Effects Of SiRNA Targeting PGC-1α Gene On SH-SH5Y Cell Induced By MPP~+

BackgroundParkinson disease（PD） is a high incidence of neurodegenerative diseases in elderlypeople, and attracted widespread attention because of the high incidence and lack ofeffective treatment. Mitochondrial dysfunction plays an important role in PDpathogenesis mechanism. Peroxisome proliferator-activated receptor1alpha (PGC-1α)is a multifunctional transcription factor and widely involved in mitochondrialbiogenesis, glucolipid metabolism as well as antioxidant pathways regulating. In recentyears, it is generally considered the PGC-1α may become an important therapeutictarget for early intervention in PD. But it is not clear that PGC-1α signal transduction inthe pathogenesis of PD activity of mitochondrial biogenesis mechanism. RNAinterference (RNAi) is an evolutionary method that effects gene post-transcriptionalregulation and expression. RNAi，having highly specific sequence and inhibiting targetgene expression accordingly,has developed as a powerful tool for experimentalapplication.ObjectiveIn this study,the adenovirus vector-based siRNA targeting PGC-1α gene was transfectedinto SH-SY5Y cell. The most efficient of interference sequence was chosen out, furthertransfected into SH-SY5Y cell and assessed the impact of down-expressed of PGC-1αgene on SH-SH5Y cell induced by MPP~+.MethodsImmunohistochemistry was used to identify the expression of tyrosine hydroxylase.SH-SY5Y cells treated with MPP~+were used as the in vitro cell model. A MTT assaywas used to investigate the effects of MPP~+. The SH-SY5Y cell were transfected withadenovirus mediating green fluorescent protein（Ad-GFP）separately at1,10,50,100,200moltiplicity of infection(MOI).The suitable MOI was determine after transfer efficiencywas tested by fluorescence microscope. PGC-1α mRNA and protein level were detected by real-time PCR and Western blot. mitochondrial membrane potential level weredetected by flow cytometry(FCM),ATP level were detected by luciferase drivenbioluminescence,H2O2level level were detected by fluorescence analysis (Amplexred hydrogen peroxide assay kit).ResultsThe SH-SY5Y Cells contain tyrosine hydroxylase. After the treat of MPP~+, cellviability was significantly decreased..Results indicated that the transfer efficiency ofthe adenovirus to SH-SY5Y was high, and50MOI was the suitable MOI．Four pairs ofPGC-1α specific siRNA can regulate down the PGC-1α gene mRNA and proteinexpression in SH-SY5Y cell. The most efficient interference sequence was chosen out,which further provide an experimental basis for PGC-1α gene silencing. siRNAinterference targeting PGC-1α gene promoted the MPP~+-induced mitochondrialdamages，including mitochondrial membrane potential, ATP content,H2O2generation.Conclusion1. Adenovirus is a high transfection rate of SH-SY5Y cells. The most efficientinterference sequence was chosen out, which further provide an experimental basis forPGC-1α gene silencing in targeting therapy of PD.2. siRNA interference targeting PGC-1αgene may cause mitochondrial dysfunction ofSH-SY5Y cell, and induced the cell apoptosis.