The Effect Of P11 Protein Expression On Autophagy In SH-SY5Y Cells

BackgroundAutophagy is a defense mechanism inherent in cells,it can maintain normal metabolism under normal circumstances,it plays an important role in keeping a stable intracellular environment when cells occur in nutritional deprivation,lacking of energy,etc.Depression is a complex and multi-factorial disease,it is reported that there is a close relationship between the pathogenesis of depression and the level of p11 protein in the brain.Furthermore,Other researchers reported that they observed autophagy up-regulation in the brain of the rat depression model,which evidence suggests that p11 may affect autophagy in a certain extent.The mechanism about how could p11 affect autophagy is unclear now.PurposeTo explore the effects of p11 protein expression on autophagyMethods1.Plasmid DNA transformation and extraction:the plasmid p11-pcDNA3.0,pcDNA3.0,p11-si,si-control were saved by our Lab,which have been verified by sequencing.The plasmids were transformed through E.coli DH5a Competent and extracted by micro-plasmid extraction kit.2.Plasmid verification:the plasmids were verified by agarose gel electrophoresis,quality control plasmid.3.Cells culture:SH-SY5Y were cultured in RPMI 1640 medium containing 10%inactivated fetal bovine serum which were transferred to the incubator containing 5%CO₂.The culture medium was changed daily,the cells were washed with PBS before changing liquid and passaged 2-3 times weekly.4.Cells transfection:the plasmids p11-pcDNA3.0,pcDNA3.0,p11-si,si-control were transfected into SH-SY5Y with FUGENE transfection plasmid.After 6 hrs the medium was replaced completely with fresh medium,the cells fluorescence was observed 24h after transfection and the cells were lyzed to extract the protein after 48h.5.Western blot detection of protein expression in cells:p11 protein expression and autophagy-related protein LC3,Atg5,p62 and other proteins.6.Statistical analysis:Quantity one analysis software and SPSS19.0 statistical software were used for data analysis.Results1.plasmids p11-pcDNA3.0,pcDNA3.0,p11-si and si-control were extracted successfully,the bands match with a molecular weight of plasmid through 1.2%agarose gel electrophoresis2.Fluorescence microscopy of green fluorescent display plasmids were thransfected in SH-SY5Y successfully.（plasmids p11-si and si-control with a green fluorescent gene）3.Western Blot:to transfect SH-SY5Y with plasmid p11-pcDNA3.0,p11 expression was up-regulated,while to transfect SH-SY5Y with plasmid p11-si,p11 expression was suppressed.4.Effect of P11 on the autophagy-related protein expression:When p11 expression is high,the expression of LC3,p62and Atg5 were affected,but none statistically significant;when p11 expression is low,the expression of p62 significantly reduced,while the expression of LC3 and Atg5 were increased,autophagy was up-regulation.ConclusionHigh p11 expression can affect the levels of p62 and LC3 in SH-SY5Y cells.Lack of p11 expression can up-regulate autophagy levels in SH-SY5Y cells.