Inhibition of MDR1 expression in hepatocellular carcinoma cell Bel-7402/R by vector-mediated RNA interference

OBJECTIVE:To construct a recombinant plasmids generating short hairpin RNA which contain multi-drug resistance gene MDR1 in mammalian cells, in order to investigate the suppression of MDR1 mRNA in hepatocellular carcinoma cells Bel-7402/R.METHODS: By using molecular cloning technique, double-stranded DNA containing MDR1 was cloned into PGE-1 vector digesting by two restricted endoenzymes according to its special orientation. Human hepatocellular carcinoma cells Bel-7402/R was transfected with recombinant plasmid by LyoVecTM, MDR1 mRNA was assessed by RT-PCR, drug sensitivity was measured by MTT assay in vitro. The effect of intracellular Rh123 accumulation and the expression of P-gp were determined by the flow cytometry (FCM). RESULTS: The size of the PCR product was 669 bp. DNA sequencing showed the sequence of recombinant vector pshRNA-MDR1 was successfully constructed, and MDR1 mRNA expression was suppressed;IC 50 of cells which transfected pshRNA-MDR1 reduced obviously, P0.05. Positive rate of P-gp receded from 77.7% to 20.4%, and the concentration of Rh123 in cells which transfected pshRNA-MDR1 increased greatly,P0.05.CONCLUSION: The recombinant plasmid constructed by the authors can suppress the expression of MDR1 mRNA and P-gp in hepatocellular carcinoma cells Bel-7402/R.