Effects Of Lentivirus Vector-mediated RNA Interference On Multi-drug Resistance In Non Small Cell Lung Cancer

Objective Multi-drug resistance gene MDR1 and multi-drug resistance related protein gene MRP are two factors generating multi-drug resistance, which were selected in this research to study the effects of multi-drug resistance in non small cell lung cancer (NSCLC). Using RNA interference, we constructed two recombinants mediated by lentivirus vector PTM-Si-MDR1 and PTM-Si-MRP to express Si-MDR1 and Si-MRP. After transfected these recombinants to human lung cancer cell A549 and DDP-resistant cell A549/DDP, we studied their resistance to Gemzer, ADM, and DDP. We expected to find a novel way to cure NSCLC by our research. This research involved three parts: (1) Screening of efficient interfering sites of Si-MDR1 and Si-MRP. (2) Construction of Si-MDR1 and Si-MRP recombinant lentivirus. (3) Drug resistance of A549-Si-MDR1 and A549-Si-MRP cells.Methods (1) Four interfering sites of MDR1 and MRP gene were designed respectively, then chemosynthesised SiRNA fragments and transfected to human lung cancer cell A549 linked with vector pSUPER. The transfection efficiency was detected by fluorescence microscope; MDR1 and MRP gene expression in A549 cell were detected by real-time PCR 48 hours after transfection. The efficient sites for packing recombinant lentivirus were screened. (2) Using the lentivirus system of PTM, pHelper1.0 and pHelper2.0 to mediate Si-MDR1 and Si-MRP expression. 293T cells were transfected for amplification and Hela cells were transfected for lentivirus titer determination. After host cells A549 and A549/DDP transfected for 2 days, transfection efficiency was determined by FACS; the suppression of MDR1 and MRP gene in mRNA level and protein level were determined by real-time PCR and Western Blot respectively. (3) A549-Si-MDR1 and A549/DDP-Si- MDR1 cell treated with three drugs (DDP, ADM and Gemzer) for 24 hours, then the morphous of EGFP positive cells were detected by fluorescence microscope. (4)One week after PTM, PTM-Si-MDR1, and PTM-Si-MRP transfected into A549 and A549/DDP cells (MOI=10), the cells treated with DDP, ADM and Gemzer with different dose for 48 hours, then cytoactive was determined using CCK-8 kit. (5) Follwing the establishment of stable transfected A549 and A549/DDP cell lines by PTM, PTM-Si-MDR1, and PTM-Si-MRP lentivirus, the primary lung cancer xenograft model was established in BALB/c nude mice. After administrating chemotheraphy drugs, the MDR phenotype was assessed in vivo by primary tumor weight calculation and multi-site metastasis assessment.Results (1) Designed SiRNA fragments of MDR1 and MRP genes, constructed screening plasmids pSUPER-Si-MDR1 and pSUPER-Si-MRP to select efficient interfering sites. (2) Transfected the recombinant plasmids to human lung cancer cell A549 and DDP-resistant cell A549/DDP. Transfection efficiency was about 47.9%, determined by fluorescence microscope. (3) Determined MDR1 and MRP gene expression in A549 cell in mRNA level by real-time PCR, 48 hours after transfection. MDR1 and MRP were silenced specificly and efficiently. Thus the plasmid pSUPER-Si-MDR1 and pSUPER-Si-MRP were successful to express SiRNA. (4) Packed two lentivirus vectors using the efficient interfering sites, transfected into 293T cell for amplification. Transfection efficiency was high to 85%, determined by fluorescence microscope and FACS. Results proved that the plasmids could transfect 293T with high efficiency and express SiRNA stably. (5) Recombinant lentivirus titers were determined, being about 5×108TU/ml. (6) A549 and A549/DDP were transfected by PTM-Si-MDR1, PTM-Si-MRP and PTM (blank vector), the transfection efficiencies of PTM-Si-MDR1 and PTM-Si-MRP were 77.3% and 80.8% respectively, determined by FACS. (7) The MDR1 and MRP gene expression in A549 were determined by real-time PCR after transfection 2 days. Suppression efficiencies of MDR1 and MRP were 72% and 69%, which were both obvious. (8) Being treated with three chemotherapy drugs: DDP (50ug/ml), ADM(1ug/ml)and Gemzer (200ug/ml) for 24 hours, A549-SiMDR1 cells (one week after transfection) shrinked and lost morphous, which were typical characters of apoptosis. (9) A549 cells (A549-PTM, A549-Si-MRP, A549-Si-MDR1) and A549/DDPcells (A549/DDP-PTM, A549/DDP-Si-MRP, A549/DDP- Si-MDR1) were treated with DDP, ADM and Gemzer with different doses, and determined cytoactive of each cell group after 48 hours. Results showed that, the sensitivity of A549/DDP to 3 drugs were all boosted. (10) As compared with PTM-transfected A549/DDP group, the primary tumer weight and metastasis ratio of contralater lung and distant organs were all decreased significantly in both the stable-transfected PTM-Si-MDR1 and PTM-Si-MRP A549/DDP group. These date indicated that the MDR phenotype was completely reversed by RNAi technology targeting MDR1 and MRP mRNAs.Conclutions The lentivirus vector-mediated RNA interference can suppress MDR1 and MRP gene expression remarkably in A549 and DDP-resistant A549/DDP cells to last, and can improve the effects on killing cancer cells. Our research offered experimental prove to raise NSCLC chemotherapy effect clinically, and to prolong the survival time of patients after chemotherapy.