RNA interference mediated silencing of α-synuclein in MN9D cells and its effects on cell viability

Abstract

Objective: To silence the expression of α-synuclein in MN9D dopaminergic cells using vector mediated RNA interference (RNAi) and examined its effects on cell proliferation and viability. Methods: We identified two 19-nucleotide stretches within the coding region of the α-synuclein gene and designed three sets of oligonucleotides to generate double-stranded (ds) oligos. The ds oligos were inserted into the pENTR™/H1/TO vector and transfected into MN9D dopaminergic cells. α-Synuclein expression was detected by RT-PCR, real-time PCR, immunocytochemistry staining and Western blot. In addition, we measured cell proliferation using growth curves and cell viability by 3-(4, 5)-dimethylthiahiazo (-z-y1)-3, 5-diphenytetrazoliumromide (MTT). Results: The mRNA and protein levels of α-synuclein gene were significantly down-regulated in pSH2/α-SYN-transfected cells compared with control MN9D and pSH/CON-transfected MN9D cells, while pSH1/α- SYNtransfected cells showed no significant difference. Silencing α-synuclein expression does not affect cell proliferation but may decrease cell viability. Conclusion: Our results demonstrated pSH2/α-SYN is an effective small interfering RNA (siRNA) sequence and potent silencing of mouse α-synuclein expression in MN9D cells by vector-based RNAi, which provides the tools for studying the normal function of α-synuclein and examining its role in Parkinson's disease (PD) pathogenesis. α-Synuclein may be important for the viability of MN9D cells, and loss of α-synuclein may induce cell injury directly or indirectly. © 2008 Shanghai Institutes for Biological Sciences, CAS and Springer-Verlag GmbH.