Construction of eukaryotic expression vector of miR-139 and its effect on proliferation of ZR75-1 cells

Objective To construct eukaryotic expression vector of miR-139 and to study its effect on ZR75-1 cells.Methods Mature miR-139 sequence was amplified from human genomic DNA.The target gene was inserted in to pMD19-T vector.Restriction digestion,ligation and sequencing were performed to evaluate the recombinant.Then the target gene and vector PIRES2-EGFP(IE) were digested at the same time,and the target gene was linked to the vector.Subsequently,miR-139 expression vector IE-139 and the blank vector IE were transfected into ZR75-1 cells.The expression of mature miR-139 was verified at the transcription level using quantitative real time PCR(qRT-PCR) while the expression of c-Myc,EIF4G2 and ZBTB34 mRNA was detected.Results Restriction enzyme digestion and plasmid sequencing confirmed the successful construction of IE-139 vector.Detected with qRT-PCR,ZR75-1 cells increased miR-139 expression remarkably 72 hours after transfection(P0.01),and miR-139 could significantly inhibit c-Myc and EIF4G2 mRNA expression(P0.05).Conclusion The recombinant eukaryotic expression vector IE-139 is successfully constructed.qRT-PCR results indicated that the mature miR139 is effectively expressed in ZR75-1 cells,and miR-139 inhibits c-Myc and EIF4G2 mRNA expression.This study sheds light on the function of miR-139 and its molecular mechanism of regulating target genes.