泛素特异性蛋白酶37通过去泛素化稳定骨髓细胞瘤病毒癌基因同源物蛋白促进肝母细胞瘤进展的机制研究

doi:10.3969/j.issn.1674-8115.2026.10.004

上海交通大学学报（医学版） ›› 2026, Vol. 46 ›› Issue (10): 1339-1354.doi: 10.3969/j.issn.1674-8115.2026.10.004

• 论著 · 基础研究 • 上一篇

泛素特异性蛋白酶37通过去泛素化稳定骨髓细胞瘤病毒癌基因同源物蛋白促进肝母细胞瘤进展的机制研究

邓睿¹^,², 方后顺¹^,², 周吉全¹^,², 陈尧¹^,²(), 李慧¹^,²()

^1.上海交通大学医学院附属上海儿童医学中心儿科转化医学研究所，国家卫生健康委员会儿童血液肿瘤重点实验室，上海 200127
^2.上海市儿科临床分子诊断重点实验室，上海 200127

收稿日期:2026-04-15 接受日期:2026-06-20 出版日期:2026-10-15 发布日期:2026-10-15
通讯作者: 李慧，研究员，博士；电子信箱：lihui@scmc.com.cn。
陈尧，实验师，硕士；电子信箱：chenyao01@scmc.com.cn。
基金资助:
国家自然科学基金(82570207)

USP37-mediated deubiquitination maintains c-MYC protein homeostasis and contributes to hepatoblastoma progression

Deng Rui¹^,², Fang Houshun¹^,², Zhou Jiquan¹^,², Chen Yao¹^,²(), Li Hui¹^,²()

^1.Key Laboratory of Pediatric Hematology and Oncology of the National Health Commission, Pediatric Translational Medicine Institute, Shanghai Children's Medical Center, Shanghai Jiao Tong University School of Medicine, Shanghai 200127, China
^2.Shanghai Key Laboratory of Clinical Molecular Diagnostics for Pediatrics, Shanghai 200127, China

Received:2026-04-15 Accepted:2026-06-20 Online:2026-10-15 Published:2026-10-15
Contact: Li Hui, E-mail: lihui@scmc.com.cn.
Chen Yao, E-mail: chenyao01@scmc.com.cn.
Supported by:
National Natural Science Foundation of China(82570207)

摘要/Abstract

摘要：

目的·探讨泛素特异性蛋白酶37（ubiquitin-specific protease 37，USP37）在肝母细胞瘤（hepatoblastoma，HB）发生发展中的生物学功能，并探索其通过调控骨髓细胞瘤病毒癌基因同源物（myelocytomatosis viral oncogene homolog，c-MYC）蛋白稳定性影响肿瘤进展的分子机制。方法·检索PubMed数据库截至2026年3月31日发表的相关文献，结合HB转录组数据及临床病理特征，系统筛选可能参与c-MYC蛋白稳定性调控的USP家族成员。采用实时定量荧光反转录聚合酶链式反应（real time fluorescent quantitative PCR，qPCR）和受试者工作特征（receiver operating characteristic，ROC）曲线分析验证USP37的表达特征及诊断效能，并利用Kaplan-Meier Plotter数据库肝细胞癌队列进行探索性生存分析。在HuH6和HepG2细胞中构建USP37稳定敲低模型，通过细胞增殖及克隆形成实验评估其生物学功能，并采用shRNA抵抗型USP37表达载体进行回补验证。结合qPCR、蛋白质印迹法（Western blotting）、放线菌酮（cycloheximide，CHX）追踪实验、蛋白酶体抑制实验、免疫共沉淀（co-immunoprecipitation，Co-IP）及去泛素化实验，解析USP37对c-MYC表达及蛋白稳定性的调控机制。结果·通过文献证据、转录组差异表达及临床病理特征的交叉筛选，确定USP37为HB中潜在调控分子。临床样本及公共数据库分析显示，USP37在HB组织中显著上调。基于8对HB及配对癌旁组织的qPCR结果进行ROC曲线分析，曲线下面积（area under the curve，AUC）为0.942 7，提示USP37具有区分HB组织与癌旁组织的能力。33例HB患者临床资料分析显示，USP37高表达与肿瘤转移（P=0.028）及肿瘤体积增大（P=0.031）显著相关。体外功能实验表明，敲低USP37显著抑制HuH6和HepG2细胞增殖及克隆形成能力，而回补USP37可有效逆转细胞增殖抑制表型。机制研究显示，USP37过表达可剂量依赖性提高c-MYC蛋白水平，但不显著影响其mRNA表达；敲低USP37则显著降低c-MYC蛋白水平，而其mRNA表达无明显变化。CHX追踪实验显示，USP37过表达使293T细胞中c-MYC蛋白半衰期由2.357 h延长至4.796 h（P=0.006）；敲低USP37使HuH6和HepG2细胞中c-MYC蛋白半衰期分别由1.504 h和1.585 h缩短至0.986 h和0.792 h（P=0.006、P=0.005）。蛋白酶体抑制剂MG132处理可恢复USP37敲低细胞中的c-MYC蛋白水平。正反向Co-IP实验进一步证实USP37与c-MYC存在相互作用；去泛素化实验显示，USP37过表达可降低c-MYC泛素化水平。结论·USP37作为去泛素化酶，通过稳定c-MYC蛋白促进HB细胞增殖及肿瘤进展，提示靶向USP37-c-MYC轴有望成为潜在干预靶点。

关键词: 泛素特异性蛋白酶37（USP37）, 骨髓细胞瘤病毒癌基因同源物（c-MYC）, 肝母细胞瘤, 蛋白稳定性, 去泛素化

Abstract:

Objective ·To investigate the biological functions of ubiquitin-specific protease 37 (USP37) in the development and progression of hepatoblastoma (HB), and to explore the molecular mechanism by which USP37 promotes tumor progression through regulation of the protein stability of myelocytomatosis viral oncogene homolog (c-MYC). Methods ·Relevant literature published in the PubMed database up to March 31, 2026, was searched. HB transcriptomic data and clinicopathological characteristics were integrated to systematically screen USP family members potentially involved in the regulation of c-MYC protein stability. Real time fluorescent quantitative polymerase chain reaction (qPCR) and receiver operating characteristic (ROC) curve analysis were performed to validate the expression pattern and diagnostic performance of USP37. Exploratory survival analysis was conducted using the hepatocellular carcinoma cohort from the Kaplan-Meier Plotter database. Stable USP37 knockdown models were established in HuH6 and HepG2 cells. Cell proliferation and colony formation assays were performed to evaluate the biological functions of USP37, and rescue experiments were conducted using an shRNA-resistant USP37 expression vector. qPCR, Western blotting, cycloheximide (CHX) chase assays, proteasome inhibition assays, co-immunoprecipitation (Co-IP), and deubiquitination assays were performed to investigate the regulatory effects of USP37 on c-MYC expression and protein stability. Results ·Through integrated screening based on literature evidence, transcriptomic differential expression, and clinicopathological characteristics, USP37 was identified as a potential key regulatory molecule in HB. Analysis of clinical samples and public databases showed that USP37 was significantly upregulated in HB tissues. ROC curve analysis based on qPCR results from eight paired HB and adjacent non-tumor tissues yielded an area under the curve (AUC) of 0.942 7, indicating that USP37 could discriminate HB tissues from adjacent non-tumor tissues. Clinicopathological analysis of 33 patients with HB showed that high USP37 expression was significantly associated with tumor metastasis (P=0.028) and increased tumor volume (P=0.031). In vitro functional assays demonstrated that USP37 knockdown significantly inhibited the proliferation and colony-forming ability of HuH6 and HepG2 cells, whereas USP37 re-expression effectively rescued the proliferation-inhibitory phenotype. Mechanistically, USP37 overexpression increased c-MYC protein levels in a dose-dependent manner but did not significantly affect c-MYC mRNA expression. Conversely, USP37 knockdown significantly reduced c-MYC protein levels without markedly altering its mRNA expression. CHX chase assays showed that USP37 overexpression prolonged the half-life of c-MYC protein in 293T cells from 2.357 h to 4.796 h (P=0.006), whereas USP37 knockdown shortened the half-life of c-MYC protein in HuH6 and HepG2 cells from 1.504 h and 1.585 h to 0.986 h and 0.792 h, respectively (P=0.006 and 0.005). MG132 treatment restored c-MYC protein levels in USP37-knockdown cells. Reciprocal Co-IP assays further confirmed the interaction between USP37 and c-MYC, while deubiquitination assays demonstrated that USP37 overexpression reduced the ubiquitination level of c-MYC. Conclusion ·USP37 functions as a key deubiquitinase that promotes HB cell proliferation and tumor progression by stabilizing c-MYC protein, suggesting that the USP37-c-MYC axis may serve as a potential therapeutic target.

Key words: ubiquitin-specific protease 37 (USP37), myelocytomatosis viral oncogene homolog (c-MYC), hepatoblastoma, protein stability, deubiquitination

中图分类号:

R73-3

邓睿, 方后顺, 周吉全, 陈尧, 李慧. 泛素特异性蛋白酶37通过去泛素化稳定骨髓细胞瘤病毒癌基因同源物蛋白促进肝母细胞瘤进展的机制研究[J]. 上海交通大学学报（医学版）, 2026, 46(10): 1339-1354.

Deng Rui, Fang Houshun, Zhou Jiquan, Chen Yao, Li Hui. USP37-mediated deubiquitination maintains c-MYC protein homeostasis and contributes to hepatoblastoma progression[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2026,(10): 1339-1354.

图/表 10

表1 shRNA序列

Tab 1 shRNA sequences

shRNA name		shRNA sequence (5'→3')
USP37 shRNA-1	Forward	CCGGTCCGGGTAGAGGATCGATTAACTCGAGTTAATCGATCCTCTACCCGGATTTTTG
USP37 shRNA-1	Reverse	AATTCAAAAATCCGGGTAGAGGATCGATTAACTCGAGTTAATCGATCCTCTACCCGGA
USP37 shRNA-2	Forward	CCGGCCGGATTTGCAGAAGATGATACTCGAGTATCATCTTCTGCAAATCCGGTTTTTG
USP37 shRNA-2	Reverse	AATTCAAAAACCGGATTTGCAGAAGATGATACTCGAGTATCATCTTCTGCAAATCCGG

表2 引物序列及用途（5′→3′）

Tab 2 Primer sequences and applications （5′→3′）

Primer	Application	Forward	Reverse
USP37-Flag	Plasmid construction	CTAGAGGATCCCCGGGTACCGGTCGCCACCATGTCTCCTCTGAAGATACATGGT	CCGTCATGGTCTTTGTAGTCGCTAGCCAAGGCCTGACGGGTAGTCTT
USP37-HA	Plasmid construction	CTAGAGGATCCCCGGGTACCGGTCGCCACCATGTCTCCTCTGAAGATACATGGT	TCTGGGACATCATAAGGGTAGCTAGCCAAGGCCTGACGGGTAGTCTT
USP37-shRNA-resistant	Plasmid construction	CCCTGGCCGTGGCTCAATCAAGACTGTAGCAGGAAGTGGAATAGCTCGGA	GATTGAGCCACGGCCAGGGTTACCAAGAACTTTTCGAAATGG
USP37	qPCR	GGAACACTCTTCTGGTGGCA	GTGAGAGGAAAGGGGCACTC
c-MYC	qPCR	GTGGTCTTCCCCTACCCTCT	TGTTCCTCCTCAGAGTCGCT
β-actin	qPCR	AACTGGGACGACATGGAGAAAA	GGATAGCACAGCCTGGATAGCA

图1 HB中c-MYC相关USP家族成员的系统性筛选Note： A. Summary of USP family members reported to be involved in the regulation of c-MYC ubiquitination and stability, based on published studies retrieved from PubMed. B. Differential expression analysis of USP family members in the GSE104766 dataset. Genes with FPKM > 1 were included in the analysis, and the significance thresholds were set at P<0.05 and fold change > 1.5. C. Venn diagram showing the overlap between reported c-MYC-associated deubiquitinases and significantly upregulated USP family members in HB, identifying candidate USP regulators. D. Clinical correlation analysis of the candidate USP genes in 33 HB samples with available clinical information. P values were calculated using Fisher's exact test. Color intensity represents -log10(P value).

Fig 1 Systematic screening of c-MYC-associated USP family members in HB

表3 c-MYC泛素化与稳定性调控相关USP家族成员

Tab 3 USP family members involved in the regulation of c-MYC ubiquitination and stability

USP	Cell model	Reference
USP1	Bladder cancer cell lines (T24, UM-UC-3)	[22]
USP3	Clear cell renal cell carcinoma cell lines (786-O, Caki-2)	[23]
USP5	Hepatocellular carcinoma cell lines (Huh-7, HepG2)	[24]
USP7	Pancreatic ductal adenocarcinoma cell lines (PANC1, CFPAC-1, AsPC1)	[25]
USP10	Osteosarcoma cell lines (143B, HOS)	[26]
USP11	Prostate cancer cell line (22Rv1)	[27]
USP13	Glioblastoma stem cells	[28]
USP16	Prostate cancer cell lines (PC3, DU145)	[29]
USP19	Hepatocellular carcinoma cell lines (Hep3b, Huh-7)	[30]
USP22	Breast cancer cell lines (MCF7, T47D)	[31]
USP26	Gastric cancer cell lines (SNU-216, HGC-27)	[32]
USP27X	Breast cancer cell lines (MCF-7, MDA-MB-231); mouse embryonic fibroblasts	[33]
USP28	Osteosarcoma cell line (U2OS), cervical cancer cell line (HeLa); mouse intestinal model, MEFs, colorectal cancer model	[34‒35]
USP29	Neuroblastoma cell line (SK-N-BE2), burkitt lymphoma cell line (Ramos), non-small cell lung cancer cell line (A549)	[36]
USP30	Oral squamous cell carcinoma cell lines (SCC4, HSC4)	[37]
USP33	Prostate cancer cell lines (22Rv1, DU145, PC3); ovarian cancer cell lines (SKOV3, ES2)	[38‒39]
USP34	Hepatocellular carcinoma cell lines (Huh-7, HepG2, HCCLM3, MHCC97H)	[40]
USP36	Cervical cancer cell line (HeLa), non-small cell lung cancer cell line (H1299), breast cancer cell line (MDA-MB-231), non-tumorigenic breast epithelial cell line (MCF10A)	[41]
USP37	Non-small cell lung cancer cell lines (H1299)	[21]
USP38	Non-small cell lung cancer cell lines (A549), cervical cancer cell lines (HeLa)	[42]
USP40	Acute myeloid leukemia cell lines (HL-60, Kasumi-1)	[43]
USP43	Bladder cancer cell lines (T24, 5637, UM-UC-3)	[44]
USP44	Meningioma cell lines (IOMM-Lee, CH157)	[45]
USP45	Cervical cancer cell lines (HeLa, SiHa)	[46]
USP47	Non-small cell lung cancer cell lines (A549, Calu-1, H358)	[47]

表3 c-MYC泛素化与稳定性调控相关USP家族成员

Tab 3 USP family members involved in the regulation of c-MYC ubiquitination and stability

USP	Cell model	Reference
USP1	Bladder cancer cell lines (T24, UM-UC-3)	[22]
USP3	Clear cell renal cell carcinoma cell lines (786-O, Caki-2)	[23]
USP5	Hepatocellular carcinoma cell lines (Huh-7, HepG2)	[24]
USP7	Pancreatic ductal adenocarcinoma cell lines (PANC1, CFPAC-1, AsPC1)	[25]
USP10	Osteosarcoma cell lines (143B, HOS)	[26]
USP11	Prostate cancer cell line (22Rv1)	[27]
USP13	Glioblastoma stem cells	[28]
USP16	Prostate cancer cell lines (PC3, DU145)	[29]
USP19	Hepatocellular carcinoma cell lines (Hep3b, Huh-7)	[30]
USP22	Breast cancer cell lines (MCF7, T47D)	[31]
USP26	Gastric cancer cell lines (SNU-216, HGC-27)	[32]
USP27X	Breast cancer cell lines (MCF-7, MDA-MB-231); mouse embryonic fibroblasts	[33]
USP28	Osteosarcoma cell line (U2OS), cervical cancer cell line (HeLa); mouse intestinal model, MEFs, colorectal cancer model	[34‒35]
USP29	Neuroblastoma cell line (SK-N-BE2), burkitt lymphoma cell line (Ramos), non-small cell lung cancer cell line (A549)	[36]
USP30	Oral squamous cell carcinoma cell lines (SCC4, HSC4)	[37]
USP33	Prostate cancer cell lines (22Rv1, DU145, PC3); ovarian cancer cell lines (SKOV3, ES2)	[38‒39]
USP34	Hepatocellular carcinoma cell lines (Huh-7, HepG2, HCCLM3, MHCC97H)	[40]
USP36	Cervical cancer cell line (HeLa), non-small cell lung cancer cell line (H1299), breast cancer cell line (MDA-MB-231), non-tumorigenic breast epithelial cell line (MCF10A)	[41]
USP37	Non-small cell lung cancer cell lines (H1299)	[21]
USP38	Non-small cell lung cancer cell lines (A549), cervical cancer cell lines (HeLa)	[42]
USP40	Acute myeloid leukemia cell lines (HL-60, Kasumi-1)	[43]
USP43	Bladder cancer cell lines (T24, 5637, UM-UC-3)	[44]
USP44	Meningioma cell lines (IOMM-Lee, CH157)	[45]
USP45	Cervical cancer cell lines (HeLa, SiHa)	[46]
USP47	Non-small cell lung cancer cell lines (A549, Calu-1, H358)	[47]

图2 USP37 在HB中的表达分析及其临床相关性Note： A. Transcriptomic analysis of USP37 expression in HB (T) and adjacent non-tumorous liver tissues (N) from the SCMC cohort (n=10) and the GSE104766 dataset (n=25). Each dot represents an individual sample. Statistical significance was assessed using a paired t test; ①P=0.003,②P<0.001. B. qPCR analysis of USP37 mRNA expression in HB patient tissue samples. Data are presented as x±s. Each sample was analyzed in triplicate. Roman numerals (Ⅰ‒Ⅷ) indicate individual paired patient samples. Statistical significance was determined using paired t test, ①P<0.001, ②P=0.003, ③P=0.002, ④P=0.004, ⑤P=0.002, and ⑥P=0.001. C. Receiver operating characteristic (ROC) curve analysis evaluating the diagnostic performance of USP37 expression in distinguishing HB tissues from non-tumorous tissues. The area under the curve (AUC) is shown. D. Kaplan-Meier survival analysis of the hepatocellular carcinoma (HCC) cohort stratified by USP37 expression levels. Overall survival was compared between the high- and low-expression groups using the log-rank test. The hazard ratio (HR) and 95% confidence interval (CI) are indicated.

Fig 2 Expression analysis and prognostic evaluation of USP37 in HB

表4 USP37 表达水平与HB临床特征的关系［n（%）］

Tab 4 Association between USP37 expression and clinical features in HB ［n（%）］

Characteristic	USP37 Expression		P value
Characteristic	Low (n=17)	High (n=16)	P value
Age at diagnosis			0.176
≤24 months	11 (33.33)	6 (18.18)
>24 months	6 (18.18)	10 (30.30)
Gender			0.514
Male	9 (27.27)	11 (33.33)
Female	8 (24.24)	5 (15.15)
Risk stratification			0.142
Low/intermediate-risk	14 (42.42)	9 (27.27)
High-risk	3 (9.09)	7 (21.21)
PRETEXT stage			1.000
Ⅰ/Ⅱ	7 (21.21)	7 (21.21)
Ⅲ/Ⅳ	10 (30.30)	9 (27.27)
Metastatic			0.028
Non-metastatic	16 (48.48)	8 (24.24)
Metastatic	1 (3.03)	6 (18.18)
NA	0	2 (6.06)
Tumor volume			0.031
≤500 cm³	11 (33.33)	3 (9.09)
>500 cm³	2 (6.06)	7 (21.21)
NA	4 (12.12)	6 (18.18)

图3 USP37 敲低对HB细胞增殖的影响Note： A. qPCR analysis of USP37 mRNA levels in HuH6 and HepG2 cells transduced with control shRNA (shCtrl) or two independent USP37-targeting shRNAs (shUSP37-1 and shUSP37-2). Data are presented as x±s from n=3 independent experiments. Statistical significance was assessed using Student's t test; ①P<0.001. B. Western blotting analysis showing USP37 protein levels in HuH6 and HepG2 cells expressing shCtrl, shUSP37-1, or shUSP37-2. β-actin was used as a loading control. Relative protein levels were quantified by densitometric analysis and normalized to β-actin. C. Cell proliferation assays of HuH6 and HepG2 cells expressing shCtrl, shUSP37-1, or shUSP37-2. Data are shown as x±s from n=6 independent experiments. Statistical significance was determined using student's t test; ①P<0.001. D. Colony formation assays performed in HuH6 and HepG2 cells following USP37 knockdown. Representative images and quantification of colony numbers are shown. Data are presented as x±s from n=3 independent experiments. Statistical significance was assessed using Student's t test; ①P<0.001. E. Western blotting analysis showing USP37 and Flag-USP37 protein levels in HuH6 and HepG2 cells expressing shCtrl, shUSP37-1/2, or shUSP37-1/2+Flag-USP37. β-actin was used as a loading control. Relative protein levels were quantified by densitometric analysis and normalized to β-actin. F. Cell proliferation assays of HuH6 and HepG2 cells expressing shCtrl, shUSP37-1/2, or shUSP37-1/2+Flag-USP37. Data are shown as x±s from n=6 independent experiments. Statistical significance was determined using one-way ANOVA; ①P<0.001.

Fig 3 Effects of USP37 knockdown on HB cell proliferation

图4 USP37 表达水平变化对 c-MYC mRNA及蛋白水平的影响Note： A. qPCR analysis of USP37 and c-MYC mRNA levels in 293T cells transfected with increasing amounts of Flag-USP37 plasmid (0, 0.5, and 1 μg). mRNA levels were calculated using the 2-ΔΔCt method, with the 0 μg group set as 1. Data are presented as x±s from n=3 independent experiments. Statistical significance was assessed using Student's t test: for USP37 mRNA. B.Western blotting analysis of c-MYC and Flag-USP37 protein levels in 293T cells transfected with increasing amounts of Flag-USP37 plasmid (0, 0.5, and 1 μg). β-actin was used as the loading control. c-MYC protein levels were quantified by densitometry, normalized to β-actin, and expressed relative to the 0 μg group (1, 1.91, and 3.25 for 0, 0.5, and 1 μg, respectively). C. qPCR analysis of c-MYC mRNA levels in HuH6 and HepG2 cells expressing shCtrl, shUSP37-1, or shUSP37-2. mRNA levels were calculated using the 2-ΔΔCt method, with the shCtrl group set as 1. Data are presented as x̄±s from n=3 independent experiments. Statistical significance was determined using Student's t test: in HuH6 cells. D. Western blotting analysis of c-MYC and USP37 protein levels in HuH6 and HepG2 cells expressing shCtrl, shUSP37-1, or shUSP37-2. β-actin was used as the loading control. c-MYC protein levels were quantified by densitometry, normalized to β-actin, and expressed relative to the shCtrl group: HuH6, 1/0.32/0.26 (shCtrl/shUSP37-1/shUSP37-2); HepG2, 1/0.40/0.35.

Fig 4 Effects of USP37 expression modulation on c-MYC mRNA and protein levels

图5 USP37 表达水平变化对c-MYC蛋白稳定性的影响分析Note： A. CHX chase assay in 293T cells transfected with control vector or Flag-USP37. Cells were treated with CHX (50 µg/mL) and harvested at 0, 0.5, 1, and 2 h. Left: representative immunoblots of c-MYC, Flag-USP37, and β-actin. Right: quantification of c-MYC protein levels. c-MYC signals were normalized to β-actin, further normalized to the 0 h time point within each group, and log₂-transformed. Data are presented as x±s from n=2 independent experiments. Statistical significance between the two degradation curves was assessed by two-way ANOVA (P=0.006). The estimated half-life of c-MYC was 2.357 h in the control group and 4.796 h in the USP37-overexpression group. B. CHX chase assay in HuH6 cells expressing shCtrl or shUSP37-1/2. Cells were treated with CHX (50 µg/mL) and harvested at 0, 0.5, 1, and 2 h. Left: representative immunoblots of c-MYC, USP37, and β-actin. Right: quantification of c-MYC protein levels, normalized to β-actin, then to the 0 h time point within each group, and log₂-transformed. Data are presented as x̄±s from n=2 independent experiments. Statistical significance between the two degradation curves was assessed by two-way ANOVA (P=0.006). The estimated half-life of c-MYC was 1.504 h in shCtrl cells and 0.986 h in shUSP37 cells. C. CHX chase assay in HepG2 cells expressing shCtrl or shUSP37-1/2. Cells were treated with CHX (50 µg/mL) and harvested at 0, 0.5, 1, and 2 h. Left: representative immunoblots of c-MYC, USP37, and β-actin. Right: quantification of c-MYC protein levels, normalized to β-actin, then to the 0 h time point within each group, and log2-transformed. Data are presented as x̄±s from n=2 independent experiments. Statistical significance between the two degradation curves was assessed by two-way ANOVA (P=0.005). The estimated half-life of c-MYC was 1.585 h in shCtrl cells and 0.792 h in shUSP37 cells.

Fig 5 Analysis of c-MYC protein stability following modulation of USP37 expression

图6 USP37 通过去泛素化调控c-MYC蛋白稳定性的机制分析Note： A. HuH6 and HepG2 cells expressing shCtrl or shUSP37 were treated with DMSO or the proteasome inhibitor MG132 (20 μmol/L for 6 h). Protein levels of c-MYC and USP37 were analyzed by Western blotting. β-actin was used as a loading control. Relative protein levels were quantified by densitometric analysis and normalized to β-actin. B. Co-immunoprecipitation (Co-IP) analysis of the interaction between USP37 and c-MYC. 293T cells were transfected with HA-c-MYC with or without Flag-USP37, as indicated. Cell lysates were immunoprecipitated with anti-Flag beads, and the precipitated proteins were immunoblotted with anti-HA and anti-Flag antibodies. Whole-cell lysates (WCL) were included as input controls to confirm protein expression. C. Reverse Co-IP analysis of the interaction between USP37 and c-MYC. 293T cells were transfected with HA-USP37 with or without Flag-c-MYC, as indicated. Cell lysates were immunoprecipitated with anti-Flag beads, and the precipitated proteins were immunoblotted with anti-HA and anti-Flag antibodies. WCL were included as input controls. D. In vivo deubiquitination assay. 293T cells were transfected with His-Ub and HA-c-MYC, with or without Flag-USP37, as indicated. At 24-36 h after transfection, cells were treated with MG132 (20 μmol/L) for 6 h before harvest. HA-c-MYC was immunoprecipitated with anti-HA beads, and the ubiquitinated c-MYC was detected by immunoblotting with an anti-His antibody. The immunoprecipitated HA-c-MYC levels are shown below as a loading control for the IP. WCL was analyzed to confirm the expression of His-Ub, HA-c-MYC, and Flag-USP37.

Fig 6 Mechanistic analysis of USP37-mediated deubiquitination and stabilization of c-MYC

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泛素特异性蛋白酶37通过去泛素化稳定骨髓细胞瘤病毒癌基因同源物蛋白促进肝母细胞瘤进展的机制研究

USP37-mediated deubiquitination maintains c-MYC protein homeostasis and contributes to hepatoblastoma progression

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