Gene Silencing Characterization And Functional Study Of PSD-95 Small Interfering RNA

Introduction:Glutamate receptor-associated postsynaptic signal transduction,which is modulated by postsynaptic density protein 95(PSD-95),participates in the central sensitization of neuropathic pain.Objective:The PSD-95 gene silencing efficacy by small interfering RNA(siRNA) and its succedent influence on glutamate-induced toxicity or signal transduction are to be illustrated.Methods:Gene-specific siRNAs of rat PSD-95 were synthesized chemically for in vitro transfection into mouse neuroblastoma and rat glioma hybridoma(NG108-15) cells.The contributions of transfection agents,transfection methods,time course,cell density and siRNAs final concentration to the silencing efficacy were evaluated by real-time PCR. PSD-95 gene silenced NG108-15 cells were further stimulated by glutamate,with either the cell viability or the expression and phosphorylation of signal proteins to be assayed.Results:The siRNA,which is exclusively identical to rat PSD-95,decreased PSD-95 mRNA level by 91.5%under appropriate conditions.PSD-95 gene silencing enhanced cellular tolerance against the glutamate toxicity,meanwhile the phosphorylation of Ca²⁺/Camodulin dependent protein kinaseⅡαisoform was attenuated.Conclusion:All results suggested the potential of PSD-95 gene specific siRNA in relieving neuropathic pain.