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Identication of ubiquitin‑specic
protease 32 as an oncogene
in glioblastoma and the underlying
mechanisms
Sifang Chen1, Xi Chen1, Zhangyu Li1, Jianyao Mao1, Weichao Jiang1, Zhi Zhu2, Yukui Li1,
Zhengye Jiang1, Wenpeng Zhao1, Guowei Tan1 & Zhanxiang Wang1,3,4*
Glioblastoma (GBM) patients present poor prognosis. Deubiquitination by deubiquitinating enzymes
(DUBs) is a critical process in cancer progression. Ubiquitin‑specic proteases (USPs) constitute the
largest sub‑family of DUBs. Evaluate the role of USP32 in GBM progression and provide a potential
target for GBM treatment. Clinical signicance of USP32 was investigated using Gene Expression
Omnibus databases. Eects of USP32 on cell growth and metastasis were studied in vitro and in vivo.
Dierentially expressive genes between USP32‑knockdown U‑87 MG cells and negative control cells
were detected using RNA sequencing and used for Gene Ontology and Kyoto Encyclopedia of Genes
and Genomic pathway enrichment analyses. Finally, RT‑qPCR was used to validate the divergent
expression of genes involved in the enriched pathways. USP32 was upregulated in GBM patients,
being correlated to poor prognosis. USP32 downregulation inhibited cell growth and metastasis
in vitro. Furthermore, USP32 knockdown inhibited tumorigenesis in vivo. In addition, UPS32 was
identied as a crucial regulator in dierent pathways including cell cycle, cellular senescence, DNA
replication, base excision repair, and mismatch repair pathways. USP32 acts as an oncogene in GBM
through regulating several biological processes/pathways. It could be a potential target for GBM
treatment.
Glioblastoma (GBM), the most aggressive type of glioma, are characterized by cellular heterogeneity, a diusive
inltration of tumor cells, and the presence of glioma stem-like cells capable of signicantly expanding and
generating new tumors. GBM patients have an average overall survival time of barely more than 15 months1–4.
Understanding the molecular mechanisms driving malignancy helps in the development of agents specically
targeting the tumor cells or tumor microenvironment5. Despite improvements in therapeutic strategies, GBM
remains a clinical challenge. ere is an urgent need to nd novel regulators or targets for exploring highly
bioactive and brain-penetrating targeted therapies.
Ubiquitination is an ATP-dependent cascade process that ligates ubiquitin to a substrate protein, catalyzed
by the E1, E2, and E3 three-enzyme cascade6. e ubiquitinated proteins can be specially recognized by the 26S
proteasome, and this leads to the proteasomal degradation of these proteins, regulating dierent cell processes.
Many studies have revealed that the ubiquitin–proteasome system is involved in the progression of GBM7–10. A
siRNA screening analysis showed that 22% of GBM-survival-relevant genes were components of the 20S and 26S
proteasome subunits11. Epidermal growth factor receptor (EGFR) amplication and mutations are commonly
observed in GBM; EGFR stability and downstream signaling are subject to the ubiquitin regulatory network10.
Homologous to E6-AP Carboxyl Terminus (HECT) E3 ligase Smurf2 suppresses TGF-β signaling by targeting
TbR-I for proteasomal degradation, promoting the progression of GBM12. HECT E3 ligase HERC3 play an essen-
tial role in autophagy-induced EMT, resulting in the chemoresistance of GBM13. In GBM, the p53 and c-Myc
levels are regulated by E3 ligases MDM2 and TRIM3, respectively14,15. e ubiquitination is oen antagonized by
deubiquitinating enzymes (DUBs) which remove the ubiquitin chains from ubiquitinated proteins16. More than
100 DUBs have been identied and categorized into 8 subfamilies, of which ubiquitin-specic proteases (USPs)
OPEN
1Department of Neurosurgery, The First Aliated Hospital of Xiamen University, No. 55 Zhenghai Road, Siming
District, Xiamen 361000, Fujian, China. 2Department of Neurosurgery, Heze Municipal Hospital, Heze 274000,
Shandong, China. 3Xiamen Key Laboratory of Brain Center, The First Aliated Hospital of Xiamen University,
Xiamen 361000, Fujian, China. 4Department of Neuroscience, Institute of Neurosurgery, School of Medicine,
Xiamen University, Xiamen 361000, Fujian, China. *email: csfsong@163.com
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constitute the largest one. DUBs not only function to reverse ubiquitination but are also involved in protein
tracking, chromatin remodeling, cell cycle regulation, base excision repair, mismatch repair, and signaling
pathway modulations, which are tightly associated with the development of cancer17,18. DUBs have emerged
as attractive targets for targeted therapy in cancer19,20 aer the clinical success of proteasomal inhibitors, such
as bortezomib, ixazomib, and carlzomib. NSC 144303 (G5) and NSC 632839 (F6), two DUBs inhibitors, are
under pre-clinical trial6. Several DUBs were reported to regulate multiple cellular processes such as apoptosis,
proliferation, and stemness in GBM7,21.
USP32 is a member of USPs discovered in recent years. Previous studies had shown that USP32 is highly
expressed in breast cancer and promotes the growth of breast tumor cells22. Hu etal. revealed that USP32
knockdown suppresses cellular proliferation and cell metastasis in small cell lung cancer23. e study of Dou
etal. reported that high expression of USP32 is signicantly associated with high T stage and poor prognosis in
gastric cancer patients24. USP32 was also identied as an oncogene in epithelial ovarian25. However, USP32 is still
rarely reported in malignant disease processes, especially in GBM. In this study, USP32 was knocked down in
GBM cells, evaluating the eect of this enzyme on cell growth and metastasis. Tumor xenogra experiments in
nude mice were also performed to validate the role of USP32 in GBM development. Transcriptional sequencing
identied the dierential expressed genes between stably USP32-knockdown U-87 MG cells and negative control
cells. Functional annotations, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes
(KEGG) pathway enrichment analyses, were used to uncover the underlying mechanism of USP32 on promoting
the progression of GBM. Our study may provide a potential target for GBM treatment.
Methods
Cell lines and cell culture. 293T, SVG p12, U-87 MG, U-118 MG, U-251 MG, T98G, and A172 cells were
purchased from Xiamen Immocell Biotechnology Co., Ltd (Xiamen, Fujian, China). All cells were maintained
using Dulbecco’s modied Eagle’s medium (DMEM, IMMOCELL) containing 15% fetal bovine serum (FBS,
Gibco) at 37°C in an incubator with an atmosphere containing 5% CO2 and 21% O2.
High‑content screening. Small interfering RNAs (siRNAs) targeting USPs were obtained from GeneP-
harma (Shanghai, China). Table1 describes these siRNAs. U-87 MG and U-251 MG cells were seeded into
96-well plates at a density of 1 × 104 per well. Lipofectamine RNAiMAX (Life Technology, Carlsbad, CA, USA)
was used for siRNA transfection at 37°C. Aer a 36h of transfection, the cells were incubated with 10μL/well
CCK-8 solution (Yeasen, Shanghai, China). e cell viability was evaluated 2h later by detecting the optical
density at 450nm (OD450) using a microplate reader (Molecular Devices, San Francisco, CA, USA).
Data mining from Gene Expression Omnibus (GEO) databases. Dataset GSE59612 (normal 17,
GBM 39) was downloaded from the GEO website (https:// www. ncbi. nlm. nih. gov/ geo/) to evaluate the dier-
ential expression of USP32 between normal tissues and tumor tissues. e mRNA expression data and survival
information of GBM patients in datasets GSE74187 (GBM 60) and GSE83300 (GBM 50) were also downloaded.
e association between USP32 expression level and prognosis was analyzed. GBM patients were divided into
two groups (high and low) using the optimal cut-o value of USP32 expression level, which was determined
using the surv_cutpoint function of the R package survminer via RStudio soware (version 2021.09.0 + 351,
https:// www. rstud io. com/).
RT‑qPCR. Total RNA from U-87 MG was obtained using an RNA Extraction Kit (Vazyme, Nanjing, Jiangsu,
China) and reverse transcribed into cDNA using HiScript II Reverse Transcriptase (Vazyme). One hundred
nanogram of cDNA was used for qPCR per well. qPCR was performed using a Bio-Rad CFX96 system (Bio-Rad
Laboratories, Hercules, CA, USA) with an AceQ qPCR SYBR Green Master Mix Kit (Vazyme). e thermocy-
cling condition was 96°C for 5min, followed by 96°C for 15s, 60°C for 25s, and 72°C for 20s, for 45 cycles.
e 2−ΔΔCt method was used to calculate the relative mRNA levels, which was calibrated to 18S RNA. e primers
for RT-qPCR are shown in Table2.
Western blotting. U-251 MG and U-87 MG cells were lysed using RIPA buer (Vazyme). e protein
concentrations of samples were measured using a bicinchoninic acid (BCA) protein quantication kit (Abcam,
Shanghai, China). Samples (12μg/lane) were loaded into a 12% SDS-PAGE gel for electrophoresis and then
transferred onto a PVDF membrane (Roche, Basel, Switzerland). e membrane was incubated with primary
antibody Anti-USP32 (1:1000, CAT#ab251903, Abcam) or Anti-GAPDH (1:2000, CAT#ab9485, Abcam) at
4°C overnight, followed by incubation with secondary antibody HRP-conjugated Goat Anti-Rabbit IgG H&L
(1:2000, CAT#ab6721, Abcam) at 28°C for 30min. e signals were visualized using the ECL detection system
(ermo Fisher Scientic, Waltham, MA, USA) and quantied by densitometry using Image J v1.48u.
USP32 knockdown by transfection. U-251 MG and U-87 MG cells were seeded into 6-well plates at
a density of 5 × 105 per well and transfected with 200pmol/well siRNA NC, siUSP32-2261, or siUSP32-386
using LipofectamineRNAiMAX. e cells were harvested and used for further experiments aer incubation for
6–48h at 37°C.
CCK‑8 assay for cell viability. U-251 MG and U-87 MG cells were tripsinized aer a 24h of transfection
and then seeded into 96-well plates at a density of 5 × 103 per well. At dierent time points (0, 24, 48, 72h), 10μL/
well CCK-8 solution was added into cells and the cell viability was evaluated by detecting OD450.
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siRNAs Sense (5′–3′) Antisense (5′–3′)
siUSP1-1780 GGU UAA AGU CUG CAA CUA AUU UUA GUU GCA GAC UUU AAC CUU
siUSP1-1501 GCA UAG AGA UGG ACA GUA UUU AUA CUG UCC AUC UCU AUG CUU
siUSP2-1339 GCG CUU UGU UGG CUA UAA UUU AUU AUA GCC AAC AAA GCG CUU
siUSP2-1921 CCU GUA CGC UGU GUC CAA UUU AUU GGA CAC AGC GUA CAG GUU
siUSP3-1109 GGG ACA GAA UCU AGA AAG UUU ACU UUC UAG AUU CUG UCC CUU
siUSP3-1569 GCU GGU UCC ACU UCA AUG AUU UCA UUG AAG UGG AAC CAG CUU
siUSP4-377 GCG UGG AAU AAA CUA CUA AUU UUA GUA GUU UAU UCC ACG CUU
siUSP4-860 GCA AAU GGU GAU AGC ACU AUU UAG UGC UAU CAC CAU UUG CUU
siUSP5-822 GGA GCU GAC GUG UAC UCA UUU AUG AGU ACA CGU CAG CUC CUU
siUSP5-1265 GCC AGA ACA GAA GGA AGU UUU AAC UUC CUU CUG UUC UGG CUU
siUSP6-2274 GGA AGG ACA UAC UUA UGA AUU UUC AUA AGU AUG UCC UUC CUU
siUSP6-2494 GCA CAG UAG CAA ACU CAU AUU UAU GAG UUU GCU ACU GUG CUU
siUSP7-2625 GUG GUU ACG UUA UCA AAU AUU UAU UUG AUA ACG UAA CCA CUU
siUSP7-603 GCA GUG CUG AAG AUA AUA AUU UUA UUA UCU UCA GCA CUG CUU
siUSP8-873 CCA AAG AGA AAG GAG CAA UUU AUU GCU CCU UUC UCU UUG GUU
siUSP8-3569 GCA AGA CAA CGG UGG UUU AUU UAA ACC ACC GUU GUC UUG CUU
siUSP9X-7849 GGG CAA UGG AGA UCU UAA AUU UUU AAG AUC UCC AUU GCC CUU
siUSP9X-2333 CCC GCA CUG AAA CAA AUU AUU UAA UUU GUU UCA GUG CGG GUU
siUSP9Y-2805 CCU UGC AAC CUA CAU GAA UUU AUU CAU GUA GGU UGC AAG GUU
siUSP9Y-8135 GCA GUU GUC CUG UUG CUU AUU UAA GCA ACA GGA CAA CUG CUU
siUSP10-630 GCU UUG GAU GGA AGU UCU AUU UAG AAC UUC CAU CCA AAG CUU
siUSP10-1175 GCA CAC CAC GGA AAG CAU AUU UAU GCU UUC CGU GGU GUG CUU
siUSP11-1415 GCA AUG UAU CUG UGA CCU UUU AAG GUC ACA GAU ACA UUG CUU
siUSP11-2088 CCU CCU GGA CAA UUG CCU UUU AAG GCA AUU GUC CAG GAG GUU
siUSP12-314 CUU CGG CAU UAG AGA AAG AUU UCU UUC UCU AAU GCC GAA GUU
siUSP12-648 CCU ACU AAA UAC AAU UGC UUU AGC AAU UGU AUU UAG UAG GUU
siUSP13-1685 CGU GCC AAG AUA CCA UUU AUU UAA AUG GUA UCU UGG CAC GUU
siUSP13-2004 GCC UGA UGA ACC AAU UGA UUU AUC AAU UGG UUC AUC AGG CUU
siUSP14-655 GCU UCA GCG CAG UAU AUU AUU UAA UAU ACU GCG CUG AAG CUU
siUSP14-1632 GCA UAU CGC UUA CGU UCU AUU UAG AAC GUA AGC GAU AUG CUU
siUSP15-249 GGA ACA CCU UAU UGA UGA AUU UUC AUC AAU AAG GUG UUC CUU
siUSP15-1150 GCA GAU GGA AGG CCA GAU AUU UAU CUG GCC UUC CAU CUG CUU
siUSP16-331 GGA AUG GAA UAU CUG CCA AUU UUG GCA GAU AUU CCA UUC CUU
siUSP16-469 GCA UGC CUU GAA GCA CUA UUU AUA GUG CUU CAA GGC AUG CUU
siUSP17-1431 CCA UCA UCC UGA ACA GCA AUU UUG CUG UUC AGG AUG AUG GUU
siUSP17-1538 GGA GAU CCA AAG GGA AGA AUU UUC UUC CCU UUG GAU CUC CUU
siUSP18-527 GCU GCC UUA ACU CCU UGA UUU AUC AAG GAG UUA AGG CAG CUU
siUSP18-1418 CUG CAU AUC UUC UGG UUU AUU UAA ACC AGA AGA UAU GCA GUU
siUSP19-2439 GCA UUC AGA ACA AGC CCU AUU UAG GGC UUG UUC UGA AUG CUU
siUSP19-2518 GCG GCA CAA GAU GAG GAA UUU AUU CCU CAU CUU GUG CCG CUU
siUSP20-249 CCA UAG GAG AGG UGA CCA AUU UUG GUC ACC UCU CCU AUG GUU
siUSP20-1041 GCC CAU CAG AAG AUG AGU UUU AAC UCA UCU UCU GAU GGG CUU
siUSP21-641 CCA ACU UAG CCC GUU CCA AUU UUG GAA CGG GCU AAG UUG GUU
siUSP21-1353 GCU AGA AGA ACC UGA GUU AUU UAA CUC AGG UUC UUC UAG CUU
siUSP22-1367 GCU ACC AGG AGU CCA CAA AUU UUU GUG GAC UCC UGG UAG CUU
siUSP22-695 GGA GAA AGA UCA CCU CGA AUU UUC GAG GUG AUC UUU CUC CUU
siUSP24-611 GGA AUU GAA UUC CCU ACA AUU UUG UAG GGA AUU CAA UUC CUU
siUSP24-719 GCA UCU ACC UAC CUA GCA AUU UUG CUA GGU AGG UAG AUG CUU
siUSP25-897 GCC AAA GAA CCC UAU GGU AUU UAC CAU AGG GUU CUU UGG CUU
siUSP25-1128 GCC GGU AUU AAC AUU UGA AUU UUC AAA UGU UAA UAC CGG CUU
siUSP26-1600 CCU UAU UGU UCA CCU CAA AUU UUU GAG GUG AAC AAU AAG GUU
siUSP26-2426 GGU UCC AAU AAG AAU CCA AUU UUG GAU UCU UAU UGG AAC CUU
siUSP27-1398 GGC GCA AGA UCA CUA CAU AUU UAU GUA GUG AUC UUG CGC CUU
siUSP27-855 CUC CUC AUG UGC CCU AUA AUU UUA UAG GGC ACA UGA GGA GUU
siUSP28-1836 GGG CCU AUA UCU AUA AUC AUU UGA UUA UAG AUA UAG GCC CUU
siUSP28-841 GCA UUC CAG CUA GCU GUU AUU UAA CAG CUA GCU GGA AUG CUU
Continued
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Table 1. Sequences of siRNAs targeting USPs.
siRNAs Sense (5′–3′) Antisense (5′–3′)
siUSP29-983 CCC AUC AAG UUU AGA GGA UUU AUC CUC UAA ACU UGA UGG GUU
siUSP29-1922 GGU GAA GAA UAA CGA GCA AUU UUG CUC GUU AUU CUU CAC CUU
siUSP30-176 CCG UCA GAU AUA AAG UCA UUU AUG ACU UUA UAU CUG ACG GUU
siUSP30-521 GCU GCU UGU UGG AUG UCU UUU AAG ACA UCC AAC AAG CAG CUU
siUSP31-954 GCC UCU CUA UGU CAC UGU AUU UAC AGU GAC AUA GAG AGG CUU
siUSP31-3922 GCU CGC AAA UCC AAG UCU UUU AAG ACU UGG AUU UGC GAG CUU
siUSP32-2261 GCG CAU UAA AGA GGA AGA UUU AUC UUC CUC UUU AAU GCG CUU
siUSP32-386 GAC CUG UGG ACU CUC AUA UUU AUA UGA GAG UCC ACA GGU CUU
siUSP33-829 CCC AGU AAU ACA ACA UUA AUU UUA AUG UUG UAU UAC UGG GUU
siUSP33-597 GGA GAA UAG AUG UUC AUA UUU AUA UGA ACA UCU AUU CUC CUU
siUSP34-1228 GCG ACU GAG UAC UCA ACA UUU AUG UUG AGU ACU CAG UCG CUU
siUSP34-3023 CCU GAU CAU UUC AGG UUA AUU UUA ACC UGA AAU GAU CAG GUU
siUSP35-3188 CCC UGC ACA AGG ACU UGA UUU AUC AAG UCC UUG UGC AGG GUU
siUSP35-1916 GCU CGG AGU AUC UGA AGU AUU UAC UUC AGA UAC UCC GAG CUU
siUSP36-741 CCA ACU ACC UGC UCU CCA AUU UUG GAG AGC AGG UAG UUG GUU
siUSP36-474 GCA AAU AUG UGU UGC UCA AUU UUG AGC AAC ACA UAU UUG CUU
siUSP37-555 CCA AGG AUA UUU CAG CUA AUU UUA GCU GAA AUA UCC UUG GUU
siUSP37-2235 GCA CAU AUG GCA AUU UCU AUU UAG AAA UUG CCA UAU GUG CUU
siUSP38-3501 GGU AAG UUG GAA AUA CAA GUU CUU GUA UUU CCA ACU UAC CUU
siUSP38-1047 GGU UCG AAC GAU AGG CCA UUU AUG GCC UAU CGU UCG AAC CUU
siUSP39-958 GGA ACC CUC GAA AUU UCA AUU UUG AAA UUU CGA GGG UUC CUU
siUSP39-1375 GCA UCA CUG AGA AGG AAU AUU UAU UCC UUC UCA GUG AUG CUU
siUSP40 -721 GCA GCA AAG UCG GCC AAA UUU AUU UGG CCG ACU UUG CUG CUU
siUSP40 -1212 GCU CCA UUC UCA GAU AUU UUU AAA UAU CUG AGA AUG GAG CUU
siUSP42-459 GCU CCA GAA UUU GGG CAA UUU AUU GCC CAA AUU CUG GAG CUU
siUSP42-750 GCA GAA AGC AUG CUU GAA UUU AUU CAA GCA UGC UUU CUG CUU
siUSP43-813 GCC ACU UUC AAG CAC AAU AUU UAU UGU GCU UGA AAG UGG CUU
siUSP43-2196 GGG CUU AUA UCC UGU UCU AUU UAG AAC AGG AUA UAA GCC CUU
siUSP44-653 GGG UAC AGG UGA UGA UUC UUU AGA AUC AUC ACC UGU ACC CUU
siUSP44-1553 CGC UCA GGA AUU UCU UUG UUU ACA AAG AAA UUC CUG AGC GUU
siUSP45-1377 GGC ACC UCG AUU UAA AGA UUU AUC UUU AAA UCG AGG UGC CUU
siUSP45-753 GCA GCU AGU ACU UAC UUC UUU AGA AGU AAG UAC UAG CUG CUU
siUSP46-204 GGU CCA GAG CAG UUU CCA AUU UUG GAA ACU GCU CUG GAC CUU
siUSP46-426 CCA CCA AAG AAG UUC AUU UUU AAA UGA ACU UCU UUG GUG GUU
siUSP47-2463 GCU GUC GCC UUG UUA AAU AUU UAU UUA ACA AGG CGA CAG CUU
siUSP47-3757 CCA GCA AUC AAG AGU UUG AUU UCA AAC UCU UGA UUG CUG GUU
siUSP48-676 GCA UCU CCA GUA CUU GUU UUU AAA CAA GUA CUG GAG AUG CUU
siUSP48-871 GCA GUU CUG UGG AGA AUA UUU AUA UUC UCC ACA GAA CUG CUU
siUSP49-1925 GGG UCC AUG UCG UCU UUG AUU UCA AAG ACG ACA UGG ACC CUU
siUSP49-1825 GAA GCU AGA AAG CAG UUA AUU UUA ACU GCU UUC UAG CUU CUU
siUSP50-571 GCU CAG GAA UUC UUG AUU UUU AAA UCA AGA AUU CCU GAG CUU
siUSP50-692 CCA CUG AGA CAU CCA UCA UUU AUG AUG GAU GUC UCA GUG GUU
siUSP51-895 CCA UUU AGC UGU AGA CCU UUU AAG GUC UAC AGC UAA AUG GUU
siUSP51-1225 CCA UAU UCC UCU ACU GAA AUU UUU CAG UAG AGG AAU AUG GUU
siUSP52-896 GCU GCA GAA UCA CAU ACU AUU UAG UAU GUG AUU CUG CAG CUU
siUSP52-1247 GCG CUU CAU UCC UAC AUA UUU AUA UGU AGG AAU GAA GCG CUU
siUSP53-3296 GAG CCA ACA UCA CUU AGA AUU UUC UAA GUG AUG UUG GCU CUU
siUSP53-1605 GUG CGG UAC AUU UCU ACA AUU UUG UAG AAA UGU ACC GCA CUU
siUSP54-3654 GCU GCC UAA UGG UGA AAC UUU AGU UUC ACC AUU AGG CAG CUU
siUSP54-2546 GAG CCC UAG UCG AUA AGA AUU UUC UUA UCG ACU AGG GCU CUU
siRNA NC CUC CGA ACG UGU CAC GUU CGU GAC ACG UUC GGA GUU
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Colony formation assay for cell growth. U-251 MG and U-87 MG cells were tripsinized and then
seeded into 6-well plates at a density of 1 × 103 per well 6h aer transfection. e cells were continuously cul-
tured at 37°C for 7–10days. e colonies were xed with 4% methanal (Sangon Biotech, Shanghai, China) in
phosphate buer saline (PBS) at 28°C for 15min. Aer washing with PBS three times, the colonies were pho-
tographed using HUAWEI Mate 40 (Huawei, Shenzhen, China) and the number of colonies was counted using
Image J v1.48u (NIH, Bethesda, MD, USA).
Microscopic analysis of EdU incorporation for cell proliferation. U-251 MG and U-87 MG cells
were tripsinized aer a 24h of transfection and then seeded into 96-well plates at a density of 2 × 104 per well.
Twelve hours later, the cells were labeled with 10μM EdU solution (Beyotime, Shanghai, China) at 37°C for 3h.
Aer xation with 4% methanal in PBS at 28°C for 20min and subsequent permeation with 0.5% Triton X-100
(Sangon Biotech) in PBS at 28°C for 10min, the cells were incubated with 50µL of 1 × Click Additive Solution
(Beyotime) at 28°C for 30min. e nucleus was stained with 5μg/mL DAPI (Yeasen). Finally, the uorescent
dots were observed and photographed using a uorescence microscope (MOTIC, Hongkong, China) and then
photographed. Image J v1.48u was used to count the number of cells.
Flow cytometry‑based cell cycle. U-251 MG and U-87 MG cells were collected aer a 24h of transfec-
tion and then xed using 70% ethanol in PBS at − 20°C for 6h. e cells were then treated with 0.5% Triton
X-100 and 10μg/mL RNase (Sangon Biotech) at 28°C for 25min. Finally, the cells were stained with 20μg/
mL propidium iodide (PI, Vazyme) in the dark at 28°C for 25min and then placed into to the ow cytometer
NovoCyte 1300 (ACEA, San Diego, CA, USA) for uorescent detection within the PE-channel (Ex: 488nm/Em:
578nm).
Transwell assay for cell migration and invasion. U-251 MG and U-87 MG cells were tripsinized aer
a 24h of transfection and then suspended in FBS-free DMEM at a density of 3 × 105/mL. In the transwell migra-
tion assay, 100μL of suspended cells were placed into the upper chambers of transwell plates (NEST, Wuxi,
Jiangsu, China). e lower chamber was supplied with 550μL 10% FBS DMEM. In the Matrigel invasion assay,
the membrane of the upper chamber was pre-coated with sixfold diluted Matrigel (Corning, Corning, NY, USA)
before seeding cells. e migrated and invasive cells were xed with 4% methanal in PBS and then stained with
1% crystal violet (Sangon Biotech) in PBS at 28°C for 20min, respectively. e stained cells were observed and
photographed using a light microscope (MOTIC). e number of migrated/invasive cells were counted using
Image J v1.48u in three random elds.
Construction of stable U‑87 MG cell lines with USP32 knockdown (shUSP32). e lentiviral
vector PLKO.1-TRC-Puro (Antihela, Xiamen, Fujian, China) was used to construct plasmid overexpressing
short hairpin RNA targeting USP32 and shctrl plasmid. e primers for shUSP32 plasmid construction were
designed based on the sequence of siUSP32-386 (Table2). For lentiviral packaging, 293T cells (4 × 106/well)
were seeded into 6-well plates and transfected with 3µg shUSP32 or shctrl plasmid, 2µg psPAX2 (Antihela),
Table 2. Primers for RT-qPCR and plasmid construction.
Name Forward primer (5′–3′) Reverse primer (5′–3′)
CCNB1 GAC CTG TGT CAG GCT TTC TCTG GGT ATT TTG GTC TGA CTG CTTGC
CDC25A TCT GGA CAG CTC CTC TCG TCAT ACT TCC AGG TGG AGA CTC CTCT
CDC45C TGG ATG CTG TCC AAG GAC CTGA CAG GAC ACC AAC ATC AGT CACG
CDK1 GGA AAC CAG GAA GCC TAG CATC GGA TGA TTC AGT GCC ATT TTGCC
MCM3 CGA GAC CTA GAA AAT GGC AGCC GCA GTG CAA AGC ACA TAC CGCA
MCM4 CTT GCT TCA GCC TTG GCT CCAA GTC GCC ACA CAG CAA GAT GTTG
MCM6 GAC AAC AGG AGA AGG GAC CTCT GGA CGC TTT ACC ACT GGT GTAG
MCM7 GCC AAG TCT CAG CTC CTG TCAT CCT CTA AGG TCA GTT CTC CACTC
FEN1 ACT AAG CGG CTG GTG AAG GTCA GCA GCA TAG ACT TTG CCA GCCT
NEIL3 AGT GGT CTC CAC CCA GCT GTTA AGA GCA AGT CCT GCT TTA CGGC
POLE ACG CTG GAA GAG GTG TAT GGCT GGA ACG GTT CTC AGA GAT GAGC
POLE2 TGC GTC CGT TTT CCT AGC AGCA GGG CAG ACA TAA AGA GGT AGGG
EXO1 TCG GAT CTC CTA GCT TTT GGCTG AGC TGT CTG CAC ATT CCT AGCC
RFC2 GTC GGG AAT GAA GAC ACC GTGA CAG AAT GCT TGT GGT CTT GCCG
RFC3 CCT GAG ACA GAT TGG GAG GTGT AGC TCA TAC AGC CTT CCA CGAAC
RFC4 GGC AGC TTT AAG ACG TAC CATGG TCT GAC AGA GGC TTG AAG CGGA
Shctrl
CCG GCT CCG AAC GTG TCA CGCTC AAT TAA AAA CTC CGA ACG TGT CAC GCT CGA GCG TGA CAC GTT CGGAG
GAG CGT GAC ACG TTC GGA GTT TTT AAT TAA AAA GAC CTG TGG ACT CTC ATA TCT CGA GAT ATG AGA GTC CAC
AGGTC
shUSP32 CCG GGA CCT GTG GAC TCT CAT ATC TCG AGA TAT GAG AGT CCA CAG GTC
TTTTT
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and 1µg pMD2.G (Antihela) using LipofectamineRNAiMAX at 37°C. Aer incubation for 48h, the lentivirus
was harvested and used to infect U-87 MG cells at a multiplicity of infection of 30 with the addition of 10µg/mL
polybrene (ermo Fisher Scientic). Forty-eight hours aer infection, U-87 MG cells were treated with 1.0μg/
mL puromycin (Yeasen) for 3days, constructing shUSP32 and shctrl U-87 MG cells.
Animal experiments. Six-week-old female BALB/c nude mice were obtained from Vitalriver (Beijing,
China). e subcutaneous injection of shctrl or shUSP32 cells (4 × 106) was performed into the right ank of six
mice. e long diameter (a) and short diameter (b) of tumors were measured every 4days. e tumor volume
(V) was calculated using the formula V = ab2/226. e mice were euthanized using isourane (RWD life science,
Shenzhen, China) at day 48. Tumors were dissected o and photographed using HUAWEI Mate 40. e tumor
was also weighed.
RNA sequencing (RNA‑Seq) and data analysis. Total RNA from shctrl or shUSP32 U-87 MG cells was
used as input material for the RNA sample preparations. mRNA was puried from total RNA using poly-T oligo-
attached magnetic beads. e RNA-Seq library was built by Novogene (Beijing, China). Aer cluster generation
using TruSeq PE Cluster Kit v3-cBot-HS (Illumia, San Diego, CA, USA) on a cBot Cluster Generation System
(Illumia), the library preparations were sequenced on a Novaseq platform (Illumina). RNA-Seq data analysis
was performed according to the protocol of Novogene. In brief, reads were aligned to the human transcriptome
and genome hg19 using T Hisat2 v2.0.5. Transcripts and genes were quantied using featureCounts v1.5.0-
p3. Dierential expression analysis was performed using DESeq2 R package v1.20.0. Genes with an adjusted
p value < 0.05 were considered as dierentially expressed. GO and KEGG pathway enrichment analyses27–29 of
dierentially expressed genes were performed using the clusterProler R package.
Figure1. High content screening based on cell viability to screen USPs may function in glioblastoma. Data are
represented as mean ± standard deviation (SD) of three biological replicates.
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Statistical analysis. All statistical analyses were performed using GraphPad Prism v8.2.1 (GraphPad So-
ware, San Diego, CA, USA). e data are presented as mean ± standard deviation (SD) unless otherwise shown.
ANOVA followed by Tukey’s post-hoc test was used for multiple comparisons among three groups. Unpaired
Student’s t test was performed to compare the dierence between two groups. Survival curves were calculated
using the Kaplan–Meier method, and the signicance was determined by the log-rank test. Statistical signi-
cance was accepted at p < 0.05.
Ethics approval. Animal experiments were conducted in accordance with the national guidelines for the
humane treatment of animals and were approved by the Institutional Animal Care and Use Committee (IACUC)
at Xiamen University. e study is reported in accordance with ARRIVE guidelines.
Results
High content screening. High content screening was performed to identify the USPs regulating GBM cell
survival. As shown in Fig.1, knockdown of USP1, USP8, or USP32 in U-87 MG cells inhibited cell viability by
at least half. Knockdown of USP32, USP9X, or USP1 suppressed the viability of U-251 MG. erefore, USP32
was chosen for further study.
USP32 expression level linked to poor prognosis. First, we investigated the clinical signicance of
USP32 using GEO dataset. As can be seen in Fig.2A, the USP32 expression level was higher in GBM tissues
compared to normal tissues. Moreover, higher USP32 expression level indicated poorer prognosis (Fig.2B).
Next, we evaluated the USP32 expression level in normal brain cells and GBM cells. e results showed that the
USP32 mRNA and protein levels in GBM cells (U-118 MG, U-87 MG, A172, T98G, and U-251 MG) were higher
than those in the normal brain cell SVG p12 (Fig.2C,D). Furthermore, U-87 MG and U-251 MG have the high-
est USP32 expression levels. Based on these ndings, we chose to knock down USP32 in U-87 MG and U-251
MG cells to study the function of USP32 in GBM.
Figure2. USP32 expression level in glioblastoma (GBM) tissues and cell lines and the association of USP32
expression level with prognosis. (A,B) USP32 is upregulated in GBM patients (A), which indicates poor
prognosis (B). e USP32 mRNA expression data and survival information of GBM patients were downloaded
from the Gene Expression Omnibus databases. (C,D) e USP32 mRNA and protein levels in GBM cells (U-118
MG, U-87 MG, A172, T198G, and U-251 MG) and normal brain cell SVG p12 were determined using RT-qPCR
(C) and western blotting (D), respectively. Data are represented as mean ± SD of three technical replicates.
Unpaired Student’s t test for (A). Kaplan–Meier method and the signicance was determined using the log-rank
test for (B). One way ANOVA followed by Tukey’s post-hoc test for (C,D): vs SVG p12, *p < 0.05, ***p < 0.001,
****p < 0.0001.
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Knockdown of USP32 inhibits cell growth. USP32 in U-87 MG and U-251 MG cells was knocked down
using siRNAs. As shown in Fig.3A,B, the mRNA and protein levels of USP32 were signicantly reduced aer
transfection with siUSP32-2261 and siUSP32-386. Moreover, siUSP32-386 had higher knockdown eciency
Figure3. Knockdown of USP32 inhibits cell growth. (A) e mRNA level of USP32 was reduced by siUSP32-
2261 and siUSP32-386. Data are represented as mean ± SD of three biological replicates. (B) Le panel:
Representative images of three independent western bolting analyses showing the knockdown eciency of
siUSP32-2261 and siUSP32-386. Right panel: statistical quantication of le panel. (C) MTT assay determining
the cell viability of U-87 MG and U-251 MG. Data are represented as mean ± SD of six biological replicates. (D)
Representative images of three independent colony formation assays showing that USP32 knockdown inhibited
cell proliferation. (E) Statistical quantication of (D). (F) USP32 knockdown reduced the number of EdU+ cells.
Bar: 10μm. (G) Statistical quantication of (F). Data are represented as mean ± SD of three technical replicates.
(H) Representative images of three independent cell cycle assays by ow cytometry. (I) Histogram showing the
percentage of each cell-cycle phase in (H). One way ANOVA followed by Tukey’s post-hoc test: vs siRNA NC,
*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
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than siUSP32-2261. Similarly, the CCK-8 assay indicated that USP32 knockdown signicantly suppressed the
viability of U-87 MG and U-251 MG cells (Fig.3C). Cellular proliferation was analyzed using colony formation
and EdU assays in U-87 MG and U-251 MG cells; Fig.3D,E show that USP32 knockdown signicantly reduced
the number of colonies. In U-87 MG cells, the number of colonies decreased from 125 ± 11 to 80 ± 14 (p = 0.0058)
for siUSP32-2261 and 46 ± 5 (p = 0.0003) for siUSP32-386. In U-251 MG cells, the number of colonies decreased
from 48 ± 8 to 24 ± 3 (p = 0.0051) for siUSP32-2261 and 8 ± 3 (p = 0.0003) for siUSP32-386. e percentage of
EdU+ cells was also signicantly reduced by silencing USP32 (Fig.3F). In U-87 MG cells, the percentage of
EdU+ cells decreased from 25.5 ± 2.5% to 16.9 ± 2.0% (p = 0.0045) for siUSP32-2261 and 8.6 ± 1.1% (p = 0.0001)
for siUSP32-386. In U-251 MG cells, the percentage of EdU+ cells decreased from 39.3 ± 1.4% to 28.3 ± 2.3%
(p = 0.0016) for siUSP32-2261 and 24.2 ± 2.3% (p = 0.0003) for siUSP32-386 (Fig.3G). Next, we investigated the
eect of USP32 on cell cycle progression. e results show that USP32 knockdown promotes the arrest of cells
in the G0/G1 phase (Fig.3H,I). ese ndings suggested that silencing USP32 may inhibit cell growth due to
cell-cycle arrest.
Knockdown of USP32 inhibits cell metastasis. Transwell migration and Matrigel invasion assays were
performed to study the eect of USP32 on cell metastasis. Figure4A shows that USP32 downregulation inhib-
ited cell migration. In U-87 MG cells, the number of migrated cells was 330 ± 26, 186 ± 17, and 123 ± 13 for
siRNA NC, siUSP32-2261 (p = 0.0003, vs siRNA NC), and siUSP-386 (p < 0.0001, vs siRNA NC), respectively. In
U-251 MG cells, the number of migrated cells was 431 ± 20, 305 ± 21, and 246 ± 13 for siRNA NC, siUSP32-2261
(p = 0.0004, vs siRNA NC), and siUSP-386 (p < 0.0001, vs siRNA NC), respectively (Fig.4B). is downregula-
tion also inhibited cell invasion (Fig.4C). In U-87 MG cells, the number of invasive cells decreased from 136 ± 22
to 53 ± 10 (p = 0.0010) for siUSP32-2261 and 24 ± 3 (p = 0.0002) for siUSP32-386. In U-251 MG cells, the number
of invasive cells decreased from 136 ± 22 to 53 ± 10 (p = 0.0010) for siUSP32-2261 and 24 ± 3 (p = 0.0002) for
siUSP32-386. In U-251 MG cells, the number of invasive cells were reduced from 156 ± 19 to 77 ± 10 (p = 0.0008)
for siUSP32-2261 and 34 ± 6 (p < 0.0001) for siUSP32-386 (Fig.4D). Taken together, these data indicate that
USP32 knockdown inhibited cell metastasis.
Figure4. Knockdown of USP32 inhibits cell metastasis. (A) Transwell migration assay showing that silencing
USP32 suppressed cell migration. Bar: 10μm. Images were representatives of three independent experiments.
(B) Statistical quantication of (A). (C) Matrigel invasion assay showing that USP32 deciency suppressed
cell invasion. Bar: 10μm. Images were representatives of three independent experiments. (D) Statistical
quantication of (C). One way ANOVA followed by Tukey’s post-hoc test: vs siRNA NC, **p < 0.01, ***p < 0.001,
****p < 0.0001.
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Knockdown of USP32 inhibits tumor growth in vivo. Next, stably USP32-knockdown U-87 MG cells
were constructed to validate the function of USP32 invivo. Figure5A–C show that the stable cell lines were suc-
cessfully constructed. e constructed cells were then subcutaneously injected into nude mice. As can be seen
in Fig.5D, the tumor in group shUSP32 grew more slowly compared to group shctrl. e dissected tumors are
shown in Fig.5E, being the weight lighter when group shUSP32 was compared to shctrl (Fig.5F). Moreover, the
mRNA and protein levels of USP32 were indeed lower in group shUSP32 than those in group shctrl (Fig.5G–I).
ese data demonstrate that USP32 knockdown inhibits tumor growth invivo.
Analysis of dierentially expressive genes (DEGs). Transcriptional sequencing was performed to
nd the dierentially expressive genes between shctrl and shUSP32 U87-MG cells. e volcano plot indicates
that 2017 genes were signicantly upregulated and 2333 genes were signicantly downregulated aer USP32
knockdown (Fig.6A). e heat map was used to show the distinguishable mRNA expression patterns between
shUSP32 and shctrl samples (Fig.6B).
GO and KEGG pathway enrichment analyses. All upregulated and downregulated genes were used for
GO and KEGG enrichment analyses. In the GO enrichment analysis, 10 molecular functions such as cadherin
binding and catalytic activity acting on DNA, 11 cellular components such as condensed chromosome and cell
cycle checkpoint, and 9 biological processes such as mitotic cell cycle phase initiation and DNA replication
were signicantly modulated (Fig.6C). KEGG pathway enrichment analysis discovers 20 pathways signicantly
linked to USP32 expression, including cell cycle and DNA replication pathways (Fig.6D). e enriched DEGs in
GO and KEGG pathway enrichment analyses are listed in Supplementary TableS1 and Supplementary TableS2,
respectively. e expression prole of several DEGs associated with cell cycle, DNA replication, base excision
repair, and mismatch repair are shown in Fig.7A. RT-qPCR analysis conrmed that USP32 knockdown reduced
the expression of these genes (Fig.7B).
Figure5. Knockdown of USP32 suppresses tumor growth invivo. (A,C) Stably USP32-knockdown U-87
MG cell lines were successfully constructed, determined using RT-qPCR (A) and western blotting (B,C). Data
are represented as mean ± SD of three technical replicates. (D) Tumor volume was tracked every 4days by
calculating using the formula: volume = long diameter × short diameter2/2. Data are represented as mean ± SD
of six mice. (E) e image of tumors. e mice were euthanized using isourane at day 48 and tumors were
dissected o. (F) USP32 knockdown reduced the weight of tumors. Data are represented as mean ± SD of six
mice. (G) RT-qPCR analysis degerming the mRNA level of USP32 in tumor tissues. Data are represented as
mean ± SD of six mice. (H) Western blotting. (I) Statistical quantication of (H). Unpaired Student’s t test: vs
shctrl, *p < 0.05, ***p < 0.001, ****p < 0.0001.
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Discussion
GBM accounts for 16% of primary brain tumors with an incidence rate of thirty-two per million population1,2.
GBM oen causes poor prognosis and the median-survival time of patients is less than 2 years30,31. It is urgent
to nd novel targets for brain-penetrating targeted therapies. Recently, more and more USPs were reported to
play important roles in GBM progression7,21. USP1, USP8, USP9x, and USP28 were identied as oncogenes in
GBM32–35. USPs with antitumor activity such as USP11 and USP286 promote cell viability aer being silenced36,37,
which is consistent with the results in Fig.1. In this study, USP32 knockdown inhibited cell growth and metastasis
invitro, and suppressed tumor growth invivo, which suggests that USP32 acts as an oncogene in GBM and may
serve as a potential target for GBM treatment.
EdU+ cells indicate the cells in DNA replication. DNA replication, occurring in the S phase of interphase
during cell cycle, is an important step for cell proliferation and division38. Results showed that the percentages of
EdU+ cells and G0/G1-phase cells were reduced and increased aer USP32 knockdown, respectively. Moreover,
GO and KEGG pathway analyses revealed that this enzyme is involved in DNA replication and cell cycle pro-
cesses or pathways. is suggests that USP32 may promote cells passing through the G0/G1 phase and initiate
the DNA replication, promoting the proliferation of cancer cells, which is consistent with the study of Hu etal.23.
Base excision repair is an essential genome-maintenance pathway by which cells repair damaged DNA bases
that arise at a high level during DNA replication. Failure to remove the damaged DNA bases causes increasing
Figure6. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomic (KEGG) pathway enrichment
analyses using dierentially expressive genes (DEGs) screened out by transcriptional sequencing. (A) Volcano
plot based on the results of RNA sequencing for transcriptomes from shctrl and shUSP32 U87-MG cells. (B)
e heat map showing the distinguishable mRNA expression patterns between the shUSP32 and shctrl samples.
(C) GO enrichment analysis showing the considerable molecular functions, cellular components, and biological
processes. (D) KEGG pathway enrichment analysis showing the considerable pathways.
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levels of mutation and chromosomal instability, nally resulting in carcinogenesis39. DNA mismatch repair is a
rescue system that conserves the DNA sequences by removing the erroneously mismatched, inserted, and deleted
bases during DNA duplication and recombination. Defects in DNA mismatch repair are also associated with
carcinogenesis40. Moreover, DUBs are oen involved in base excision repair and mismatch repair processes17,18.
erefore, USP32 eect on the expression of molecules functioning in these processes was validated using RT-
qPCR, although the adjusted p-values for base excision repair and mismatch repair pathways in KEGG pathway
analysis were greater than 0.05. e results indicate that USP32 regulates the expressions of MCM3, MCM4,
MCM6, MCM7, FEN1, NEIL3, and POLE, suggesting that USP32 plays an important role in base excision repair
and mismatch repair.
ere are few reports about elements of the ubiquitin–proteasome system involved in GBM metastasis. USP18
was reported to promote epithelial-mesenchymal transition in GBM cells by deubiquitinating and stabilizing
Twist141. UBE2T, a ubiquitin-conjugating enzyme, stabilizes GRP78 to promote the metastasis of GBM cells42.
Our study demonstrates that USP32 facilitates the migration and invasion of GBM cells, which supports that
the ubiquitin–proteasome system plays an important role in the GBM metastasis.
Further experiments are needed to conrm the mechanism of action by which USP32 upregulates the expres-
sion of several genes (Fig.7B). Further, the eect of USP32 modulation on cell function in normal glial cells
and a broader panel of GBM cells will be evaluated. In addition, a collection of clinical samples to analyze the
expression of this enzyme in GBM and normal tissue will also be included.
In conclusion, our study demonstrates that USP32 acts as an oncogene in GBM through regulating cell
cycle, DNA replication, base excision repair, and mismatch repair. USP32 could be a potential target for GBM
treatment.
Data availability
e data in this study will be made available from the corresponding author on reasonable request.
Figure7. USP32 is involved in cell cycle, DNA replication, base excision repair, and mismatch repair processes/
pathways. (A) e expression prole of several DEGs. (B) RT-qPCR analysis conrming the regulatory eect of
USP32 on cell cycle, DNA replication, base excision repair, and mismatch repair processes/pathways. Data are
represented as mean ± SD of three technical replicates.
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Received: 26 October 2021; Accepted: 7 March 2022
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Acknowledgements
e study is supported by National Natural Science Foundation of China (No. 82072777).
Author contributions
W.Z.X. conceived and designed the study. C.S.F. collected the data. W.Z.X. and C.S.F. veried and analyzed the
data. C.X., L.Z., M.J.Y., J.W., Z.Z., L.Y.K., J.Z.Y., Z.W.P., and T.G.W. performed the experiments and draed the
manuscript. All authors read and approved the nal manuscript.
Competing interests
e authors declare no competing interests.
Additional information
Supplementary Information e online version contains supplementary material available at https:// doi. org/
10. 1038/ s41598- 022- 09497-y.
Correspondence and requests for materials should be addressed to Z.W.
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