Lentivirus-mediated RNA interference suppresses DNA binding protein A expression and inhibits proliferation of colorectal cancer cells

Abstract

Objective To investigate the effects of lentivirus-mediated RNA interference(RNAi)targeting DNA binding protein A(dbpA)on the proliferation of colorectal cancer cell line SW620. Methods The lentiviral vector shRNA-dbp A was constructed and verified by polymerase chain reaction(PCR)and DNA sequencing. SW620 cells were transfected with shRNA- dbp A plasmid, nontargeting shRNA plasmid, or empty plasmid.Forty- eight hours after the transfection, the cells were examined for dbp A expression using Western blotting. Seventy- two hours after transfection, flow cytometry was used to detect the cell apoptosis and cell cycle changes. The cell growth inhibition rate was detected by methyl thiazol tetrazolium(MTT)assay,and then clone formation was detected. Results PCR analysis and DNA sequencing demonstrated that the RNAi sequence targeting dbpA gene was successfully inserted into the lentiviral vector.shRNA- dbpA transfection resulted in obviously reduced expression of dbpA in SW620 cells. After transfection,the apoptosis rate of shRNA- dbpA- transfected cells were increased to(26.60±0.38)%,significantly higher than that in cells transfected with the nontargeting plasmid or the empty plasmid[(12.54±0.25)% and(4.46±0.19)%, respectively,P<0.01]; the cell number in G0/G1 phase increased while that in G2/M phase decreased in shRNA- dbpA- transfected cells. The growth inhibition test indicate that the OD value of the fifth day in shRNA- dbpA group was 1.183±0.226, significantly lower than that in the other two groups(5.295± 0.282 and 10.207±0.383, respectively,P<0.01).The colony formation number is(37±3)、(64±5)and (175±10) respectively,shRNA-dbpA is significantly higher than that in the other two groups(P<0.01). Conclusion Lentivirus-mediated RNAi targeting dbpA can effectively suppress the expression of dbpA in SW620 cells and decrease the proliferation of the colorectal cells. Key words: DNA binding protein A; RNA interference; SW620 cells; Proliferation