Inhibition Of Nogo-A Gene Expression By RNAi And Its Effect On Dopamine Release And Apoptosis Of PC12 Cells

PartⅠConstruction of Eukaryotic Expressing Vector of shRNA Targeting Nogo-A GeneObjective To construct eukaryotic expression vector of short hairpin RNA(shRNA) targeting Nogo-A gene. Methods Genome sequences of Nogo-A gene was retrieved from Genbank.Two short hairpin RNA DNA sequence was designed and chemical synthesized.After annealing,the shRNA was inserted into the downstream of U6 promoter of plasmid pGenesil-1 and construct the recombinant vector,then verified by the restriction map and gene sequence analysis. Result The gene sequence analysis results proved that the shRNA coding sequences had been inserted the designed site. Conclusion The eukaryotic expression vector of shRNA targeting Nogo-A gene has been constructed successfully and provide a new way to study the function of Nogo-A protein and gene therapy of axonal regeneration inhibition.PartⅡA study of Inhibiting Nogo-A gene expression in PC12 cells by Short hairpin RNAObjective To study the inhibitive effect of Nogo-A shRNA in PC12 cells and lay base for further study of Nogo-A gene function.Methods The recombinant plasmid was transfected into PC12 cells by lipofecamine 2000,the EGFP expression was detected by fuorescent microscopy and flow cytometry,the mRNA and protein expression level of Nogo-A gene was detected by RT-PCR and Western blot methods after 48h repectively.Results There are no significant change of the Nogo-A mRNA and protein expression level in empty vector-transfected group in comparison to the control group (p>0.05),while the expression level in shRNA-transfected group decreased significantly(p<0.05).ConclusionThe pGenesil-1/Nogo–A shRNA recombinant plasmid can effective suppress the expression of Nogo-A gene in PC12 cells.PartⅢInhibition of Nogo-A gene expression by RNAi and its effects on Dopamine release in PC12 CellsObjectiveTo study the effects on dopamine release of PC12 cells after knocking down Nogo-A gene and explore the fuction of Nogo-A in neuroendocrine process. Methods The short hairpin RNA(shRNA) eukaryotic expression vector targeting Nogo-A gene was constructed and transfected into cultured PC12 cells by lipofecamine 2000.The dopamine release of each group was detected by HPLC(high performance liquid chromatography) technique in 24h,48h after transfection.Results HPLC showed the release of dopamine in PC12 cells was significantly lower than those in control group in 48h after transfection (P<0.05),while there was no significantly change in the empty vector group (P>0.05).Conclusion Nogo-A gene silence can inhibite the realese of dopamine in PC12 cells. It suggested that Nogo-A may involved in the mechanism of dopamine realese. Part IV Inhibition of Nogo-A gene expression by RNAi and its effects on the apoptosis of PC12 CellsObjective To study the effects of Nogo-A gene silence on the apoptosis of PC12 cells. Methods The short hairpin RNA(shRNA) eukaryotic expression vector targeting Nogo-A gene was constructed and transfected into cultured PC12 cells by lipofecamine 2000.At different time point after transfection,the cell viability of each group were assayed by MTT colorimetry and the cell apoptosis was studied by TUNEL assay and flow cytometry.Results Compared with the control group,the cell viability of Nogo-A shRNA transfected group increased significantly(P<0.05),the cell apoptosis rate decreased significantly(P<0.05).Conclusion Nogo-A gene might relate to the tumor cell apoptosis.