The Effects Of Lentivirus-mediated Coexpression Of Trail And ShRNA Against Bcl-2on The Growth Of Lymphoma Cells

TRAIL (tumor necrosis factor-related apoptosis inducing ligand) is a member of tumor necrosis factor family that can induce apoptosis selectively of tumor cells, transformed cells and infected cells by virus without cytotoxicity to normal cells Death receptor pathway can be activated by TRAIL binding to death receptor DR4and DR5through both mitochondrial roads and non-mitochondrial roads. TRAIL-based cancer therapy has been applied in clinic research and many evidences have showed that TRAIL will be potential and ascendant drug for cancer treatment. Howerever, there are still some limitations for TRAIL in malignant hematological disease. The over-expression of Bcl-2and P53mutation often make tumor sensitivity blunt to TRAIL and leads to drug resistance. Bel-2gene, the most important anti-apoptosis factor, was originally derived from B cells follicular lymphoma and mainly distributed in tissue of liver, pancreas, colon, prostate cancer, leukemia. Bcl-2can protect cells avoid to enter apoptosis program by regulate the molecular mechanism of drug-induced apoptosis.We supposed that the combination of TRAIL expression and Bcl-2inhibition may have a synergistic effect on the growth of tumor cells. First, the mechanism of TRAIL and Bcl-2inducing apoptosis is different. TRAIL can bind to death receptor to activate the exogenous pathway, and then activate Caspase-8to enhance the mitochondria pathway. While Bcl-2induced apoptosis through mitochondria pathway directly, Bcl-2protein can lead to lose of membrane potential and increase in membrane permeability of mitochondria. Second, recent studies have demonstrated that the overexprssion of Bcl-2is correlated to the chemotherapy and radiation resistance of tumor cells, and the inhibition of Bcl-2can increase the drug-induced sensitivity of tumor.Lentiviruses are an exciting gene delivery tool because of their ability to efficiently transduce both dividing and nondividing cells. Additionally, their capacity to establish long-lasting transgene expression due to chromosomal integration of the proviral DNA makes this technique very attractive in the cancer research field such as lymphoma.Therefore, we use lentivirus vector and RNAi technology to construct a recombinant lentivirus system that can coexpress the exogenous gene and shRNA expression box. In this study, recombinant lentivirus lenti-trail/Bcl-2-shRNA was constructed to regulate the expression of TRAIL and Bcl-2synchronously. While the infection efficiency and gene regulation ability of lenti-trail/Bcl-2-shRNA was evaluated. We deeply analysis the inhibition effect and apoptosis induction of lenti-trail/Bcl-2-shRNA targeting different lymphoma cell lines to elucidate the synergism relationship between TRAIL and Bcl-2gene expression in lymphoma, and to explore a new model of lymphoma treatment.Object:1. To construct the recombinant lentivirus system that can coexpress the TRAIL gene and Bcl-2-shRNA. To evaluate the infection efficiency and regulation ability to TRAIL and Bcl-2gene of recombinant lentivirus lenti-trail/Bcl-2-shRNA.2. To investigate the the inhibition effect and apoptosis induction of lenti-trail/Bcl-2-shRNA targeting different lymphoma cell lines and elucidate the synergism relationship between TRAIL and Bcl-2gene expression in lymphoma. To explore the bystander effect of difference of TRAIL-sensitivity among lymphoma cells with different level of recombinant lentivirus lenti-trail/Bcl- 2-shRNA to lymphoma cells.Main Content:The first chapter:Construction of lentiviral expression vector pWPI-trail/bcl-2-shRNAMethod:1. Anneal bcl-2-shRNA oligonucleotide and clone the duplexes into the mU6promoter downstream Lentivirus vector pLL3.7. Construct the Lentiviral expression vector pll-bcl-2-shRNA.2. Extract human total RNA from placental tissue, amplify TRAIL cDNA by RT-PCR and then clone the fragment to pGM-T cloning vector. Cut TRAIL gene from pGM-T-trail cloning vector, and clone to EF-1a promoter downstream on pWPI lentivirus expression vector (IRES-GFP upstream), screen positive colonies and construct pWPI-trail expression vector.3. Get the mU6/bcl-2-shRNA expression box from the plasmid pll-bcl-2-shRNA, and clone into pWPI-trail plasmid upstream EF-1a promoter, screen positive colonies and construct pWPI-trail/bcl-2-shRNA expression vector.Results:1. Restriction enzyme digestion and sequencing showed pll-bcl-2-shRNA was constructed successfully.2. Restriction enzyme digestion and sequencing showed pWPI-trail was constructed successfully.3. Restriction enzyme digestion showed pWPI-trail/bcl-2-shRNA was constructed successfully. The second chapter:Preparation of recombinant lentivirus lenti-trail/bcl-2-shRNA and evaluation of infection efficiency to lymphoma cells.Method:1. package recombinant lentiviruses lenti-bcl-2-shRNA, lenti-trail and lenti-trail/bcl-2-shRNA by pWPI (pLL3.7)/vsvg/δ8.0system. Concentrate and purify viruses, then determine viral titer.2. Evaluate infection efficiency of recombinant lentiviruses to different lymphoma cells (human follicular lymphoma cell line DoHH2, human TCL cell line Jurkat and human NK cell lymphoma cell line YTS),assess the virus-sensitivity of lymphoma cell lines.Results:1. Construct the three recombinant lentiviruses successfully, and the purified virus titer was up to109IFU/ml.2. Recombinant lentiviruses lenti-bcl-2-shRNA, lenti-trail and lenti-trail/bcl-2-shRNA can infect the lymphoma cell lines effectively.The third chapter:Function assessment of recombinant lentivirus lenti-trail/bcl-2-shRNA and antitumor effection in vitro.Method:1. Lymphoma cell lines were infected by recombinant lentiviruses lenti-bcl-2-shRNA, lenti-trail and lenti-trail/bcl-2-shRNA,bcl-2and TRAIL mRNA were assayed by RT-PCR, while protein were assayed by western blot.2. The antitumor effects of lenti-bcl-2-shRNA, lenti-trail and lenti-trail/bcl-2-shRNA were evaluated by MTT measurement. Synergism relationship between bcl-2-shRNA and TRAIL was estimated.3. The impact of recombinant lentiviruses on lymphoma cell lines apoptosis rate and cell cycle distribution and the bystander effect were analyzed by Flow cytometry (FCM).Results:1. Recombinant lentivirus lenti-bcl-2-shRNA can inhibit the Bcl-2expression on the mRNA and protein levels. While lenti-trail can express TRAIL efficiently. lenti-trai^cl-2-shRNA can coexpress TRAIL and bcl-2-shRNA successfully。2. lenti-bcl-2-shRNA,lenti-trail and lenti-trail/bcl-2-shRNA can inhibit the growth of lymphoma cells with different inhibition rates (P＜0.05), while markedly in lenti-trail/bcl-2-shRNA group(P＜0.05). The downregulation of Bcl-2and upregulation of TRAIL have a commendable synergism effects.3. Recombinant lentivirus lenti-trail/bcl-2-shRNA could induce effectively bystander effect against lymphoma cells.Conclusion:1. The lentivirus expression vector pWPI-trail/bcl-2-shRNA coexpressing TRAIL gene and bcl-2-shRNA was successfully constructed.2. Recombinant lentivirus system that can carry exogenous gene and shRNA expression box was successfully established. Recombinant lentivirus lenti-trail/bcl-2-shRNA packaged using this system can infect lymphoma cell lines and regulate TRAIL and Bcl-2gene expression effectively.3. Recombinant lentivirus lenti-trail/bcl-2-shRNA can inhibit the growth of lymphoma cells more effective than lenti-bcl-2-shRNA and lenti-trail.4. Recombinant lentivirus lenti-trail/bcl-2-shRNA could induce effectively bystander effect against lymphoma cells.