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. 2022 Oct 20;42(10):1462–1469. [Article in Chinese] doi: 10.12122/j.issn.1673-4254.2022.10.04

Numb通过上调V1G1的表达激活近端肾小管细胞mTORC1信号通路

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

Ze LIU 1, Da YOU 2, Yong LI 1, Yongmei HE 1, Afang LI 1, Pan LI 1,*, Chunyan LI 1
PMCID: PMC9637490  PMID: 36329579

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.

Keywords: Numb, mTOR complex 1, AMP activated protein kinas, V1G1, NRK-52E cells

哺乳动物雷帕霉素靶蛋白（mTOR）是一种高度保守的丝氨酸-苏氨酸蛋白激酶，属于磷酸肌醇3-激酶相关激酶家族[1-3]。mTOR在细胞内与其他蛋白相互作用形成两种结构和功能不同的复合物：mTOR复合物1（mTORC1）和mTOR复合物2[4-6]。mTORC1通过整合营养因素、生长因子等信号，直接磷酸化其下游关键效应因子蛋白核糖体S6激酶1（S6K1）和4E结合蛋白1（4EBP1）促进蛋白质合成、细胞生长和增殖[7, 8]。在正常肾脏中，mTORC1在近端肾小管上皮细胞及其他肾实质细胞均有表达，对于维持细胞稳态具有重要作用[9-11]。近端肾小管上皮细胞mTORC1信号通路活性异常参与多种肾脏疾病的发生发展[11-14]。因此深入阐明近端肾小管上皮细胞中mTORC1信号通路的调控机制，有望为以mTORC1信号通路作为靶点治疗的肾脏疾病提供新思路和新靶点。

mTORC1受多种信号调节，如生长因子、氨基酸和细胞能量。AMP活化蛋白激酶（AMPK）作为一种关键的生理能量传感器，可通过磷酸化TSC2或直接通过磷酸化Raptor抑制mTORC1的活化[15, 16]。溶酶体作为氨基酸介导mTORC1活化的主要场所，其功能的维持对于mTORC1信号通路的活化具有重要作用[17, 18]。空泡型H+-ATP酶（V-ATPase）负责细胞内溶酶体等细胞器的酸化，对于维持溶酶体功能及mTORC1的活化具有重要作用[19, 20]。下调V-ATPase亚基V1G1的表达可抑制V-ATPase活性及mTORC1的活化[21]。Numb基因具有控制细胞不对称分裂和细胞命运选择的功能[22]。哺乳动物Numb有3个主要的功能结构域，通过与不同的蛋白分子相互作用，参与p53、Notch等多条信号通路的调控[23-26]。研究证明，沉默MCF-7细胞中Numb基因的表达抑制溶酶体功能[27]。但是，Numb在近端小管上皮细胞mTORC1信号通路活化中的作用及作用机制尚不清楚。

本研究采用近端肾小管上皮细胞株NRK-52E细胞、正常小鼠肾脏组织及发生AKI的小鼠肾脏组织为研究对象，证明了Numb具有促进mTORC1活化的作用，并阐明其机制可能与上调V1G1的蛋白表达有关。本研究为阐明近端肾小管上皮细胞mTORC1信号通路的调控机制提供新的线索，为mTORC1活性异常介导的肾脏疾病的治疗提供新靶点。

1. 材料和方法

1.1. 材料与试剂

实验用野生型雄性BALB/c小鼠（南方医科大学动物中心），共25只，7周龄，体质量20~25 g。实验细胞株：正常大鼠近端肾小管上皮（NRK-52E）细胞由南方医科大学南方医院慢性肾病国家临床医学研究中心提供，人肾上皮细胞（293T）由郴州市第一人民医院转化医学研究所提供，小鼠用NC-siRNA（阴性对照siRNA）、Numb-siRNA（广州锐博），细胞用NC-siRNA、Numb-siRNA（上海吉玛），100X氨基酸混合液、Lipofectamine2000（Invitrogen）、顺铂、IV型胶原酶（sigma），高糖型DMEM培养基、DMEM-F12培养、胎牛血清、胰蛋白酶、EBSS培养基（Gibco），ECL化学发光试剂盒（碧云天），Percoll分离液（Amersham Biosciences），原代肾小管上皮细胞培养基(Lifeline Cell Technology)，Numb、S6、p-S6、AMPK、p-AMPK、S6K1、p-S6K1、4EBP1、p-4EBP1、Megalin抗体（Cell Signaling Technology）、V1G1及GAPDH抗体（Proteintech），硝酸纤维素膜（Biorad），免疫组化试剂盒、辣根过氧化物酶（HRP）标记的山羊抗兔、兔抗鼠二抗（北京中杉金桥），pSIN-EF2-puromycin空载病毒质粒、pSPAX2质粒及pMD2.G质粒由郴州市第一人民医院转化医学研究所提供，pSIN-EF2-V1G1-puromycin慢病毒质粒（南京金斯瑞），大肠杆菌DH5α感受态（康体生命）、质粒抽提试剂盒（天根），SureENTRY（Qiagen），阳离子聚合物聚乙烯亚胺（PEI）（Polyscience）。所有动物处理方案均经湘南学院动物实验伦理委员会批准。

1.2. 实验方法

1.2.1. 小鼠小干扰RNA（SiRNA）转染

所有动物都在湘南学院动物实验室22~24 ℃、50%~60%的湿度、12 h明暗循环环境下饲养。适应1周后将小鼠随机分为NC-siRNA处理组和Numb-siRNA处理组，5只/组。NC-siRNA处理组小鼠给予尾静脉注射NC-siRNA，Numb-siRNA处理组小鼠给予尾静脉注射Numb-siRNA。具体方法为将Numb-siRNA或NC-siRNA（5nmol/L溶于0.2mLDEPC水中）于10 s内经小鼠尾静脉注射至体内，连续注射4 d，1次/d，第5天收集小鼠肾脏组织用于检测分析。Numb-siRNA序列为5'-CAGCCUGUUUAGAGCGU AAdtdt-3'。

1.2.2. 细胞siRNA转染

NRK-52E细胞用含10% 胎牛血清的高糖DMEM培养基（完全培养基）在37 ℃、5% CO2的加湿培养箱中常规培养。培养融合度达80%~90% 时，用0.25%的胰蛋白酶消化，传代接种到6孔培养板，待细胞生长融合至60%~70%时，按照Lipofectamine2000转染试剂盒说明书进行NC-SiRNA及Numb-siRNA的转染，转染48 h后收集细胞通过Western blot检测Numb的敲除效率。NC-siRNA序列为：GCGACGAU CUGCCUAAGAUdT，Numb-siRNA序列为：GCACC UGCCCAGUGGAUCCTT。

1.2.3. 氨基酸诱导mTORC1活化的细胞模型

NRK-52E细胞用完全培养基在37 ℃、5% CO2的加湿培养箱中常规培养。待培养融合度达80%~90%或者转染siRNA 48 h后，弃除完全培养基，给予1 mL EBSS培养基孵育50 min，使细胞处于氨基酸饥饿状态，然后加入10 µL/孔100×氨基酸混合液至1×工作浓度刺激10 min。

1.2.4. 顺铂诱导的AKI小鼠模型

雄性BALB/c小鼠随机分为生理盐水对照组、顺铂处理组及Numb干预组。对照组给予NC-siRNA及生理盐水注射；顺铂处理组给予NC-siRNA及顺铂注射；Numb干预组给予Numb-siRNA及顺铂注射。顺铂处理组和对照组模型建立方法为腹腔单次注射顺铂/生理盐水25 mg/（kg·d）。具体建模方法为：NC-siRNA或Numb-siRNA注射完成后，第2天进行顺铂/生理盐水注射构建AKI小鼠模型，在建模期间隔天注射1次NC-siRNA或Numb-siRNA，第3天收集小鼠肾脏组织及血液标本。

1.2.5. 免疫组化染色检测Numb、Megalin的表达与分布

采用SP二步法，将4 μm厚的组织切片行脱蜡、水化、微波炉加热进行抗原修复；30 mL/L H2O2去离子水孵育，一抗Numb（1∶100）、Megalin（1∶100），4 ℃孵育过夜；PBS洗涤后，加相应二抗孵育，DAB显色，自来水充分冲洗、复染、脱水、封片，光镜下观察并拍照。

1.2.6. 原代近端肾小管上皮细胞分离及培养

雄性BALB/c小鼠按照上述方法注射NC-siRNA或NumbsiRNA后，第2天小鼠经5% 水合氯醛麻醉后，75%乙醇中浸泡5 min，勿将小鼠口鼻浸入乙醇中。超净台中剖开小鼠胸腹部，取双肾置于冷PBS中去除肾蒂及包膜，取薄层肾皮质于PBS中剪碎至1 mm3，PBS洗涤800 r/min离心2次，5 min/次。弃上清液，加入0.75 mg/mL的IV型胶原酶消化液(含5% 胎牛血清），终质量浓度为1 g/L，37 ℃振荡消化15 min，用1 mL移液枪充分抽吸组织消化液使组织充分裂解，然后用等体积含10% 胎牛血清的PBS中止消化，将组织消化液置于4 ℃以50×g离心2 min收集肾小管。DMEM-F12培养基洗涤后，用32% Percoll分离液重悬沉淀，以2000×g、4 ℃离心10 min。离心后可见液体分层，吸取近管底第2层细胞悬液，即为近端肾小管节段及其游离的肾小管上皮细胞。15 mL DMEM-F12培养基洗涤，并以1000 r/min离心10 min以去除残留的Percoll分离液。纯化的近端小管使用原代近端肾小管上皮细胞培养基重悬后接种到60 mm的培养皿中，于37 ℃、5% CO2细胞孵箱静置培养，隔天换液。原代培养待5~6 d细胞长满后，用0.05% 胰蛋白酶消化并传代培养。

1.2.7. 慢病毒载体的包装

委托南京金斯瑞生物科技有限公司构建V1G1过表达的慢病毒重组质粒pSIN-EF2-V1G1-puromycin，将制备好的pSIN-EF2-V1G1-puromycin质粒、对照空载病毒质粒pSIN-EF2-puromycin及其2种辅助病毒包装原件载体质粒pSPAX2、pMD2.G分别进行高纯度无内毒素抽提。将上述得到质粒，pSIN-EF2-V1G1-puromycin/pSIN-EF2-V1G1-puromycin、pSPAX2、pMD2.G与PEI转染试剂混匀并在室温中孵育20 min，将上述混合液分别缓慢加入到293 T细胞中混匀后继续培养24 h，然后将基础培养基更换为2%血清的培养基，继续培养48 h后收集转染后的293T细胞上清液，再于7000 r/min离心5 min，收集的上清即为V1G1过表达慢病毒颗粒及空载慢病毒颗粒，将其按照500 μL/管进行分装，冻存于-80 ℃备用。

1.2.8. 慢病毒感染

NRK-52E细胞用含10%胎牛血清的高糖DMEM培养基在37 ℃、5% CO2的加湿培养箱中常规培养，将其接种于6孔培养板，待其细胞融合度60%~70%时，按照SureENTRY转染试剂盒说明书进行V1G1过表达慢病毒及空载慢病毒的感染，感染48 h后收集细胞通过Western blot检测V1G1的过表达情况。

1.2.9. siRNA转染、慢病毒感染和氨基酸饥饿/再刺激共处理

将NRK-52E细胞随机分为对照组、Numb沉默组以及V1G1干预组。对照组细胞进行NC-siRNA转染及空载慢病毒感染；Numb沉默组进行Numb-siRNA感染及空载慢病毒感染；V1G1干预组进行Numb-siRNA感染及V1G1过表达慢病毒感染。待细胞融合度为60%~70%进行siRNA转染，12 h后进行慢病毒感染，培养48 h后再构建氨基酸诱导mTORC1活化的细胞模型。

1.2.10. Western blot法检测相关蛋白的表达

收集的肾脏及细胞使用RIPA蛋白裂解液提取总蛋白，采用蛋白质二甲喹啉甲酸法（BCA法）进行蛋白定量分析；加入2X SDS上样缓冲液至终浓度为1X，蛋白于100 ℃煮10 min；用SDS-PAGE凝胶电泳分离蛋白后将蛋白由凝胶转至硝酸纤维素膜上；使用5%脱脂奶粉于室温封闭1 h后，剪出所需蛋白条带用相应的一抗于4 ℃孵育过夜；次日采用相应的HRP标记二抗于室温孵育1 h，加入化学发光试剂，使用化学发光仪进行拍照，Fluorchem 8900软件分析蛋白条带的灰度值。

1.3. 统计学分析

采用统计学软件SPSS 20.0软件进行数据分析。定量资料以均数±标准差表示，两组间比较采用t检验；3组比较采用单因素方差分析；4组比较采用两因素方差分析，组间两两比较则采用SNK-q检验。P < 0.05为差异具有统计学意义。

2. 结果

2.1. Numb对小鼠近端肾小管上皮细胞中mTORC1信号通路活性的影响

Western blot结果显示：与NC-siRNA处理组相比，Numb-siRNA处理组小鼠肾脏Numb、p-S6的蛋白表达水平下调（P < 0.05，图 1A、B、D、E）。免疫组织化学染色结果显示：Numb基因主要在肾脏近端肾小管上皮细胞表达，与NC-siRNA处理组相比，Numb-siRNA处理组小鼠肾脏近端肾小管上皮细胞Numb蛋白表达水平降低，但不影响近端肾小管标记蛋白Megalin的表达与分布（图 1C）。

图 1.

图 1

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Numb对小鼠近端肾小管上皮细胞中mTORC1信号通路活性的影响

Effect of Numb knockdown on activity of mTORC1 pathway in mouse proximal tubules. A, B: Western blotting for detecting Numb protein expression in the kidneys of Numb-siRNA-treated mice and NC-siRNAtreated mice (n=5, *P < 0.05). C: Immunohistochemistry for detecting Numb (upper panel) and megalin (lower panel) expressions in sequential sections of the kidney tissues from Numb-siRNA treated mice and NC-siRNA-treated mice (Scale bar: 50 µm). Upper arrows indicate an decreased expression of Numb, and lower arrows indicate an unchanged expression of Megalin. D, E: Western blotting for detecting the protein level of p-S6 in the kidneys of Numb-siRNA-treated mice and NC-siRNA-treated mice. S6 was used to verify equivalent loading (n=5, *P < 0.05).

2.2. Numb对AKI诱导的肾脏mTORC1信号通路活化的影响

Western blot结果显示：与对照组小鼠相比，NC-siRNA+顺铂处理组小鼠肾脏p-S6K1和p-4EBP1的蛋白表达水平上调（P < 0.05），而与NC-siRNA+顺铂处理组小鼠相比，Numb-siRNA+顺铂处理组小鼠肾脏p-S6K1和p-4EBP1的蛋白表达水平下调（P < 0.05，图 2A~C）。

图 2.

图 2

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Numb对AKI诱导的肾脏mTORC1信号通路活化的影响

Effect of Numb knockdown on ctivity of mTORC1 pathway in proximal tubules in cisplatin-induced AKI mouse model. A: Western blots of p-S6K1, S6K1, p-4EBP1, and 4-EBP1 in the kidneys of the mice in different groups. S6K1 and 4-EBP1 were used to verify equivalent loading for p-S6K1 and p-4EBP1, respectively. B: Quantitative analysis of p-S6K1 to total S6K1 ratio. C: Quantitative determination of p-4EBP1 to total 4-EBP1 ratio. *P < 0.05 vs NC-siRNA-treated mice with saline treatment; #P < 0.05 vs NC-siRNA-treated mice with cisplatin treatment (n=5).

2.3. Numb对NRK-52E细胞中mTORC1信号通路活性的影响

Western blot显示：细胞给予PBS培养50 min后，p-S6的蛋白表达水平降低，而再给予1×氨基酸刺激10 min后，p-S6的蛋白表达水平上调（P < 0.05，图 3A、B）；在NC-siRNA转染的细胞中，氨基酸再刺激促进S6的磷酸化，而转染Numb-siRNA的细胞中，Numb及p-S6的蛋白表达水平较对照组均显著下调（P < 0.05，图 3C~E）。

图 3.

图 3

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Numb对NRK-52E细胞中mTORC1信号通路活性的影响

Effect of Numb knockdown on the activity of mTORC1 pathway in NRK-52E cells. A: Western blotting for detecting phosphorylation of S6 in NRK-52E cells after amino acid starvation and readdition. B: Quantitative determination of p-S6 in the cells (*P < 0.05 vs -/- controls; #P < 0.05 vs starved cells. n=3). C: Amino acids readdition failed to restore the phosphorylation of S6 in cells with Numb-siRNA transfection compared with cells with NC-siRNA transfection. D: Quantitative determination of Numb and S6 in each group (**P < 0.01 vs NC-siRNA-transfected cells with starvation; #P < 0.05 vs NC-siRNA-transfected cells with amino acid mixture readdition). E: Quantitative determination of p-S6 in each group (**P < 0.01 vs NC-siRNA-transfected cells with starvation. #P < 0.05 vs NC-siRNAtransfected cells with amino acid mixture readdition. n=3).

2.4. Numb对近端肾小管上皮细胞中AMPK活性的影响

Western blot结果显示：在NRK-52E细胞中，与NC-siRNA处理组相比，Numb-siRNA处理组细胞中p-AMPK的蛋白表达水平下调（P < 0.05，图 4A）；与NC-siRNA处理组小鼠相比，Numb-siRNA处理组小鼠肾脏p-AMPK的蛋白表达水平下调（P < 0.05，图 4B）；为了进一步证实Numb促进近端肾小管上皮细胞中AMPK的活化，我们从Numb-siRNA处理组小鼠和NC-siRNA处理组小鼠中提取了原代近端肾小管细胞。Western blot结果显示：与NC-siRNA处理的小鼠原代近端肾小管细胞相比，Numb-siRNA处理的小鼠原代近端肾小管细胞中的Numb和p-AMPK表达水平均下调（P < 0.05，图 4C、D）。

图 4.

图 4

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Numb对近端肾小管上皮细胞中AMPK活性的影响

Effect of Numb knockdown on the activity of AMPK in proximal tubular cells. A: Western blotting for detecting p-AMPK and AMPK in Numb-siRNA-or NC-siRNA-transfected NRK-52E cells and quantitative determination of p-AMPK to total AMPK ratio (n=3. *P < 0.05). B: Western blotting for detecting p-AMPK and AMPK in the kidneys of Numb-siRNA-treated mice and NC-siRNA-treated mice and quantitative determination of the p-AMPK to total AMPK ratio (n=5. *P < 0.05). C: Western blotting for detecting Numb expression in the primary proximal tubular cells from Numb-siRNA-treated mice and NC-siRNA-treated mice and quantitative determination of Numb to GAPDH ratio. D: Western blotting for detecting p-AMPK and total AMPK expressions in the primary proximal tubular cells (n=5. *P < 0.05).

2.5. V1G1是Numb的功能靶点，V1G1可逆转Numb沉默导致的mTORC1活化的抑制

Western blot显示：在NRK-52E细胞中，与NC-siRNA处理组相比，Numb-siRNA处理组细胞中V1G1的蛋白表达水平下调（P < 0.05，图 5A、B）；感染V1G1过表达慢病毒的NRK-52E细胞V1G1蛋白表达上调（图 5C）；与NC-siRNA处理组相比，转染Numb-siRNA后抑制氨基酸介导的S6的磷酸化（P < 0.01），而感染V1G1过表达慢病毒可逆转Numb沉默介导的pS6的蛋白表达水平的降低（P < 0.05，图 5D~F）。

图 5.

图 5

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Numb通过上调V1G1促进氨基酸介导的NRK-52E细胞中mTORC1的活化

Numb promotes amino acid-induced activation of mTORC1 by upregulating V1G1 protein expression in NRK-52E cells. A, B: Western blotting for detecting the protein levels of V1G1 in Numb siRNA or NC-siRNA-transfected NRK-52E cells (n=3. *P < 0.05). C: Western blotting for detecting protein levels of V1G1 in NRK-52E cells with V1G1 overexpression. D: V1G1 overexpression restores the phosphorylation of S6 in cells with Numb-siRNA transfection. E: Quantitative determination of V1G1 and S6 in each group (**P < 0.01 vs NC-siRNA-transfected cells with amino acid mixture readdition; #P < 0.05 vs Numb-siRNA transfected cells with amino acid mixture readdition). F: Quantitative determination of p-S6 in each group (**P < 0.01 vs NC-siRNA transfected cells with amino acid mixture readdition; #P < 0.05 vs Numb-siRNA transfected cells with amino acid mixture readdition).

3. 讨论

mTORC1是一个从酵母到哺乳动物都保守存在的复合体，通过促进蛋白质的翻译以及合成等方式，促进细胞的生长和增殖[2, 28]。在肾脏近端肾小管上皮细胞中mTORC1具有丰富表达，其活性异常是AKI等肾脏疾病发生发展的重要原因[11-14]。因此，阐明近端肾小管上皮细胞中mTORC1信号通路的调控机制，将为mTORC1活性异常相关性肾脏疾病的治疗提供新靶点。Numb是一种多功能蛋白，参与调控多种信号通路的传导[24-26]。我们前期研究证明，Numb在肾脏主要表达于近端肾小管上皮细胞，在AKI、CKD的发生发展中均发挥着重要作用[29-31]。关于Numb在近端肾小管上皮细胞mTORC1信号通路中的调控作用和机制的研究尚未见报道，因此，本研究旨在探讨Numb对近端肾小管上皮细胞中mTORC1信号通路活化的影响及其潜在的分子机制。

mTORC1是一种营养传感器，当细胞接受到氨基酸、生长因子等信号刺激后，mTORC1从细胞胞浆转移至溶酶体而被激活[32]。研究发现，溶酶体功能受损抑制氨基酸诱导mTORC1信号通路的活化[17, 18]。最近的一项研究表明，在MCF-7细胞中，沉默Numb基因的表达抑制Rab7活性和溶酶体功能[27]。提示Numb可能参与近端肾小管上皮细胞中mTORC1信号通路活性的调控。近有研究报道，同时敲除成骨细胞中Numb和Numbl基因通过抑制PTEN蛋白的降解抑制mTORC1信号通路的活化[33]。本研究表明，在Numb表达水平下调的NRK-52E细胞株、正常小鼠肾脏及顺铂诱导的AKI小鼠模型肾脏中，mTORC1下游信号分子S6、S6K1和4EBP-1的磷酸化水平显著降低，说明在生理情况及AKI病理情况下，Numb均具有促进近端肾小管细胞中mTORC1信号通路活化的作用，与既往研究一致。接下来，我们进一步探讨Numb激活mTORC1信号通路的潜在分子机制。

AMPK作为机体代谢的重要调控分子，是mTORC1的负性上游调控分子，直接参与对mTORC1活性的调节[34]。研究证明，AMPK可以通过磷酸化TSC2或直接通过磷酸化Raptor抑制mTORC1信号通路的活化[15, 16]。本研究结果显示，在Numb表达水平下调的NRK-52E细胞、小鼠肾脏或者原代近端肾小管上皮细胞中，p-AMPK的蛋白表达水平均是下调的，说明下调近端肾小管上皮细胞中Numb的表达抑制AMPK的活化，与我们预想结果相反。另有研究报道，溶酶体V-ATPase-ragulator复合物是AMPK和mTORC1活化的共同激活剂，V-ATPase酶活性降低，导致AXIN/LKB1-AMPK复合物和mTORC1从溶酶体外膜解离，从而分别抑制LKB1和Rheb-GTP对AMPK和mTORC1的激活[35]。因此，我们认为Numb可能通过稳定V-ATPase的酶活性从而激活mTORC1信号通路。

V-ATPase是一类特殊的膜转运蛋白，是由14个亚基组成的质子泵，只有V0和V1亚单位相结合才能发挥其蛋白酶的作用[36]。V-ATPase位于溶酶体等多种细胞器的膜上，通过与Ragulator形成复合物，促进mTORC1的溶酶体定位及其活化[33]。研究发现，下调宫颈癌（Hela）细胞中V-ATPase亚型V1G1的蛋白表达抑制V-ATPase活化以及溶酶体功能[37]。有研究证明，小分子天然产物Verucopeptin靶向V-ATPase的V1G亚基，有效的抑制V-ATPase的活性和mTORC1信号通路的活化[21]。本研究结果显示，沉默NRK-52E细胞中Numb的表达，V1G1蛋白表达水平显著下调，V1G1过表达可以逆转Numb沉默所介导的mTORC1活化的抑制作用，该结果表明Numb可以通过上调NRK-52E细胞中V1G1的蛋白表达激活mTORC1信号通路，与既往研究一致[21]。

综上所述，本研究表明，Numb可通过上调V1G1的蛋白表达，促进近端肾小管上皮细胞中mTORC1信号通路的活化。这揭示了mTORC1信号通路的新分子调控机制，为由mTORC1活性异常介导的肾脏疾病的治疗提供了新的治疗靶点。

Biography

刘泽，博士，讲师，E-mail: liuze0113@xnu.edu.cn

Funding Statement

湖南省教育厅优秀青年项目（19B525）；湘南学院高层次人才研究基金（063，6011901）；湘南学院校级青年英才计划（063，6212001）

Contributor Information

刘 泽 (Ze LIU), Email: liuze0113@xnu.edu.cn.

李 潘 (Pan LI), Email: 582842343@qq.com.

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