荧光素酶报告系统检测各组荧光素酶活性。结果 :Targetscan、PicTar、miRanda、PITA、miRDB数据库预测结果显示,miR-148a-3p与CUL5基因3’UTR存在互补结合位点。酶切及测序结果表明,双荧光素酶报告载体构建成功;荧光素酶活性实验表明,miR-148a-3p mimics能够与CUL5基因3′UTR结合并抑制荧光素酶活性;对其预测靶位点进行突变后,突变型载体中的报告荧光活性有所上升。结论 :miR-148a-3p能够与CUL5靶向性结合,CUL5是miR-148a-3p新的靶基因。

Objective To construct CUL5 3’-UTR dual luciferase reporter vector,and verify the targeted relationship between miR-148 a-3 p and CUL5.Methods Bioinformatics software(Targetscan,PicTar database,miRanda and PITA,miRDB)was used to predict the target genes of miR-148 a-3 p;PCR was used to amplified CUL5 gene 3’-UTR sequence;Dual luciferase report plasmid pmiR-RB-ReportTM was used to construct CUL5 wild type and mutant dual luciferase report vector.Cotransfection of miR-148 a-3 p or negative control with wild type hsa-CUL5-WT or hsa-CUL5-MUT dual luciferase report plasmid to 293 T cells,respectively.Dual luciferase reporter system was used to detect the luciferase activity.Results There is a complementary binding site in CUL5 gene 3’UTR for miR-148 a-3 p by bioinformatics prediction(Targetscan,PicTar,miRanda,PITA,and miRDB database).The enzyme digestion and sequencing results showed that the CUL5 dual luciferase reporter vector was successfully constructed;luciferase activity analysis results showed that over expression of miR-148 a could inhibit the luciferase activity with wild-type 3’-UTR of CUL5,but increse that of mutant 3’-UTR of CUL5.Conclusion CUL5 is a novel target for hsa-miR-148 a-3 p.