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


CONCLUSIONS
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.
Liu
Dong-Mei
DM
Beijing Institute for Neuroscience, Capital Medical University, Beijing Center of Neural Regeneration and Repair, Key Laboratory for Neurodegenerative Disease of the Ministry of Education, Beijing 100069, China.
Jin
Ling
L
Wang
Hao
H
Zhao
Huan-Ying
HY
Zhao
Chun-Li
CL
Yang
Hui
H
D000818:Animals; D002460:Cell Line; D049109:Cell Proliferation; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D015536:Down-Regulation / Q000187:drug effects / Q000235:genetics; D020868:Gene Silencing; D005822:Genetic Vectors / Q000235:genetics; D006825:Hybridomas; D051379:Mice; D008810:Mice, Inbred C57BL; D009410:Nerve Degeneration / Q000235:genetics / Q000378:metabolism; D009474:Neurons / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D009841:Oligonucleotides / Q000235:genetics; D010300:Parkinson Disease / Q000235:genetics* / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000235:genetics / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000235:genetics; D014162:Transfection / Q000379:methods; D051844:alpha-Synuclein / Q000235:genetics* / Q000378:metabolism
D009841:Oligonucleotides; D012330:RNA, Double-Stranded; D034741:RNA, Small Interfering; D051844:alpha-Synuclein