Construction Of Lentiviral Vector Of SiRNA Specific For Osteopontin And Characterization Of Its Efficency In U251 Glioma Cell Line

Objective:To observe the influence of lentiviral vector-mediated small interference RNA on expression of human osteopontin gene in human glioma cell line U251,so as to pave a way for OPN gene-targeted gene therapy of glioma.Ethods:Gene engineering technique was used to screen small interference RNA sequences targeting OPN gene,the sequences were separately cloned into the pGCL-GFP vector to construct LV-OPNshRNA,which was subsequently confirmed by PCR and DNA sequencing analysis.The titer of 1entivirus was determined after 293T cells were cotransfected with LV-OPNshRNA,pHelper 1.0,pHelper 2.0.The recombinant 1entiviruse was injected into U251 cells and the mRNA and protein expression were examined by RT-PCR and Western blotting,The proliferation of U251 was detected by MTT assay.The effects of LV-OPNshRNA on invasion of the transfected cells were investigated by reconstituted matrigel invasion and polycarbonate filters migration experiments.Result:1. Lentiviral siRNA vectors of OPN gene have been successfully constructed.2. After transfection with LV-OPNshRNA,OPN expression in U251 cells was significantly inhibited at both mRNA and protein levels compared with the non-transfected and empty vector transfected U251 cells.3. After transfection with LV-OPNshRNA,MTT assay showed that the proliferation of U251 cells was significantly inhibited compared with the non-transfected and empty vector transfected U251 cells.4. After transfection with LV-OPNshRNA,the invasion of U251 cells was significantly inhibited compared with the non-transfected and empty vector transfected U251 cells.Conclusion:The OPN gene was successfully silenced in U251 cells by siRNA,the proliferation and invasion of U251 cells were obviously inhibited. And it implies that OPN gene knockdown as a new strategy in the clinical treatment of human glioma.