RNA interference mediated silencing of alpha-synuclein in MN9D cells and its effects on cell viability.
- Author:
Dong-Mei LIU
1
;
Ling JIN
;
Hao WANG
;
Huan-Ying ZHAO
;
Chun-Li ZHAO
;
Hui YANG
Author Information
- Publication Type:Journal Article
- MeSH: Animals; Cell Line; Cell Proliferation; Cell Survival; drug effects; genetics; Down-Regulation; drug effects; genetics; Gene Silencing; Genetic Vectors; genetics; Hybridomas; Mice; Mice, Inbred C57BL; Nerve Degeneration; genetics; metabolism; Neurons; drug effects; metabolism; pathology; Oligonucleotides; genetics; Parkinson Disease; genetics; metabolism; Plasmids; genetics; RNA Interference; RNA, Double-Stranded; genetics; pharmacology; RNA, Small Interfering; genetics; Transfection; methods; alpha-Synuclein; genetics; metabolism
- From: Neuroscience Bulletin 2008;24(2):96-104
- CountryChina
- Language:English
-
Abstract:
OBJECTIVETo silence the expression of alpha-synuclein in MN9D dopaminergic cells using vector mediated RNA interference (RNAi) and examined its effects on cell proliferation and viability.
METHODSWe identified two 19-nucleotide stretches within the coding region of the alpha-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. alpha-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-di- phenytetrazoliumromide (MTT).
RESULTSThe mRNA and protein levels of alpha-synuclein gene were significantly down-regulated in pSH2/alpha-SYN-transfected cells compared with control MN9D and pSH/CON-transfected MN9D cells, while pSH1/alpha-SYN-transfected cells showed no significant difference. Silencing alpha-synuclein expression does not affect cell proliferation but may decrease cell viability.
CONCLUSIONOur results demonstrated pSH2/alpha-SYN is an effective small interfering RNA (siRNA) sequence and potent silencing of mouse alpha-synuclein expression in MN9D cells by vector-based RNAi, which provides the tools for studying the normal function of alpha-synuclein and examining its role in Parkinson's disease (PD) pathogenesis. alpha-Synuclein may be important for the viability of MN9D cells, and loss of alpha-synuclein may induce cell injury directly or indirectly.
