Construction of lentiviral vector of siRNA specific for osteopontin and its expression in U251 glioma cell line

AIM: To observe the influence of lentiviral vector-mediated RNA interference on expression of human osteopontin(OPN) gene in human glioma cell line U251 so as to pave a way for OPN gene-targeted gene therapy of glioma.METHODS:Gene engineering technique was used to screen 2 RNA interference sequences targeting OPN gene.The sequences were separately cloned into the pGCL-GFP vector to construct LV-OPNshRNA1 and LV-OPNshRNA2,which were subsequently confirmed by PCR and DNA sequencing analysis.The titer of lentivirus was determined after 293T cells were cotransfected with LV-OPNshRNA,pHelper 1.0 and pHelper 2.0.The 2 kinds of recombinant 1entiviruses were injected into U251 cells,the OPN mRNA and protein expression were examined by real-time PCR and Western blotting and the results were compared with those of the non-transfected and blank vector transfected U251 cells.RESULTS: PCR analysis and DNA sequencing confirmed that the 2 OPN shRNA sequences were successfully inserted into the lentiviral vectors.The titers of concentrated virus were 8×1010 and 5×1010 TU/L.After transfection with LV-OPNshRNA,OPN expression in U251 cells was significantly inhibited at both mRNA and protein levels compared with that in non-transfected and empty vector transfected U251 cells.CONCLUSION: Two lentiviral RNAi vectors of OPN gene have been successfully constructed and they can effectively inhibit the expression of OPN gene in U251 cells in vitro.