LIU Cuihua
1. College of Basic Medicine, Inner Mongolia Medical University, Hohhot 010059, ChinaDENG Linlin
1. College of Basic Medicine, Inner Mongolia Medical University, Hohhot 010059, ChinaZHAO Fangxin
1. College of Basic Medicine, Inner Mongolia Medical University, Hohhot 010059, ChinaHONG Mei
1. College of Basic Medicine, Inner Mongolia Medical University, Hohhot 010059, ChinaWU Jianqiang
1. College of Basic Medicine, Inner Mongolia Medical University, Hohhot 010059, ChinaZHANG Xuan
1. College of Basic Medicine, Inner Mongolia Medical University, Hohhot 010059, ChinaLI Yijun
2. the First Clinical Medical College, Inner Mongolia Medical University, Hohhot 010059WEN Qi
2. the First Clinical Medical College, Inner Mongolia Medical University, Hohhot 010059GUO Ran
2. the First Clinical Medical College, Inner Mongolia Medical University, Hohhot 010059LIU Yitong
2. the First Clinical Medical College, Inner Mongolia Medical University, Hohhot 010059MA Qihao
2. the First Clinical Medical College, Inner Mongolia Medical University, Hohhot 0100591. College of Basic Medicine, Inner Mongolia Medical University, Hohhot 010059, China. 2. the First Clinical Medical College, Inner Mongolia Medical University, Hohhot 010059
R-33
Objective To construct a recombinant luciferase reporter gene vector for wildtype and mutant 3’-untranslated regions (UTRs) of the LGALS3BP gene to study regulation of the LGALS3BP gene and miRNAs. Methods TargetScan was used to predict the binding site of miRNAs in the 3’UTR of the LGALS3BP gene. The wildtype LGALS3BP 3’UTR was amplified by PCR using genomic DNA from SMMC-7721 hepatocellular carcinoma cells as a template. PCR products were digested by two restriction enzymes and inserted into the luciferase reporter vector pGL3-control. Recombinant plasmids were verified by colony PCR and sequencing. Primers for LGALS3BP 3’ UTR with binding site mutations were designed. Single and double site mutant luciferase reporter gene vectors were prepared by overlapping PCR and site-directed mutagenesis PCR using the wildtype LGALS3BP 3’UTR recombinant plasmid as a template. Results Colony PCR showed a target gene fragment of 871 bp, which was consistent with the expected size. The inserted sequence was consistent with the LGALS3BP 3’ UTR in sequencing. TargetScan indicated two miRNA-binding sites in the LGALS3BP 3’UTR. The single and double site mutant sequences were successfully inserted into the LGALS3BP 3’UTR. Conclusions Wildtype LGALS3BP 3’UTR (pGL3-LGAL-W-3’UTR), two single site mutants (pGL3-LGAL-M1-3’UTR and pGL3-LGAL-M2-3’UTR), and a double site mutant (pGL3-LGAL-M-3’UTR) luciferase reporter gene vectors were successfully constructed, which may facilitate studies of LGALS3BP gene regulation through miRNAs.
