Neuroscience Bulletin

, Volume 24, Issue 2, pp 96–104 | Cite as

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

  • Dong-Mei Liu (刘冬梅)
  • Ling Jin (金玲)
  • Hao Wang (王浩)
  • Huan-Ying Zhao (赵焕英)
  • Chun-Li Zhao (赵春礼)
  • Hui Yang (杨慧)
Original Article
First Online:
Received:

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.

Keywords

α-Synuclein RNA interference Parkinson’s disease 

RNA 干扰介导的 MN9D 细胞中 α-synuclein 表达抑制及其对细胞活力的影响

摘要

目的

使用载体介导的RNA 干扰(RNA interference, RNAi)方法抑制MN9D 细胞中的α-synuclein 基因表达, 并检测其对细胞增殖和活力的影响.

方法

在α-synuclein 的开放读码区选择两段19 个核苷酸的片断, 并选择一段与小鼠基因无同源性的阴性对照序列, 分别设计合成单链寡核苷酸, 退火融合成双链 寡核苷酸. 将双链 寡核苷酸克隆入pENTR/H1/TO 质粒载体, 鉴定正确后, 转染MN9D 细胞, 通过筛选获得稳定的细胞克隆. 使用RT-PCR、Real-time PCR、免疫细胞化学染色和Western blot 方法检测细胞内的α-synuclein 的表达水平, 鉴定RNAi 的效率. 利用生长曲线和MTT法分别检测比较各组细胞的增殖和活力.

结果

转染pSH2/α-SYN的细胞与正常和转染pSH/CON的MN9D细胞相比, α-synuclein mRNA及蛋白水平均明显减少; 而转染pSH1/α-SYN的细胞α-synuclein mRNA及蛋白水平与两组对照细胞没有明显差别. 抑制gα-synuclein 表达不影响细胞增殖, 但可以降低细胞活力.

结论

结果表明, SH2/α-SYN是有效的小干扰RNA (small interfering RNA, siRNA)序列, 通过载体介导的RNAi方法可以有效抑制MN9D细胞中的α-synuclein表达, 这为进一步研究α-synuclein蛋白的生理功能及其在帕金森病(Parkinson’s disease, PD)发病中的作用提供了良好的细胞模型; α-synuclein在维持MN9D细胞活力中起到重要作用, α-synuclein功能缺失会导致细胞直接或间接的损伤.

关键词

α-Synuclein RNA 干扰 帕金森病 

CLC number

Q71 
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Copyright information

© Shanghai Institutes for Biological Sciences, CAS and Springer-Verlag GmbH 2008

Authors and Affiliations

  • Dong-Mei Liu (刘冬梅)
    • 1
  • Ling Jin (金玲)
    • 1
  • Hao Wang (王浩)
    • 2
  • Huan-Ying Zhao (赵焕英)
    • 1
  • Chun-Li Zhao (赵春礼)
    • 1
  • Hui Yang (杨慧)
    • 1
  1. 1.Beijing Institute for NeuroscienceCapital Medical University, Beijing Center of Neural Regeneration and Repair, Key Laboratory for Neurodegenerative Disease of the Ministry of EducationBeijingChina
  2. 2.Beijing Tiantan HospitalCapital Medical UniversityBeijingChina

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