Reversal Of Multi-drug Resistance In Human Hepatocellular Carcinoma Cells By Short Hairpin RNA Of MDR1 Gene

ObjectiveThe aim of this study was to induce RNAi in mammalian cells by short hairpin RNAs (shRNAs) generated from a DNA vector. To construct a recombinant plasmids generating short hairpin RNA containing multi-drug resistance gene MDRl segment in mammalian cells, It was processed to transfect the vector into the hepatocellular carcinoma cell line SMMC-7721/ADM and BEL-7402/AMD by LyoVec?' to investigate the influence of the shRNA to the effect of the transcription of the MDRl mRNA and P-glycoprotein(P-gp) expression in hepatocellular carcinoma cells.Methods1. The MDRl sequence-specific shRNA was designed by the authors according to the designation principle of shRNA. Two pairs of oligos for hairpin RNA expression which targeted MDRl gene were chemically synthesized and annealed. PGE-1 vector was linearized with BamHI and Xbal. The annealed oligos were inserted into the downstream of treated PGE-l's PoLlII U6 promoter to construct RNAi plasmid(psh21-MDRl).2. Recombinant psh21MDRl vector was identified by PCR and confirmed by sequencing analysis. It was then transfected into hepatocellular carcinoma cell lines SMMC-7721/ADM and BEL-7402/AMD by LyoVec?. Drug sensitivity was measured by MTT assay, we determined the effect of intracellular Rh123 accumulation and the expression of P-gp by flow cytometry(FCM). Rh123 retention was also assayed by loser scanning confocal microtechnic(LSM).Results1. Recombinant psh21-MDRl vector was identified by PCR and confirmed by sequencing analysis. The results demonstrated that 62bp had been inserted the expected site. Furthermore, the insertion sequence was exactly correct.2. Treatment of SMMC-7721/ADM and BEL-7402/AMD cells with the 2 kinds of sh-MDR1 resulted in a reversal of MDR of different extent(P<0.05); IC₅0 of cells transfected psh21-MDR1 were reduced obviously both in SMMC-7721 and BEL-7402 cells(P<0.05); Drug sensitivity was increased significantly.3. Positive rate of P-gp receded from 75.1% to 24.6% while pshRNA-MD-1 was transfected into BEL-7402/ADM cells (P<0.05), and transfection of pshRNA-MD-2into BEL-7402/ADM cells, the positive rate of P-gp receded to 39.4% (P<0.05). But the positive rate of P-gp receded from 91.3% to 45.7% while pshRNA-MD-1 was transfected into SMMC-7721/ADM cells (P<0.05), and transfection of pshRNA-MDRl-2 into SMMC-7721/ADM cells, the positive rate of P-gp receded to 60.0% (/><0.05).4. Fluorescence intensity of cells was determined by flow cytometric analysis. The intracellular accumulation of Rhl23 increased greatly after psh21-MDRl treatment in both SMMC-7721/ADM and BEL-7402/AMD cells (P<0.05).5. The intracellular accumulation of Rhl23 was also assayed by confocal scanning laser microscopy. Same as the results of flow cytometric analysis, the intracellular accumulation of Rhl23 increased greatly after psh21-MDRl treatment in SMMC-7721/ADM and BEL-7402/AMD cells.6. ShRNAs generated from the DNA vector induced RNAi in human cells. The inhibitory effect was highly related to the target sites. The sh-MDRl-1 increase the inhibitory effect is different with the sh-MDRl-2.Conclusion1. Psh21-MDR1 RNAi system has been constructed successfully. This will facilitate the study of inhibit multi drug resistance MDR1 gene expression. Based on the results, we can synthesis the shRNA in vivo instead of in vitro. This technique not only facilitates a wide range of gene functional analysis and cell culture but also offers therapeutic means for treatment of tumors.2. The results of the study showed that the recombinant plasmid constructed by the authors could suppress the expression of MDR1 mRNA and the P-gp expression in SMMC-7721/ADM and BEL-7402/AMD cells. The psh21-MDRl designed by the authors, was not only effective but also specific on the degradation of MDR1 mRNA, the inhibition of the protein expression and the elimination of the effects of MDR1 in hepatocellular carcinoma cells, which made the base on the reversal of mdrl-induced MDR in tumor.3. The inhibitory effect of the shRNA generated from the DNA vector was highly related to the target sites and to the different cell lines. This approach may be used for gene function study.