神经前体细胞表达发育下调样蛋白4调控Wnt/β-catenin通路对急性髓系白血病细胞增殖和凋亡的影响

doi:10.3969/j.issn.1006-5725.2026.14.006

摘要/Abstract

摘要：

目的探讨神经前体细胞表达发育下调样蛋白4（NEDD4L）调控Wnt/β-catenin通路对急性髓系白血病（AML）细胞增殖和凋亡的影响。方法利用慢病毒转染技术构建稳定过表达及敲低NEDD4L的Kasumi-1和U937细胞系；通过RT-qPCR和Western blot验证NEDD4L过表达及敲低效率；CCK-8实验检测Kasumi-1和U937细胞的增殖情况；流式细胞术检测Kasumi-1和U937细胞凋亡改变；Western blot检测凋亡蛋白BAX、Bcl2及Wnt/β-catenin通路中关键蛋白β-catenin、c-Myc、cyclin D1的表达。结果 RT-qPCR和Western blot结果提示，Kasumi-1和U937细胞系中NEDD4L成功过表达和敲低（P < 0.05）。CCK-8结果提示NEDD4L过表达显著抑制Kasumi-1和U937细胞增殖能力（P < 0.05）。流式细胞术结果表明，NEDD4L过表达后Kasumi-1和U937细胞凋亡率显著升高。Western blot结果提示，过表达NEDD4L可上调凋亡蛋白BAX、下调抗凋亡蛋白Bcl2表达（P < 0.05）。机制研究表明，过表达NEDD4L可降低β-catenin、c-Myc、cyclin D1的表达，而敲低NEDD4L则上调上述蛋白。此外，Wnt/β-catenin通路激动剂SKL2001可部分逆转过表达NEDD4L所致的通路抑制效应（P < 0.05）。结论 NEDD4L通过抑制Wnt/β-catenin通路活性，从而抑制AML细胞增殖、诱导AML细胞凋亡。提示NEDD4L可能是AML潜在的治疗靶点。

关键词: 神经前体细胞表达发育下调样蛋白4, Wnt/β-catenin通路, 急性髓系白血病, 细胞增殖, 细胞凋亡

Abstract:

Objective To investigate the effect of NEDD4L on the proliferation and apoptosis of acute myeloid leukemia （AML） cells and its underlying mechanism via the regulation of the Wnt/β-catenin pathway. Methods Stable NEDD4L-overexpressing and NEDD4L-knockdown Kasumi-1 and U937 cell lines were successfully established via lentiviral transduction technology. The efficiency of NEDD4L overexpression and knockdown was rigorously verified by RT-qPCR and Western blot. Cell proliferation was accurately assessed using the CCK-8 assay， and apoptosis was precisely detected through flow cytometry. The expression levels of the apoptosis-related proteins BAX and Bcl2， along with the key proteins of the Wnt/β-catenin pathway （β-catenin， c-Myc， and cyclin D1）， were measured by Western blot. Results Results from RT-qPCR and Western blot assays demonstrated that NEDD4L was successfully overexpressed and knocked down in Kasumi-1 and U937 cell lines （P < 0.05）. CCK-8 assay results indicated that overexpression of NEDD4L significantly inhibited the proliferative capacity of Kasumi-1 and U937 cells （P < 0.05）. Flow cytometry results showed that overexpression of NEDD4L significantly increased the apoptosis rate in Kasumi-1 and U937 cells. Western blot analysis results revealed that overexpression of NEDD4L upregulated the pro-apoptotic protein BAX and downregulated the anti-apoptotic protein Bcl2 （P < 0.05）. Mechanistic studies results showed that overexpression of NEDD4L decreased the expression of β-catenin， c-Myc， and cyclin D1， whereas knockdown of NEDD4L upregulated these proteins. Furthermore， the Wnt/β-catenin pathway agonist SKL2001 partially reversed the pathway inhibition induced by overexpression of NEDD4L （P < 0.05）. Conclusion NEDD4L inhibits the proliferation of AML cells and induces the apoptosis of AML cells by suppressing the activity of the Wnt/β-catenin pathway， which suggests that NEDD4L may serve as a potential therapeutic target for AML.

Key words: NEDD4L, Wnt/β-catenin pathway, acute myeloid meukemia, cell proliferation, cell apoptosis

中图分类号:

R733.7

刘远程,胡承龙,周武,张静馨,张清琳,胡华丽,韦四喜. 神经前体细胞表达发育下调样蛋白4调控Wnt/β-catenin通路对急性髓系白血病细胞增殖和凋亡的影响[J]. 实用医学杂志, 2026, 42(14): 2533-2541.

Yuancheng LIU,Chenglong HU,Wu ZHOU,Jingxin ZHANG,Qinglin ZHANG,Huali HU,Sixi WEI. Effect of NEDD4L on proliferation and apoptosis of acute myeloid leukemia cells via the Wnt/β-catenin pathway[J]. The Journal of Practical Medicine, 2026, 42(14): 2533-2541.

图/表 7

表 1

图1

图2

图3

图4

图5

图6

参考文献 27

[1]	PALMIERI R， CANDONI A， DI RAIMONDO F， et al. Navigating acute myeloid leukemia towards better outcomes： Treatment pathways and challenges for patients ineligible for intensive chemotherapy ［J］. Blood Rev， 2025， 73： 101288.doi：10.1016/j.blre.2025.101288 . doi: 10.1016/j.blre.2025.101288
[2]	ZHANG T Y， DUTTA R， BENARD B， et al. IL-6 blockade reverses bone marrow failure induced by human acute myeloid leukemia ［J］. Sci Transl Med， 2020， 12（538）：eaax5104.doi：10.1126/scitranslmed.aax5104 . doi: 10.1126/scitranslmed.aax5104
[3]	SHALLIS R M， WANG R， DAVIDOFF A， et al. Epidemiology of acute myeloid leukemia： Recent progress and enduring challenges ［J］. Blood Rev， 2019， 36： 70⁃87.doi：10.1016/j.blre.2019. 04.005 . doi: 10.1016/j.blre.2019. 04.005
[4]	INOUE Y， CIOCCIO J， MINEISHI S， et al. Evolution of Allogeneic Stem Cell Transplantation： Main Focus on AML ［J］. Cells， 2025， 14（8）：572.doi：10.3390/ cells14080572 . doi: 10.3390/ cells14080572
[5]	CHAER F EL， BEWERSDORF J P， STAHL M， et al. The global epidemiology of acute myeloid leukaemia ［J］. Nat Rev Clin Oncol， 2026， 23（2）： 107-120. doi：10.1038/s41571-025-01099-7 . doi: 10.1038/s41571-025-01099-7
[6]	GOEL P， MANNING J A， KUMAR S. NEDD4-2 （NEDD4L）： The ubiquitin ligase for multiple membrane proteins ［J］. Gene， 2015， 557（1）： 1-10.doi： 10.1016/j.gene.2014.11.051 . doi: 10.1016/j.gene.2014.11.051
[7]	ZOU X， LEVY-COHEN G， BLANK M. Molecular functions of NEDD4 E3 ubiquitin ligases in cancer ［J］. Biochim Biophys Acta， 2015， 1856（1）： 91-106. doi：10.1016/j.bbcan.2015.06.005 . doi: 10.1016/j.bbcan.2015.06.005
[8]	MANNING J A， SHAH S S， NIKOLIC A， et al. The ubiquitin ligase NEDD4-2/NEDD4L regulates both sodium homeostasis and fibrotic signaling to prevent end-stage renal disease ［J］. Cell Death Dis， 2021， 12（4）： 398.doi：10.1038/s41419-021-03688-7 . doi: 10.1038/s41419-021-03688-7
[9]	EKBERG J， ADAMS D J. Neuronal voltage-gated sodium channel subtypes： Key roles in inflammatory and neuropathic pain ［J］. Int J Biochem Cell Biol， 2006， 38（12）： 2005–2010.doi：10.1016/j.biocel.2006.06.008 . doi: 10.1016/j.biocel.2006.06.008
[10]	KURATOMI G， KOMURO A， GOTO K， et al. NEDD4-2 （neural precursor cell expressed， developmentally down-regulated 4-2） negatively regulates TGF-beta （transforming growth factor-beta） signalling by inducing ubiquitin-mediated degradation of Smad2 and TGF-beta type I receptor ［J］. Biochem J， 2005， 386（Pt 3）： 461-470.doi：10.1042/bj20040738 . doi: 10.1042/bj20040738
[11]	DEBONNEVILLE C， FLORES S Y， KAMYNINA E， et al. Phosphorylation of Nedd4-2 by Sgk1 regulates epithelial Na（+） channel cell surface expression ［J］. EMBO J， 2001， 20（24）： 7052-7059.doi：10.1093/emboj/20.24.7052 . doi: 10.1093/emboj/20.24.7052
[12]	LEE I H， DINUDOM A， SANCHEZ-PEREZ A， et al. Akt mediates the effect of insulin on epithelial sodium channels by inhibiting Nedd4-2 ［J］. J Biol Chem， 2007， 282（41）： 29866-29873.doi：10.1074/jbc.M701923200 . doi: 10.1074/jbc.M701923200
[13]	GAO P， MA X， YUAN M， et al. E3 ligase Nedd4l promotes antiviral innate immunity by catalyzing K29-linked cysteine ubiquitination of TRAF3 ［J］. Nat Commun， 2021， 12（1）： 1194.doi：10.1038/s41467-021-21456-1 . doi: 10.1038/s41467-021-21456-1
[14]	ZHANG M， ZHANG Z， TIAN X， et al. NEDD4L in human tumors： Regulatory mechanisms and dual effects on anti-tumor and pro-tumor ［J］. Front Pharmacol， 2023， 14： 1291773.doi：10.3389/fphar.2023.1291773 . doi: 10.3389/fphar.2023.1291773
[15]	FAN B， WANG L， HU T， et al. Exosomal miR-196a-5p Secreted by Bone Marrow Mesenchymal Stem Cells Inhibits Ferroptosis and Promotes Drug Resistance of Acute Myeloid Leukemia ［J］. Antioxid Redox Signal， 2025， 42（16-18）： 933-953.doi：10.1089/ ars.2024.0882 . doi: 10.1089/ ars.2024.0882
[16]	刘秋岑，张祝琴，饶书权. LncRNA HOTAIRM1通过与ENO1蛋白相互作用促进急性髓系白血病进展［J］. 天津医科大学学报， 2026， 32（1）： 6-13，22.doi：10.20135/j.issn.1006-8147.2026. 01.0006 . doi: 10.20135/j.issn.1006-8147.2026. 01.0006
[17]	LÁINEZ-GONZÁLEZ D， ALONSO-AGUADO A B， ALONSO-DOMINGUEZ J M. Understanding the Wnt Signaling Pathway in Acute Myeloid Leukemia Stem Cells： A Feasible Key against Relapses ［J］. Biology （Basel）， 2023， 12（5）：683.doi：10.3390/biology12050683 . doi: 10.3390/biology12050683
[18]	FRENQUELLI M， TONON G. WNT Signaling in Hematological Malignancies ［J］. Front Oncol， 2020， 10： 615190.doi：10.3389/ fonc. 2020. 615190 . doi: 10.3389/ fonc. 2020. 615190
[19]	DING Y， ZHANG Y， XU C， et al. HECT domain-containing E3 ubiquitin ligase NEDD4L negatively regulates Wnt signaling by targeting dishevelled for proteasomal degradation ［J］. J Biol Chem， 2013， 288（12）： 8289-8298. doi： 10.1074/jbc.M112. 433185 . doi: 10.1074/jbc.M112. 433185
[20]	SHORT N J， RYTTING M E， CORTES J E. Acute myeloid leukaemia ［J］. Lancet， 2018， 392（10147）： 593-606.doi：10.1016/s0140-6736（18）31041-9 . doi: 10.1016/s0140-6736（18）31041-9
[21]	DAVER N， SENAPATI J， KANTARJIAN H M， et al. Azacitidine， Venetoclax， and Magrolimab in Newly Diagnosed and Relapsed Refractory Acute Myeloid Leukemia： Phase Ib/II Study and Correlative Analysis ［J］. Clin Cancer Res， 2025， 31（12）： 2386–2398. doi：10.1158/1078-0432.Ccr-25-0229 . doi: 10.1158/1078-0432.Ccr-25-0229
[22]	SHI Y， FANG N， WU Y， et al. NEDD4L mediates ITGB4 ubiquitination and degradation to suppress esophageal carcinoma progression ［J］. Cell Commun Signal， 2024， 22（1）： 302.doi：10.1186/s12964-024-01685-9 . doi: 10.1186/s12964-024-01685-9
[23]	ZHANG E， WANG Y， ZHANG H， et al. Resveratrol induces ferroptosis in triple-negative breast cancer through NEDD4L-mediated GPX4 ubiquitination and degradation ［J］. Free Radic Biol Med， 2025， 235： 231-247.doi：10.1016 /j.freeradbiomed. 2025.04.052 . doi: 10.1016 /j.freeradbiomed. 2025.04.052
[24]	HUANG X， CAO W， YAO S， et al. NEDD4L binds the proteasome and promotes autophagy and bortezomib sensitivity in multiple myeloma ［J］. Cell Death Dis， 2022， 13（3）： 197.doi：10.1038/s41419-022-04629-8 . doi: 10.1038/s41419-022-04629-8
[25]	CZABOTAR P E， LESSENE G， STRASSER A， et al. Control of apoptosis by the BCL-2 protein family： Implications for physiology and therapy ［J］. Nat Rev Mol Cell Biol， 2014， 15（1）： 49-63.doi：10.1038/nrm3722 . doi: 10.1038/nrm3722
[26]	STAAL F J， FAMILI F， GARCIA PEREZ L， et al. Aberrant Wnt Signaling in Leukemia ［J］. Cancers （Basel）， 2016， 8（9）：78.doi：10.3390/cancers8090078 . doi: 10.3390/cancers8090078
[27]	SAKODA T， KIKUSHIGE Y， MIYAMOTO T， et al. TIM-3 signaling hijacks the canonical Wnt/β-catenin pathway to maintain cancer stemness in acute myeloid leukemia ［J］. Blood Adv， 2023， 7（10）：2053-2065.doi：10.1182/bloodadvances. 2022008405 . doi: 10.1182/bloodadvances. 2022008405