Construction And Identification Of Multidrug Resistance Gene MDR1 Short Hairpin Expression Plasmids

Objective: RNA interference (RNAi) is an evolutionarily conserved post-transcriptional gene silencing, in which the introduction of double-stranded RNA into a cell leads to specific suppression of gene expression. RNAi has become an important tool for the study on gene expression. The aim of this study was to induce RNAi in mammalian cells by short hairpin RNA (shRNA) generated form a DNA vector. A recombinant plasmids generating short hairpin RNA in mammalian cells were constructed, and to investigate suppression of MDR1 mRNA in hepatocellular carcinoma cells Bel-7402/R.And in order to establish the feasibility of using RNA interference technique to study genes function.Methods: Two oligonucleotides for MDR-lmRNA of Genebank were Designed, double-stranded DNA which dropped temperature. Then, using cloning technique, double-stranded DNA which contained MDR1 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 LyoVec^TM, MDR1 mRNA was assessed by RT-PCR, drug sensitivity was measured by MTT assay in vitro. We determined the effect of intracellular Rhl23 accumulation and the expression of P-gP by flow-cytometry (FCM).Results: The size of the PCR product was 669bp. DNA sequencing showed the sequence of recombinant vector pshRNA-MDR1 was successfully constructed, and suppressed MDR-1 mRNA expression; IC₅0 of cells which transfected pshRNA-MDR1 were reduced obviously, P<0.05; positive rate of P-gP receded from 77.7% to 20.4%; the concentration of Rhl23 in cells whichtransfected pshRNA-MDRl increased greatly, j°<0. 05.Conclusion: The recombinant vector pshRNA-MDRl has been successfully constructed, the result showed that the recombinant plasmid constructed by the authors can suppress the expression of MDR-1 mRNA and P-gP in hepatocellular carcinoma cells Bel-7402/R, in order to find an effective method to reversal MDR in hepatocellular carcinoma cells mediated by MDR1.