Inhibitory effects of lentiviral vector-mediated RNA interference on proliferation-inducing ligand expression in human pancreatic cancer in vitro

Objective:To observe the influence of lentiviral vector-mediated RNA interference on expression of human APRIL(a proliferation-inducing ligand)gene in human pancreatic cancer cell line CFPAC-1,so as to pave a way for APRIL gene-targeted gene therapy of pancreatic cancer.Methods:Gene engineering technique was used to screen 3 RNA interference sequences targeting APRIL gene;the sequences were separately cloned into the pGCL-GFP vector to construct LV-APRIL shRNA1,LV-APRIL shRNA2 and LV-APRIL shRNA3,which were subsequently confirmed by PCR and DNA sequencing analysis.The titer of lentivirus was determined after 293T cells were cotransfected with LV-APRIL shRNA,pHelper 1.0 and pHelper 2.0.The 3 kinds of recombinant lentiviruses were injected into CFPAC-1 cells and the APRIL mRNA and protein expression were examined by real-time RT-PCR and Western blotting,respectively,and the result was compared with those of the non-transfected and blank vector transfected CFPAC-1 cells.Results:PCR analysis and DNA sequencing confirmed that the 3 APRILshRNA sequences were successfully inserted into the lentiviral vectors.The titers of concentrated virus were 5×107 TU/ml,6×107 TU/ml and 4×107 TU/ml,respectively.APRIL expression in CFPAC-1 cells was significantly inhibited at both mRNA and protein levels compared with the non-transfected and empty vector transfected CFPAC-1 cells(P0.05).After transfection with LV-APRIL shRNA1 and LV-APRIL shRNA2,APRIL mRNA expression decreased by 73% and 68%,APRIL protein expression decreased by 66% and 59%(P0.05),respectively;there was no significantly difference between the non-transfected and empty vector transfected CFPAC-1 cells.Conclusion:Three lentiviral RNAi vectors of APRIL gene have been successfully constructed,and they can effectively inhibit the expression of APRIL gene in CFPAC-1 cells in vitro.