Construction Of The Recombinant Adenovirus Ad-pre-MiR-193B And Its Anti-Leukemia Mechanism

ObjectiveTo construct recombinant adenovirus vector containing pre-miR-193b, and identify its expression level in chronic myelogenous leukemia K562 cells. To further investigate the inhibitory effect and mechanism of over-expression of miR-193b on K562 cells. To provide experimental basis of the anti-leukemia effect for study of miR-193b in animal models of CML.Methods1.The cDNA of pre-miR-193b obtained by chemical synthesis was inserted into the adenoviral shuttle vector pAdTrack-CMV.The recombinant shuttle plasmid linearized by Pmel was transformed into Adeasier cell for homologous recombinant in E. coli BJ5183 with the adenoviral backbone pAdEasy-1. The recombinant vector linearized by PacI was transfected into HEK293 cells, packed and amplified adenovirus Ad-pre-miR-193b. K562 cells infected with virus were collected including group Ad-pre-miR-193b, blank group and empty virus group. The expression of mature miR-193b was detected by Real-time PCR.2. K562 cells infected with virus were collected including Ad-pre-miR-193b group, blank group and empty virus group. The expression of bcr/abl fusion gene was detected by Real-time PCR and BCR/ABL fusion protein Western blot. The proliferation of K562 cells was measured by CCK-8 assay. The cell apoptosis was tested by flow cytometry.3.K562 cells were infected recombinant adenovirus to detect the expression level of apoptotic protein casepase-3 by Western Blot.Results1.The pAdTrack-CMV-pre-miR-193b recombinant shuttle plasmid and recombinant adenovirus pAd-pre-miR-193b plasmid was successfully constructed, which was validated by digestion identification and sequencing.The recombinant adenovirus plasmid was successfully package d in HEK293 cells. Recombinant adenovirus Ad-pre-miR-193b could infect K562 cells efficiently. Compared with the control group, miR-193b was significantly up-regulated in K562 cells infected with recombinant adenovirus Ad-pre-miR-193b detected by QRT-PCR(P<0.01).2.Compared with the control group, miR-193b was significantly up-regulated in K562 cells infected with Ad-pre-miR-193b.The expression of bcr/abl fusion gene was significantly decreased, and BCR/ABL fusion protein was decreased(P<0.05).The proliferation of K562 cells was significantly reduced compared with control group(P<0.05).The apoptosis rate was significantly increased(P<0.05). The expression level of casepase-3 was increased(P<0.01).Conclusions1.The recombinant adenovirus pAd-pre-miR-193b were constructed successfully and could infect K562 cells efficiently. miR-193b was highly expressed in K562 cells.2.MiR-193b could down-regulate the expression of bcr/abl and inhibit the biological effects of K562 cell. The Over-expression of miR-193b may be an effective target for treatment of CML, which mechanism may be through the up-regulation of casepase-3 expression, thereby inhibit the proliferation and promote apoptosis of K562 cells.