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| 敲除TMEM206基因可降低由ataxin-1中polyQ过度延伸所致的细胞凋亡 |
| Knockout of the TMEM206 gene in SH-SY5Y cells alleviate cell apoptosis induced by polyQ-expanded ataxin-1 |
| 投稿时间：2024-06-26 |
| DOI：10.3969/j.issn.1000-0399.2025.07.002 |
| 中文关键词: 人神经母细胞瘤细胞 凋亡 TMEM206基因 共济失调蛋白-1 |
| 英文关键词: Neuroblastoma cells Apoptosis TMEM206 gene Ataxin-1 |
| 基金项目:无锡市卫生健康委员会科研项目（编号：M202301） |
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| 中文摘要: |
| 目的 研究ataxin-1分子中polyQ过度延伸对细胞凋亡的作用，探讨敲除TMEM206基因对由ataxin-1分子中polyQ过度延伸所致细胞凋亡的影响。方法 通过DNA合成法分别获得携带29Q、65Q、82Q重复序列的ATXN1基因，将合成的ATXN1基因分别插入慢病毒表达载体pMT440，携带合成基因ATXN1的慢病毒载体pMT440再与pCMV-dR8.9和pCMV-VSV-G载体共转染，构建成ATXN1基因慢病毒表达载体，并分别导入购买的3组人神经母细胞瘤培养细胞（SH-SY5Y）中表达，通过流式细胞技术检测细胞凋亡率。另通过短发夹RNA技术（shRNA）沉默SH-SY5Y细胞TMEM206基因，再向3组细胞中分别导入能表达含29Q、65Q、82Q重复序列的ATXN1基因慢病毒表达载体，培养24 h，通过流式细胞技术检测细胞凋亡率。结果 SH-SY5Y细胞中ataxin-1分子中polyQ重复序列过度延伸至82Q和65Q时，两组过度延伸细胞的凋亡率分别为（16.70±1.40）%和（11.70±0.90）%，均高于polyQ重复序列为29Q时的细胞凋亡率（8.07±0.67）%，3组不同polyQ重复序列细胞凋亡率的改变有统计学意义（F=34.892，P＜0.001）。敲除SH-SY5Y细胞TMEM206基因后，ataxin-1分子中polyQ重复序列过度延伸至82Q和65Q时，两组SH-SY5Y细胞的凋亡率分别为（14.00±1.44）%和（6.71±0.53）%，也高于polyQ重复序列为29Q时细胞凋亡率（6.47±0.69）%，三者凋亡率的差异有统计学意义（F=38.680，P＜0.001）。敲除SH-SY5Y细胞TMEM206基因，可降低ataxin-1分子中82Q、65Q或29Q重复序列引起的凋亡，但凋亡率的降低多无统计学意义（t=2.347、6.734、1.902；P=0.079、0.003、0.130）。结论 Ataxin-1分子中polyQ重复序列过度延伸可使细胞凋亡增加，敲除TMEM206基因，可降低ataxin-1分子中polyQ重复序列过度延伸所致的细胞凋亡。 |
| 英文摘要: |
| Objective To investigate the role of polyQ-expanded ataxin-1 on apoptosis in neuroblastoma cells, and to evaluate the effect of TMEM206 knockout in polyQ-expanded ataxin-1-induced apoptosis of SH-SY5Y cells.Methods The polyQ-expanded (65Q and 82Q) and wild (29Q) ATXN1 genes were constructed by DNA synthesis. The ATXN1 gene expression vectors were constructed by insertion the ATXN1 genes into lentivirus vectors pMT440, then were transduced into SH-SY5Y cells with pCMV-dR8 vectors and pCMV-VSV-G vectors. The TMEM206 gene was silenced using short hairpin RNA technology in SH-SY5Y cells. Apoptosis in SH-SY5Y cells was investigated using flow cytometry after cells were transfected with the ATXN1 genes for 24 h.Results The results showed that the apoptotic rate was (16.70± 1.40)% in SH-SY5Y cell lines stably expressing 82Q-expanded ataxin-1 and (11.70±0.90)% in SH-SY5Y cell lines stably expressing 65Qexpanded ataxin-1, and the apoptotic rate was (8.07±0.67)% in SH-SY5Y cells expressing wild ataxin-1 (29Q-expanded). The apoptotic rate increased in SH-SY5Y cells expressing 82Q-expanded and 65Q-expanded ataxin-1 compared with in those cells expressing wild ataxin-1. The changes in the apoptosis rate reached statistical difference among three cell lines (F=34.892, P＜0.001). The apoptotic rate was (14.00± 1.44)% in cells expressing 82Q-expanded ataxin-1 and (6.71±0.53)% in cells expressing 65Q-expanded ataxin-1, the apoptotic rate was (6.47±0.69)% in cells expressing wild ataxin-1 when the TMEM206 gene was knocked out in SH-SY5Y cells. The changes in the apoptosis rate also reached statistical difference among three SH-SY5Y cell lines when the TMEM206 gene was knocked out (F=38.680, P＜0.001). The TMEM206 gene knockout alleviated the SH-SY5Y cell apoptosis induced by polyQ-expanded ataxin-1. Most of the decrease in most apoptosis rate did not reach statistical difference among three cell lines (t=2.347, 6.734, 1.902； P=0.079, 0.003, 0.130).Conclusion The polyglutamine tract expansion of ataxin-1 can induce apoptosis in SH-SY5Y cells, the TMEM206 gene knockout can alleviate SH-SY5Y cell apoptosis induced by polyQ-expanded ataxin-1. |
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