Construction Of Lentiviral Vector-mediated RNA Interference Of Angiopoietin-2 Gene And Its Inhibition On Human Glioma In Vitro And In Vivo

In our research ,angiopietin-2 gene should be a target gene.The lentiviral vector-mediated RNA interference(RNAi) of Ang-2 gene has been constructed and expressed successfully in the transfected U251 cell line. We investigated whether RNAi of Ang-2 gene could inhibit the growth of U251 cells in vitro and in vivo.The possible mechanism was discussed. Therefore we performed the following three part experiments.PART ONEConstruction and Identification of Lentiviral Vector-mediated RNA Interference of Ang-2 GeneObjective To construct a lentiviral vector-mediated RNA interference(RNAi) of Ang-2 gene and provide the basis for further experiments in vivo and in vitro. Methods An Ang-2 gene targeting miRNA was designed and synthesized.a plasmid pcDNA?6.2-GW/EmGFPmiR-Ang2 was constructed and comfirmed by PCR and sequencing. A lentiviral vector-mediated RNA interference(RNAi) of Ang-2 gene cloned into lentiviral expression vector pLenti6/v5-Dest.The recombined vector pLenti6/v5-Dest miRNA-Ang2 was cotransfected with the VIRAPOWER MIX packaging plasmids mixture into 293T cells by Lipofectamine 2000.Virus in the supernatant was collected and the virus titer was measured.Result PCR and DNA sequencing demonstrated that the inserted sequence were correct.The titer of virus was 8.26×107TU/ml. Conclusion The lentivirus RNAi vector targeting Ang-2 has been successfully constructed,which will provide a tool for the further study on function of Ang-2 gene in glioma cells and gene therapy.PART TWOGrowth Inhibition of Human Glioma Cell Line U251 in Vitro by stable transfer of Lentiviral Vector-mediated RNA Interference of Ang-2 Gene Objective To evaluate the expression of Ang-2 and VEGF protein in human glioma cells U251 transfected by the lentivirus RNAi vector targeting Ang-2 gene and investigate whether Ang-2 RNAi could inhibit the growth of human glioma cell line in vitro. Methods A lentivirus miRNAi expression vector targeting Ang-2 gene was constructed and packaged.U251 cell line ,an cultured human glioma cell line in vitro,was transfected lentivirus Ang-2 miRNAi vector and lentivirus vector separately.After transfection,the cells were selected by blasticidin.Then resistant clones were chosen and cultured,with parental U251 cells as a control.MTT assay was used to detect cell viability.ELISA was determined protein of Ang-2 expression and Western blot was done to determine whether protein of VEGF had expressed.Result MTT assay showed the group transfected with a lentivirus Ang-2 miRNAi vector had a much lower cell viability than other groups. ELISA showed that the group transfected with a lentivirus Ang-2 miRNAi vector had a much lower protein expression of Ang-2 than other groups and western blot showed the similary result of protein expression of VEGF in three groups.Conclusion The lentivirus miRNAi vector targeting Ang-2 gene shows high efficiency on infecting glioma U251 cells line and significantly inhibits the expression and activation of Ang-2 and VEGF in cells in vitro. Ang-2 silencing by lentivirus vector significantly suppressed proliferation and maybe inhibit the expression of VEGF in cells. Lentivirus vector mediated miRNAi targeting Ang-2 gene may serve as a novel therapeutic strategy for treatment of glioma .PART THREEEffects of Lentiviral Vector-mediated RNA Interference of Ang-2 Gene transfection on tumorigenicity and angioenesis of human glioma cellsobjective To investigate the biological effects of expression of lentivirus Ang-2 miRNAi vector in human glioma U251 cell line and explore the role of Ang-2 gene in the growth and angiogenesis of glioma.Methods U251 cell line was transfected lentivirus Ang-2 miRNAi vector and lentivirus vector separately. Parental U251 cells were used as a control .24 nude mice were divided into three groups freely. Then the U251 cells were subcutaneously injected into these nude mice. After injection, the tumor mass grew and could be detected in 1 week. Result The tumor grew faster and was bigger in the nontransfected mice than in transfected ones with lentivirus Ang-2 miRNAi vector .The average number of newly formed vessels in the transfected mice with lentivirus Ang-2 miRNAi vector was 26.93±4.24/HP at the end of the 4th week. In the non-transfected group and transfected with lentivirus vector group, it was 51.23±7.35/HP and 48.16±6.71/HP, respectively . There was significant difference between transfected with lentivirus Ang-2 miRNAi vector group and the other groups (p <0.05). Conclusion The lentivirus Ang-2 miRNAi vector may suppress growth and angiogenesis in the human U251 glioma apparently and Ang-2 might have the effect of promoting the angiogenesis of the tumor.