RNA interference mediated silencing of alpha-synuclein in MN9D cells and its effects on cell viability.
Neurosci Bull
; 24(2): 96-104, 2008 Apr.
Article
en En
| MEDLINE
| ID: mdl-18369389
ABSTRACT
OBJECTIVE:
To 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.METHODS:
We 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).RESULTS:
The 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.CONCLUSION:
Our 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.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Enfermedad de Parkinson
/
Silenciador del Gen
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Interferencia de ARN
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Alfa-Sinucleína
/
Neuronas
Límite:
Animals
Idioma:
En
Revista:
Neurosci Bull
Asunto de la revista:
NEUROLOGIA
Año:
2008
Tipo del documento:
Article
País de afiliación:
China