OBJECTIVES: Reticulon proteins, which are localized in the endoplasmic reticulum, have recently been shown to be involved in hormone secretion, in particular RTN1 and RTN3. The aim of the present study was to examine the effects of Nogo-A gene expression knockdown by RNA interference (RNAi) on dopamine release in PC12 cells. METHODS: A small hairpin RNA (shRNA) eukaryotic expression vector, targeting the Nogo-A gene, was constructed and transfected into cultured PC12 cells by lipofecamine2000. Inhibition of Nogo-A gene expression was detected by semi-quantitative reverse transcription PCR and Western blot analysis. Following transfection, dopamine release was detected by high performance liquid chromatography. RESULTS: The pGenesil-1-Nogo-A-2 plasmid was identified by gene sequencing. After transfection of the recombinant vector in PC12 cells, Nogo-A gene expression was significantly inhibited (p<0.01). Compared with the empty vector control group, dopamine release significantly decreased within 48 hours after transfection. CONCLUSION: Results from this study suggest that Nogo-A might be involved in the mechanism of DA release in PC12 cells.
OBJECTIVES: Reticulon proteins, which are localized in the endoplasmic reticulum, have recently been shown to be involved in hormone secretion, in particular RTN1 and RTN3. The aim of the present study was to examine the effects of Nogo-A gene expression knockdown by RNA interference (RNAi) on dopamine release in PC12 cells. METHODS: A small hairpin RNA (shRNA) eukaryotic expression vector, targeting the Nogo-A gene, was constructed and transfected into cultured PC12 cells by lipofecamine2000. Inhibition of Nogo-A gene expression was detected by semi-quantitative reverse transcription PCR and Western blot analysis. Following transfection, dopamine release was detected by high performance liquid chromatography. RESULTS: The pGenesil-1-Nogo-A-2 plasmid was identified by gene sequencing. After transfection of the recombinant vector in PC12 cells, Nogo-A gene expression was significantly inhibited (p<0.01). Compared with the empty vector control group, dopamine release significantly decreased within 48 hours after transfection. CONCLUSION: Results from this study suggest that Nogo-A might be involved in the mechanism of DA release in PC12 cells.
Authors: James B Summerville; Joseph F Faust; Ethan Fan; Diana Pendin; Andrea Daga; Joseph Formella; Michael Stern; James A McNew Journal: J Cell Sci Date: 2016-02-23 Impact factor: 5.285