Construction,packaging and expression activity of miR-30a expression vector

Objective To construct a lentiviral miR-30a expression vector for the study of the mechanism of miR-30a in chronic myeloid leukemia and the impact to imatinib resistance.Methods Human bone marrow genomic DNA was extracted,miR-30a precursor sequence was amplificated and cloned into the lentivirus expression vector pIVTHM,then sended sequencing.The recombinant vector was packaged in 293T cells and tittered in K562cells.GFP-positive K562 cells with green fluorescence were sorted by flow cytometry,miR-30a expression level in sorted cells was further detected by RT-PCR.The proliferation of different cells was detected by CCK-8 assay.Results Sequencing of the recombinant lentiviral expression vector was completely correct;viral titer was6×10~6 TU/mL;160 μL virus solution /1mL medium was selected to infect K562 cells;the expression level of miR-30a in infected K562 cells was significantly enhanced(P 0.05);the proliferation levels of cells overexpressed miR-30a decreased significantly(P 0.05).Conclusions miR-30a lentivirus vector is successfully constructed and miR-30a is stably expressed in K562 cells,and miR-30a may inhibit the proliferation level of K562 cells.