mTOR 通路的激活通过上调 CD44 的表达促进神经突生长。
Activation of the mTOR pathway promotes neurite growth through upregulation of CD44 expression.
机构信息
Department of Pathology, Dushu Lake Hospital Affiliated to Soochow University, Suzhou, Jiangsu, 215123, China.
Department of Histology and Embryology College of Basic Medical Sciences, Jilin University, No. 828, Xinmin Road Changchun 130061, China.
出版信息
J Int Med Res. 2023 Jun;51(6):3000605231178510. doi: 10.1177/03000605231178510.
OBJECTIVE
To explore the intrinsic mechanism of the mammalian target of rapamycin (mTOR) pathway activation and promotion of neuronal axon growth.
METHODS
Human neuroblastoma cells, SH-SY5Y, were induced with all-trans retinoic acid (ATRA; 10 μM for three days) which differentiated the cell line into a neuronal-like state. Immunohistochemical staining was used to detect the differentiation status of the neuronal-like cells. Phosphatase and tensin homolog (PTEN) RNA interference (RNAi) experiments were performed on the differentiated cells; reverse transcription-polymerase chain reaction (RT-PCR) detected transcriptional levels of PTEN following 24 h of interference. After 36 h, western blot assay was used to detect expression levels of ribosomal protein S6 kinase (pS6k) and mTOR. To downregulate the expression of PTEN and cluster of differentiation 44 (CD44), a cell-surface glycoprotein, simultaneously, PTEN siRNA and CD44 siRNA sequences were mixed in equal proportions in co-interference experiments. RT-PCR detected the transcription level of CD44, and the relationship between the CD44 and axonal growth was observed after 48 h of interference.
RESULTS
Microtubule-associated protein 2 (MAP2) expression was enhanced after three days of induction in SH-SY5Y cells. RT-PCR showed the transcription level of PTEN was significantly downregulated after 24 h of PTEN knockdown. mTOR and pS6k protein expression levels were significantly upregulated after 36 h of interference. CD44 transcription levels were upregulated after PTEN gene interference. The neurite length of the cells in the experimental interference group was significantly longer than that in the control group, and the expression level of CD44 was positively correlated with neurite growth. The neurite length of the PTEN-only interference group was significantly greater than that of the co-interference and ATRA groups.
CONCLUSION
Activation of the mTOR pathway promoted neurite growth through upregulation of CD44 expression, thus promoting neuronal regeneration.
目的
探讨哺乳动物雷帕霉素靶蛋白（mTOR）通路激活促进神经元轴突生长的内在机制。
方法
用全反式视黄酸（ATRA；10 μM，3 天）诱导人神经母细胞瘤细胞 SH-SY5Y 向神经元样细胞分化，免疫组织化学染色检测神经元样细胞的分化状态。对分化细胞进行磷酸酶和张力蛋白同源物（PTEN）RNA 干扰（RNAi）实验；干扰 24 h 后，反转录-聚合酶链反应（RT-PCR）检测 PTEN 的转录水平。36 h 后，采用 Western blot 检测核糖体蛋白 S6 激酶（pS6k）和 mTOR 的表达水平。为了同时下调 PTEN 和细胞表面糖蛋白 CD44 的表达，在共干扰实验中，将 PTEN siRNA 和 CD44 siRNA 序列以相等的比例混合。RT-PCR 检测 CD44 的转录水平，干扰 48 h 后观察 CD44 与轴突生长的关系。
结果
在 SH-SY5Y 细胞诱导 3 天后，微管相关蛋白 2（MAP2）表达增强。RT-PCR 显示，PTEN 敲低 24 h 后，PTEN 的转录水平显著下调。干扰 36 h 后，mTOR 和 pS6k 蛋白表达水平显著上调。PTEN 基因干扰后 CD44 转录水平上调。实验组细胞的神经突长度明显长于对照组，CD44 的表达水平与神经突生长呈正相关。PTEN 单独干扰组的神经突长度明显大于共干扰组和 ATRA 组。
结论
mTOR 通路的激活通过上调 CD44 的表达促进轴突生长，从而促进神经元再生。
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