title: RNA interference mediated silencing of alpha-synuclein in MN9D cells and its effects on cell viability.
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.
  
  
  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.
authors: 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.
authors: Dong-Mei
authors: DM
authors: Liu
authors: Ling
authors: L
authors: Jin
authors: Hao
authors: H
authors: Wang
authors: Huan-Ying
authors: HY
authors: Zhao
authors: Chun-Li
authors: CL
authors: Zhao
authors: Hui
authors: H
authors: Yang
mesh_terms: 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
chemical_list: D009841:Oligonucleotides; D012330:RNA, Double-Stranded; D034741:RNA, Small Interfering; D051844:alpha-Synuclein