Construction of nuclear factor of activated T-cells 5 mRNA 3'-untranslated region reporter vector and targeting verification between NFAT5 and miR-155 / 重庆医学

ABSTRACT

Objective To construct a Luciferace reporter vector containing the 3'untranslated region (3'UTR) of NFAT5 and measure the correlation between NFAT5 and miR-155.Methods The miR-155 targeting NFAT5 3'UTR was predicted by Target Scan,Mir Base and Pic Tar.NFAT5 and mutant NFAT5 sequence(NFAT5-mu) were then designed and synthesized,and they were cloned into pMIR-REPORTTM Luciferace reporter vector.Human embryonic kidney-293AD (HEK-293AD) cells of the 4th passage were divided into 4 groups according to the random number table.cells in plasimd +miR-155 mimics groups were transfected with pMIR-NFAT5 recombinant plasimid,pRL-Tk plasmid and miR-155 mimics;cells in plasimd + miR-155 mutated groups were transfected with pMIR-NFAT5-mu recombinant plasimid,pRL-Tk plasmid and miR-155 mimics;cells in plasimd + miR-155 control groups were transfected with pMIR-NFAT5 recombinant plasimid,pRL-Tk plasmid and miR-155 Negative control;cells in plasimd +miR-155 inhibitor were transfected with pMIR-NFAT5 recombinant plasimid,pRL-Tk plasmid and miR-155 inhibitor;and were respectively transfected into together by liposome.After culture for 24 h,the luciferase activity was detected by dual luciferase reporter assay system.Results TargetScan,Miranda and PicTar shared the results that NFAT5 has the complementary binding sites with 3'UTR of miR-155.And luciferase reporter vectorwas constructed.Therefore the result of sequencing and double digesting of recombined plasmid were completely correct.Dual-luciferase reporter assay showed that miR-155 possesses a target effect on 3'UTR of NFAT5.Compared to the pMIR-NFAT5 + miR-control group,the luciferase activity of the pMIR-NFAT5 + miR-1 5 5 mimics group was decreased,with statistically significant difference(P＜0.01),while there was no significant difference at other time points(P＞0.05).Conclusion The pMIR-NFAT5 recombinant plasmid and pMIR-NFAT5 recombinant mutated plasmid were confirmed with successful construction.and it was found that miR-155 can target NFAT5 mRNA 3'-UTR.The results provide the experiment data for further disclosing the mechanism of inhalation injury on the level of gene expression.