| Objective(1)To construct a expression vector plasmid pSUPER-shRNA/mrpl carrying small interfering RNA, provide theoretical and experimental evidence for inhibitory of hepatocellular carcinoma(HCC) multidrug resistance; (2) To observe the effect of pSUPER-shRNA/mrp1 on HepG2/mrp1, and investigate the function of mrp1; (3)To explore the inhibitory effect of pSUPER-shRNA/mrp1 on HepG2 multidrug resistance mediated by mrp1; (4) To test the application of constructed plasmid pSUPER-shRNA/mrp1 in vivo transfection for reversing the MDR of HCC, initiatory explore the side effect of pSUPER-shRNA/mrp1.Methods Five duplex stranded short RNA(dsRNA) targeting mrp1 gene(dsRNA/mdr1) including one negative control were designed and synthesized. By using BLAST software, homologization sequences were excluded. The five dsRNA were recombinated with pSUPER vector who named P1, P2, P3, P4 and P5 respectively, the recon were preliminary screen by PCR, base sequence were checked by plasmid sequencing. HepG2 and HepG2/mrp1 cloned strain were transfected expression vector pSUPER-shRNA/mrp1, the nude mice were inoculated the cloned strain subcutaneous. (1)To observe the effect of transfection of pSUPER-shRNA/mrp1 on HepG2/mrpl cell; (2) Mrp1 mRNA and MRP1 protein were detected by RT-PCR and Western blot hybridization respectively; (3) The expression of MRP1 protein and the accumulation of daunorubicin(DNR) were dectected by flow cytometry(FCM); (4)Drug tolerance of transfected HepG2/mrp1 was analyzed by MTT method; (5)HepG2/mrp1 and HepG2 were transplanted to nude mice, and the transplanted tumors were observed every day. (6).pSUPER-shRNA/mrp1 vectors and pSUPER-shRNA/mrp1 negative vectors were injected into the tumor respectively when the tumor sized achieved 5 mm in diameter. Seventy-two hours after injection, adriamycin of 1.5mg/kg were injected into the tumor, the information about the nude mice and the transplanted tumor were collected; (7) Blood examination and histological examination of group D, E and F nude mice were conducted, compare the data of the three groups, and evaluate the side-effect of pSUPER-shRNA/mrp1 on using in reversal of MDR.Results(1)Five duplex stranded RNAs targeting mrp1 gene(dsRNA/mrp1) were synthesized. Plasmid vector of pSUPER-shRNA/mrp1 was constructed successfully by duplex incision enzyme technique. Transfected by the recombinant plasmid, HepG2/mrp1 showed its MDR was reversed notablely in vivo and in vitro. Compared with dsRNA, pSUPER-shRNA/mrp1 could synthesize and express shRNA intra-cellular, its stability was better than the former. The effect of group of P4 was the best of the five group; (2)Transfected by pSUPER-shRNA/mrp1, the transcription of mrp1 mRNA decreased, it could be obsevered especially in group P4. the transcription of mrp1 mRNA decreased(88.03±2.04)％. The expression of MRP1 protein was(10.2±1.3)％, the accumulation of DNR was increased evidently. (3)HCC model of nude mice carried HepG2/mrp1 was established; (4) In HCC model of nude mice, the tumodgenes of both groups was identified. Transfected by pSUPER-shRNA/mrp1, pre-chemotherapy and post-chemotherapy, tumor volume difference of the experimental group was 613.24±28.58mm~3, it was significantly smaller than that of the control group(1120.04±90.76, P＜0.05). The tumor growth rate of the experimental group was(10.35±1.26)％, it was significantly lower than that of the control group(17.90±4.58)％. The tumor inhibition ratio was 45.0％; (5)Blood and pathologic examination were conducted on group D, E and F. No significant damage about liver and kidney function were detected, and no pathologic damage were detected in the three group. The inhibitory effect of the expression of mrp1 by pSUPER-shRNA/mrp1 transfection in vivo will be an safe method. Conclusions(1) Mrp1 gene was a multidrug resistance gene, it has important effect on the multidrug resistance of HCC. (2) pSUPER-siRNA/mrp1 could down-regulate mrp1 mRNA, suppress the expression of MRP1 protein. (3)Injection pSUPER-siRNA/mrp1 in vivo could reverse multidrug resistance of HCC effectively, and it has better security. (4)The experiment provide a good theoretical evidence for the use of RNAi technology on reverseing multidrug resistance of HCC on clinic.
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