Effects Of Lentivirus-mediated ShRNA Targeting HOXA9Gene On The Proliferation,Apoptosis And Drug Resistance Of U937Cells

ObjectiveTo screen lentivirus-mediated RNAi (RNA interference) vector targeting HOXA9(homeobox A9) gene and investigate the effects of the proliferation,apoptosis and drug resistance of U937cells.Methods1. RNAiusing a plasmid construct expressing shRNA targeting HOXA9was detected by Western blotting. The plasmid expressing shRNA with highest level of knockdown was determined from four different plasmids and it was used to construct lentiviral vector named HOXA9/GV118RNAi-LV#2.After U937cells were infected with HOXA9/GV118RNAi-LV#2,the silence efficiency was examined by real-time fluorogenic quantitative PCR,and the expression of HOXA9protein was detected by Western blotting.2. After U937cells were infected with lentivirus-mediated shRNA targeting HOXA9,the effect of cell proliferation inhibition was assessed by MTT and cell growth curve.The apoptosis rate and cell cycle were measured by flow cytometry. c-myc mRNA and p27mRNA expression were determined by RT-PCR.3. The sensitivity of U937cells to VCR (vincristine) and DNR (daunorubicin) after lentivirus infection was detected by MTT assay, and the apoptosis rate of U937cells in each group was measured by flow cytometry. The expression of MDR-1mRNA after DNR treatment was determined by RT-PCR.Results1.The effective plasmid pGC-LV-shHOXA9#2was successfully screened by Western blotting,and it was produced packaged lentivirus HOXA9/GV118RNAi-LV#2.The silence efficiency of lentivirus-mediated shRNA targeting HOXA9in U937cells was more than or equal to55%in knockdown group,and the expression level of HOXA9protein was decreased obviously.2.Lentivirus-mediated shRNA targeting HOXA9could obviously increased IR value(40.469±1.756)%in U937cells and decreased cell growth.The apoptosis rate of U937cells infected with HOXA9/GV118RNAi-LV#2was significantly increased to (26.760±2.253)%and the cell cycle G0/G1phase in that group was raised apprently compared to contol group and negative contol group (P<0.05).Lentiviral-shRNA vector could reduce c-myc expression,but raise p27mRNA expression of U937cells by RT-PCR.3. The IC50(half inhibitory concentration) values of VCR/DNR were significantly reduced of U937cells after infection with HOXA9/GV118RNAi-LV#2(P<0.05), and the drug sensitivity of U937cells was increased. The apoptosis rate of U937cells infected with HOXA9/GV118RNAi-LV#2was remarkably increased after VCR/DNR intervention (P<0.05). The expression level of MDR-1mRNA of U937cells infected with HOXA9/GV118RNAi-LV#2was significantly reduced after DNR intervention (P<0.05).Conclusions1. We successfully constructed HOXA9/GV118RNAi-LV#2,which could effectively silence HOXA9gene expression.2. HOXA9/GV118RNAi-LV#2can effectively inhibit the proliferation and induce apoptosis of U937cells.It can reduce c-myc mRNA expression,but raise p27mRNA expression,which make cell cycle arrested in G0/G1phase.3. HOXA9/GV118RNAi-LV#2can increase the sensitivity of U937cells to VCR/DNR and reduce MDR-1expression. HOXA9gene is become a target of leukemia gene therapy in prospect.