Lentivirus-mediated RNA Interference Reversing The Multidrug Resistance Of Single-factor Durg-resistance Cell Line K562/MDR1

BackgroundOne of the main reasons for chemotherapeutic treatment failure is multidrug resistance. The mechanisms responsible for drug resistance include many directions. The most representative one is expression of P-glycoprotein encoded by MDR1 gene. In studies of reversing drug resistance, the researchers always choose the drug-resistance cell lines induced by chemotherapeutic drugs as research modles. Many factors result in the drug resistance of these cell lines, and the character of drug resistance is not very stable.There are many ways to reverse the drug resistance caused by MDR1 gene. The most efficient method is reversing MDR1 gene directly. The RNA interference technology supplies a high-efficient and specific method to silence MDR1 gene. Lentivirus vector is a kind of hopeful transgenic vector,which can infect non-dividing cells. The exogenous gene can achieve a sustained expression by the stable integration of lentivirus vector into the host cell genome. So the lentivirus-mediated RNA interference will make it possible to silence the target gene persistently.Objective1. To establish a single-factor durg-resistance cell line K562/MDR1 whose resistance to drugs caused only by MDR1 gene.2. To construct a lentiviral vector encoding short hairpin RNA targeting MDR1 gene.3. To test the efficiency of lentivirus-mediated RNA interference to silence MDR1 gene.Method1. Establishing a single-factor durg-resistance cell line K562/MDR1: Retroviral virions carring the complete sequence of MDR1 gene cDNA were produced, and infected drug-sensitive leukemia cell line K562. The K562 cells transfected by retroviral virions were screened by colchicines. Then, a monoclone transgenic durg-resistance cell line K562/MDR1 was achieved. The MDR1 gene of K562/MDR1 was identified by Q-PCR. The expression of P-gp was detected by flow cytometry. The function of P-gp was measured by daunorubicin efflux experiment, and the drug sensitivity of K562/MDR1 was detected by MTT test.2. Lentivirus-mediated RNA interference reversing the drug resistance caused by MDR1 gene: We constructed a lentiviral vector encoding shRNA targeting MDR1 gene. The lentiviral virions were produced, and infected durg-resistance cell lines K562/MDR1,K562/A02. The efficiency of lentivirus-mediated RNA interference to silence MDR1 gene was evaluated by four tests: The change of MDR1 gene quantity was identified by Q-PCR. The variation of P-gp expression was detected by flow cytometry. The function of P-gp was measured by daunorubicin efflux experiment. MTT test detected the change of drug sensitivities of K562/MDR1,K562/A02 after RNA interference.Results1. We established a single-factor durg-resistance cell line K562/MDR1 successfully: The results of Q-PCR showed that the MDR1 gene quantity of K562/MDR1 was 2.79×10~3, much higher than K562 cells'. The expression of P-gp was 96.10%. The daunorubicin efflux rate was 90.93%. K562/MDR1 cells showed the powerful ability of drug efflux. The results of MTT test confirmed K562/MDR1 cells were resistant to chemotherapeutic drugs.2. We constructed a lentiviral vector encoding shRNA targeting MDR1 gene successfully: We synthesized a DNA sequence encoding shRNA targeting MDR1 gene, and subcloned it into a lentiviral vector plentilox 3.7 correctly.3. Silencing MDR1 gene efficiently by lentiviral vector encoding shRNA targeting MDR1 gene: The lentiviral virions showed a high efficiency of infecting durg-resistance cells. This is advantageous for the following RNA interference tests. The results of Q-PCR demonstrated that the marked downregulation of the MDR1 gene quantity in two kind of drug-resistance cell line (K562/MDR1 downregulated 100%, K562/A02 downregulated 96.15%). The results of flow cytometry also demonstrated the significant reduction of the P-gp expression(two kind of cells both downregulated 100%). After RNA interference, the daunorubicin efflux rate of drug-resistance cells downregulated markedly. The increased drug sensitivities of K562/MDR1,K562/A02 after RNA interference were detected by MTT test. All the datas documented that RNA interference can reverse the multidrug resistance caused by MDR1 gene.Conclusion1. We successfully established a single-factor durg-resistance cell line K562/MDR1 by retrovirus-mediated MDR1 gene transfer, and supply a more stable,more specific cell modle for the researches about MDR1 gene.2. The lentiviral vector which we constructed to encode shRNA targeting MDR1 gene, could efficiently downregulate the quantity of MDR1 gene and the expression of P-gp. It successfully reversed K562/MDR1 and K562/A02 cells'resistance to chemotherapeutic drugs. But it took different time to downregulate MDR1 gene quantity and P-gp expression.3. Lentivirus-mediated RNA interference demonstrated a stable and high-efficiency effect, and will make it possible to silence the MDR1 gene persistently...