Inhibitory effects of lentiviral vector-mediated RNA interference on proliferation-inducing ligand expression in human pancreatic cancer in vitro
10.3724/SP.J.1008.2008.00053
- Author:
Feng WANG
1
Author Information
1. Department of Laboratory Medicine
- Publication Type:Journal Article
- Keywords:
A proliferation-inducing ligand;
Lentivirus;
Pancreatic cancer;
RNA interference
- From:
Academic Journal of Second Military Medical University
2010;29(1):53-58
- CountryChina
- Language:Chinese
-
Abstract:
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 (P<0.05). 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% (P<0.05), respectively; there was no significantly difference between the nontransfected 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.
