南方医科大学学报

南方医科大学学报 ›› 2022, Vol. 42 ›› Issue (10): 1462-1469.doi: 10.12122/j.issn.1673-4254.2022.10.04

• • 上一篇    下一篇

Numb通过上调V1G1的表达激活近端肾小管细胞mTORC1信号通路

刘 泽，尤 达，李 勇，何咏梅，李阿芳，李 潘，李春艳   

  1. 湘南学院护理学院，临床学院，湖南 郴州 423000；湖南中医药大学护理学院，湖南 长沙 410000
  • 出版日期:2022-10-20 发布日期:2022-10-31

Numb activates the mTORC1 signaling pathway in proximal tubular epithelial cells by upregulating V1G1 expression

LIU Ze, YOU Da, LI Yong, HE Yongmei, LI Afang, LI Pan, LI Chunyan   

  1. School of Nursing, School of Clinical Medicine, Xiangnan University, Chenzhou 423000, China; School of Nursing, Hunan University of Chinese Medicine, Changsha 410000, China
  • Online:2022-10-20 Published:2022-10-31

RichHTML

59

PDF

407

摘要： 目的 探讨Numb对哺乳动物雷帕霉素靶蛋白（mTOR）复合物1（mTORC1）信号通路活性的影响及潜在的分子机制。方法 建立Numb-siRNA转染和/或顺铂诱导的雄性BALB/c 小鼠急性肾损伤模型，分为4组：NC-siRNA、Numb-siRNA、NCsiRNA+顺铂、Numb-siRNA+顺铂。免疫组化检测肾脏Numb、Megalin的表达与分布；Western blot检测各组动物肾脏中Numb、S6、p-S6、S6K1、p-S6K1、4EBP1、p-4EBP1的蛋白表达。分离培养Numb-siRNA转染的小鼠原代近端肾小管上皮细胞。体外建立Numb-siRNA转染和/或氨基酸刺激的大鼠肾小管上皮NRK-52E细胞模型，分为NC-siRNA组、Numb-siRNA组、NC-siRNA+氨基酸刺激组和 Numb-siRNA+氨基酸刺激组。Western blot检测各组细胞Numb、S6、p-S6的蛋白表达，检测沉默Numb表达前后的NRK-52E细胞、小鼠肾脏及原代近端肾小管上皮细胞中AMPK、p-AMPK的蛋白表达。V1G1过表达及空白对照慢病毒感染、Numb-siRNA转染和/或氨基酸刺激NRK-52E细胞，分为NC-siRNA+空载慢病毒+氨基酸刺激组、Numb-siRNA+空载慢病毒+氨基酸刺激组及Numb-siRNA+V1G1过表达慢病毒+氨基酸刺激组；Western blot检测V1G1、S6、p-S6的蛋白表达。结果 与NC-siRNA组相比，Numb-siRNA组小鼠肾脏近端肾小管上皮细胞Numb及p-S6的蛋白表达均下调（P<0.05），但不影响近端小管标记蛋白Megalin的表达与分布；与对照组小鼠相比，顺铂处理组小鼠肾脏p-S6K1及p-4EBP1的蛋白表达均上调（P<0.05），而Numb-siRNA处理组小鼠抑制顺铂介导的p-S6K1及p-4EBP1的蛋白表达水平的上调（P<0.05）。在NRK-52E细胞中，与氨基酸饥饿组相比，氨基酸刺激组细胞p-S6的蛋白表达上调（P<0.05），而转染Numb-siRNA的细胞中Numb及p-S6的蛋白表达较对照组均下调（P<0.05）；在Numb表达水平下调的NRK-52E细胞、小鼠肾脏及小鼠原代近端肾小管上皮细胞中，p-AMPK的蛋白表达均下调（P<0.05）；在Numb表达水平下调的NRK-52E细胞中，V1G1的蛋白表达下调（P<0.05），上调NRK-52E细胞中V1G1的蛋白表达，可以逆转Numb沉默介导的p-S6的降低（P<0.05）。结论 Numb可通过上调V1G1的蛋白表达促进近端肾小管上皮细胞mTORC1信号通路的活化。

关键词: Numb；mTOR复合物1；AMP活化蛋白激酶；V1G1；NRK-52E细胞

Abstract: Objective To investigate the role of Numb in regulating mammalian target of rapamycin (mTOR) complex 1 (mTORC1) signaling pathway. Methods Male BALB/C mouse models of acute kidney injury (AKI) were subjected to intravenous injections of Numb-siRNA or NC-siRNA with or without intraperitoneal cisplatin injections. After the treatments, the expressions and distribution of Numb and megalin in the renal tissues of the mice were detected with immunohistochemistry, and the renal expressions of Numb, S6, p-S6, S6K1, p-S6K1, 4EBP1 and p-4EBP1 were examined with Western blotting. The proximal renal tubular epithelial cells were isolated from the mice transfected with Numb-siRNA for in vitro culture. In NRK-52E cells, the effects of amino acid stimulation, Numb knockdown, and V1G1 overexpression, alone or in combination, on expressions of Numb, S6 and p-S6 were detected with Western blotting; the expressions of AMPK and p-AMPK were also detected in transfected NRK-52E cells, mouse kidneys and cultured mouse renal tubular epithelial cells. Results In BALB/C mice, injection of Numb-siRNA caused significant reductions of Numb and p-S6 expressions without affecting megalin expression in the renal proximal tubules (P<0.05). Cisplatin treatment obviously upregulated p-S6K1 and p-4EBP1 expressions in the kidneys of the mice (P<0.05), and this effect was significantly inhibited by treatment with Numb-siRNA (P<0.05). In NRK-52E cells, amino acid stimulation significantly upregulated the expression of p-S6 (P<0.05), which was strongly suppressed by transfection with Numb- siRNA (P<0.05). Numb knockdown inhibited AMPK activation in NRK-52E cells, mouse kidneys and primary proximal tubular epithelial cells (P<0.05). Numb knockdown significantly downregulated V1G1 expression in NRK-52E cells (P<0.05), and V1G1 overexpression obviously reversed the inhibitory effect of Numb-siRNA on S6 phosphorylation (P<0.05). Conclusion Numb promotes the activation of mTORC1 signaling in proximal tubular epithelial cells by upregulating V1G1 expression.

Key words: Numb; mTOR complex 1; AMP activated protein kinas; V1G1; NRK-52E cells