The e ﬀ ect of ubiquitin-speci ﬁ c peptidase 21 on proliferation, migration, and invasion in DU145 cells

Objectives: Although ubiquitin-speci ﬁ c peptidase 21 (USP21) has been shown to promote the development of various cancers, its role in prostate cancer has been poorly reported. Therefore, this study attempted to investigate the involvement of USP21 in prostate adenocarcinoma (PRAD) progression. Methods: Information from public databases was used to evaluate the pattern of USP21 expression in PRAD tissues and its e ﬀ ect on patient prognosis. Subsequently, we either upregulated or knocked down USP21 expression in the human PRAD cell line DU145 to assess cell growth, migration, invasiveness, and apoptosis. Results: The transcript levels of USP21 in PRAD tissues were low, indicating a poor prognosis. In DU145 cells, USP21 silencing impaired cell proliferation, colony formation, cell cycle progression, migratory capacity, and invasiveness, while it increased rates of apoptosis. Furthermore, cell proliferation, migration, and invasion were all induced by upregulating USP21. In addition, gene enrichment analysis revealed that USP21 had the potential to regulate cell adhesion and the cell cycle. This observation was further validated by the detection of expression of related genes in cells with either knockdown or increased USP21 expression levels. The expression and copy numbers of USP21 were signi ﬁ cantly correlated with the in ﬁ ltration levels of immune cells. Conclusions: Expression level of USP21 is associated with PRAD progression and poor prognosis, and may have a role in potential therapeutic strategies for patients with PRAD.


Introduction
Prostate tumors are some of the most frequently diagnosed tumors. Globally, prostate cancer is the second-leading cause of cancer-related death in males [1,2]. Epidemiological research has linked the etiology of prostate cancer to multiple factors, such as age, ethnicity, genetics, geography, diet, and inflammation [1]. A thorough understanding of the molecular pathways underlying prostate cancer progression is essential in order to increase the success rates of available treatments.
Deubiquitinases (DUBs) can prevent the ubiquitination of target molecules [3]. With over 50 members, the ubiquitin-specific peptidase (USP) family is the largest and most abundant group of DUBs [4]. Studies have shown that some DUBs play an oncogenic role and some play a tumorsuppressive role. For example, USP7 promotes cell migration and invasion by stabilizing the enhancer of zeste homolog 2 (EZH2) [5]. In contrast, USP28 overexpression reduced breast cancer cell metastasis [6]. Studies have also shown that knockdown of USP17 can prevent the spread of breast and lung cancers [7,8]. Despite recent improvements in the characterization of this family of DUBs, the majority of the individual substrates remain poorly understood in terms of their cellular roles and regulation. Therefore, much effort is still needed to unravel their activities and clinical consequences. USP21 is a nuclear/cytoplasmic shuttle protein that deubiquitinates RIPK1, FOXM1, STING, and histone H2A, and has a catalytic DUB domain at its C-terminus [9]. Studies have shown that USP21 is located on chromosome 1, 1q21, a region that contains various oncogenes, including MDM2 and creb3l4 [10]. Elevated copy numbers of these genes have been attributed to reduced survival in several types of human cancers [9][10][11]. Although recent reports suggest that USP21 promotes the development of a variety of cancers [9,12], its role in prostate cancer remains unclear.
In this study, we knocked down or overexpressed USP21 in order to explore its effects on the malignant behavior of DU145 cells. We subsequently conducted a bioinformatics analysis to investigate the expression pattern of USP21 in prostate adenocarcinoma (PRAD) tissue, its impact on the prognosis of patients with PRAD, and its effect on immune infiltration in tumors. Gene enrichment analysis was used to preliminarily explore and verify the pathways that USP21 and its related molecules may use to regulate DU145 cells. This study aimed to identify new targets for prostate cancer detection and treatment by exploring the role of USP21 in PRAD cells.

Kaplan-Meier survival curve analysis
Patients were sorted into TCGA-PRAD groups with USP21 highexpression or USP21 low-expression. The surv CUTpoint function from the survMiner package was used to obtain the best segmentation values. Kaplan-Meier survival curves for overall survival (OS), progressionfree interval (PFI), disease-free interval (DFI), and disease-specific survival (DSS) were plotted using the R survminer package (version 0.4.8).

Gene set enrichment analysis
The patients from the TCGA-PRAD group were divided into high-and low-expression groups according to the median values of USP21. Gene set enrichment analysis (GSEA) software (version 4.2.1) was used to study cancer-related pathways associated with USP21 levels in PRAD cells. Pearson's correlation analysis was used to rank genes. The six genes with the highest correlation were selected for each pathway, and R (version 4.0.5) and ggplot2 (version 3.3.5) software were used to compare the mRNA levels of these genes under high-or low-USP21 expression levels.

Immune infiltration analysis
Tumor Immune Estimation Resource (TIMER) (https://cistrome. shinyapps.io/timer/) was used to analyze the effect of USP21 expression level on the immune infiltration of prostate cancer cells and the effect of USP21 copy level on the infiltration of other immune cells. The relationship between USP21 expression and methylation or copy number was analyzed using Gene Set Cancer Analysis (http://bioinfo.life. hust.edu.cn/GSCA/#/).

Cell culture
The human PRAD DU145 cell line was purchased from IMMOCELL (Xiamen, China) and was maintained in Minimum Essential Medium (Thermo Scientific, Logan, UT) containing 10 % (v/v) fetal bovine serum, 100 g/mL streptomycin, and 100 U/mL penicillin in an incubator at 37°C with 5 % CO 2 .

Construction of plasmids
Plasmids expressing short hairpin (sh) USP21 (shUSP21-1 and shUSP21-2, Gene ID:27,005) or a scrambled control (shNC), and USP21 expression plasmid (USP21 OE), were created using the pLV-sh-puro and pcDNA3.3 N-3HA vectors, which were purchased from XIAMEN Anti-HeLa Biological Technology Trade Co. Ltd (Xiamen, China). Appropriate primer sequences were developed according to the specifications of the vectors and are detailed in Table 1.

Quantitative PCR
Using an RNA isolation kit (Omega) and a reverse transcription kit (Vazyme), RNA was extracted from the cells and reverse transcribed in accordance with the manufacturer's recommendations. The Real-Time PCR System (Bio-Rad Laboratories, Inc.) and SYBR qPCR kit (Vazyme) were used to determine the mRNA expression levels of the target genes (Vazyme). The quantitative PCR (qPCR) primers are listed in Table 2.

Cell proliferation
Ten thousand DU145 cells per well were planted in 96-well plates, and were transfected with 0.2 μg of shUSP21, shNC, vector, or USP21 OE plasmid.
At 24, 48, and 72 h after transfection, cells were treated with methylthiazolyldiphenyl-tetrazolium bromide (MTT) (Beyotime Biotechnology, Shanghai, China) for 4 h. Cells were agitated for 10 min with 100 µL of dimethylsulfoxide (DMSO) (added after the supernatant had been discarded), in order to dissolve the crystals. An absorbance reader (SpectraMax, San Francisco, CA, USA) was used to measure cell viability at 490 nm.
For EdU assay, 48 h after transfection, MEM with 50 µM EdU from Guangzhou RiboBio Co., Ltd. was incubated with the cells for 4 h at 37°C.
Next, the cells were fixed with 4 % formaldehyde for 20 min and incubated with 2 mg/mL glycine for 5 min at the same temperature. The cells were treated with EdU (Guangzhou RiboBio Co., Ltd.) for 20 min at 28°C after being exposed to 0.5 % Triton X-100 for 10 min. The nuclear DNA was stained for 10 min at room temperature with DAPI (5 μg/mL). Fluorescence imaging was performed using a microscope (Motic).

Colony formation assay
After the DU145 cells in 6-well plates were transfected with 5 μg of shUSP21, shNC, vector, or USP21 OE plasmid for 24 h, they were uniformly dispersed into 2.0 × 10 3 cells per well. After 2 weeks, the cells underwent a 15-min methanol fixation process. They were then stained with 0.5 % crystal violet (Yeasen, Catalog Number: 60505ES25) in phosphate-buffered saline (PBS) for 15 min. Finally, the colonies were imaged and the numbers of colonies were counted using Image J 1.52v (NIH, Bethesda, MD, USA).

Cell cycle assay
Three hundred thousand cells per well were grown in 6-well plates and were transfected with 5 μg of shUSP21, shNC, vector, or USP21 OE plasmid for 48 h. The cells were then collected and fixed in 70 % ethanol. The fixed cells were washed using PBS before being incubated at 37°C for 30 min in 0.2 % Triton X-100 and 10 μg/mL RNase. After being treated with 7-AAD (Beyotime) for 30 min in the dark, the cells were examined using the NovoCyte setup and NovoExpress ® software 1.4.1.

Apoptosis assay
A total of 1 × 10 6 DU145 cells per well were seeded in 6-well plates and transfected with 5 μg of shUSP21, shNC, vector, or USP21 OE plasmid. After 48 h, the cells were centrifuged at 200 g for 5 min. After washed with PBS, the cells were stained using an Annexin V-FITC/PI kit, in accordance with the manufacturer's instructions. The data were examined using the NovoCyte and NovoExpress ® software 1.4.1.

Statistical analysis
The R Survival package was subjected to a log-rank test. Mann-Whitney U and Wilcoxon tests were performed for non-parametric data. Student's t-test was used for parametric data to identify significant differences between the two groups. In GraphPad Prism 8.2.1, Tukey's test and one-way ANOVA were performed to determine which differences across groups were significant. The threshold for statistical significance was set at p<0.05. Data are expressed as mean ± standard deviation.

Results
USP21 mRNA level is upregulated in PARD USP21 mRNA expression was considerably higher in patients with PRAD and at stages T3 and T4 than in those without and at stages T1 and T2 ( Figure 1A). In addition, there was no significant difference in expression levels of USP21 mRNA in the residual tumors ( Figure 1A). These findings suggest that USP21 contributes to the development of PRAD. Analysis of high-throughput RNA sequencing data from TCGA-PRAD and GEO showed that the expression level of USP21 mRNA was considerably higher in tumor tissues than in non-tumor tissues ( Figure 1B). The OS, PFI, and DFI of patients with PRAD with high USP21 levels were worse than those of patients with low USP21 levels; however, there was no significant difference in DSS ( Figure 1C). Thus, these individuals have poor prognosis when USP21 mRNA expression is upregulated in PRAD tissues. Therefore, USP21 mRNA, which is highly expressed in tissues of patients with PRAD, may be closely linked to the development of PRAD.

Silencing USP21 suppresses cell proliferation
To further explore the function of USP21 in PRAD, we knocked down USP21 in DU145 cells. The results showed that shUSP21-1 and shUSP21-2 successfully reduced the levels of USP21 in DU145 cells (Figure 2A and B). The proliferation of DU145 cells after USP21 knockout was significantly lower than in those without USP21 knockout, which indicated that USP21 downregulation inhibited cell proliferation ( Figure 2C). Additionally, USP21-knockdown cells had a considerably lower EdU + rate than control cells, which suggested that USP21 deletion reduced the proportion of EdU + cells ( Figure 2D). Moreover, USP21 silencing suppressed colony formation ( Figure 2E). Cell cycle assay demonstrated that USP21-knockdown enhanced the proportion of cells in the G 0 /G 1 phase ( Figure 2F).

Decreased USP21 levels of DU145 cells resulted in increase of apoptosis and reduced migration and invasion abilities
After USP21-knockdown, there was a dramatic increase in the proportion of apoptotic cells according to an Annexin V/PI staining experiment performed using flow cytometry ( Figure 3A). These observations indicated that a decrease in USP21 expression upregulates apoptosis. Moreover, the number of migratory and invading DU145 cells was considerably reduced after the downregulation of USP21 ( Figure 3B). This phenomenon suggested that knockdown of USP21 led to a decrease in the migration ability, colonization of new spaces, and invasiveness of DU145 cells.

Overexpressing USP21 enhances proliferation
As shown in Figure 4A and B, the USP21 expression plasmid enhanced the expression of USP21 in cells. Proliferation, colony formation, and cell cycle alterations of the tested cells were subjected to MTT and EdU assays, crystal violet staining, and PI staining, respectively. The formation of cell colonies was accelerated and cell proliferation improved after USP21 ( Figure 4C-E). In addition, the G 0 /G 1 phase of the cell cycle was halted by overexpression of USP21 ( Figure 4F). Therefore, DU145 cell proliferation was more rapid when USP21 was expressed at higher levels.

Increasing USP21 augments migration and invasion ability of DU145 cells
Next, annexin V/PI labeling in conjunction with flow cytometry was employed to identify apoptosis in DU145 cells after USP21 overexpression. There had no discernible effect on the apoptosis of DU145 cells ( Figure 5A) after overexpression of USP2. Laboratory Transwell assays confirmed that USP21 overexpression in DU145 cells increased their migration and invasion abilities ( Figure 5B). Thus, USP21 overexpression enhanced DU145 cell migration and invasiveness.

USP21 in PRAD was of the capacity to control cell cycle and adhesion molecules
GSEA was performed to examine the impact of USP21 mRNA expression on cell adhesion molecules and cell cycle pathways in patients with PRAD. As expected, USP21 expression was significantly correlated with the cell cycle and cell adhesion molecules ( Figure 6A and B). Cell adhesion molecules were mostly negatively correlated with USP21, whereas most cell cycle-related molecules were positively correlated with USP21 ( Figure 6A and B). SELP, CNTN1, ICAM1, and NEGR1 showed considerably higher mRNA expression after USP21 knockdown ( Figure 6C and D). The mRNA levels of these genes decreased following USP21 overexpression ( Figure 6C and D). Furthermore, silencing USP21 led to a decrease in the mRNA expression levels of MCM7, ORC6, PCNA, and ANAPC2, whereas USP21 upregulation promoted an increase in the mRNA levels of these genes ( Figure 6C and D).

Correlations between immune infiltration in PRAD and USP21 expression
Immune cells in the tumor microenvironment have been demonstrated to affect tumor development [13]. The results of the present study indicate that USP21 may promote the development of PRAD. Therefore, additional tests were conducted to determine whether USP21 expression was associated with immune infiltration. The data showed a correlation between USP21 expression and B cell, CD8+ T cell, CD4+ T cell, macrophage, neutrophil, and dendritic cell infiltration ( Figure 7A). Subsequently, we investigated whether there was any connection between invading immune cells and USP21 copy number. Deletion of USP21 significantly increased the infiltration levels of neutrophils ( Figure 7B), which suggested that USP21 may be able to encourage immunological infiltration in patients with  PRAD. Moreover, USP21 copy number positively correlated with USP21 expression, whereas USP21 methylation negatively correlated with USP21 expression ( Figure 7C).

Discussion
Prostate cancer is the most prevalent type of cancer and the main cause of cancer-related mortality in men [3]. The cornerstone of clinical prostate cancer treatment is androgen deprivation therapy, which can be administered via gonadotropin-releasing hormone agonists or bilateral orchiectomy [14]. Nevertheless, castration-resistant prostate cancer almost always advances within 2-3 years of initiating androgen deprivation therapy [15]. In addition, patients undergoing androgen deprivation therapy have higher risks of experiencing cardiovascular events such as myocardial infarction or stroke [16,17]. Therefore, additional therapeutic options and alternative targets are required for improved treatment of prostate cancer. Some DUBs are overexpressed in tumor tissues, and others are emerging as novel anticancer strategy targets or indicators [18]. One 19S proteasome-associated DUBs is USP14, which is implicated in stabilizing androgen receptor proteins and promoting the G 0 /G 1 to S phase transition in human prostate cancer cells, is a viable target for prostate cancer therapy [18]. Another study showed that prostate cancer and cell lines exhibit significantly elevated USP17 expression, which leads to poorer patient OS [3]. The proliferation, migration, and invasion of prostate cancer cells are dramatically reduced when USP17 expression is inhibited, and apoptosis is markedly increased [3]. A different study reported that USP2a, USP7, and USP10 were involved in the occurrence and development of prostate cancer [19]. However, the present study is the first to reveal the role of USP21 in PRAD cells.
Non-small cell lung cancer growth can be accelerated when USP21 boosts proliferative activity, migration, and invasion. In addition, USP21 is highly expressed in bladder and liver cancers in which it exerts an oncogenic role [20,21]. Our results are consistent with those of previous studies, and support that the RNA level of USP21 is also upregulated in patients with prostate cancer. Furthermore, USP21 silencing in DU145 cells in patients with prostate cancer inhibited cell proliferation and malignant behavior, and enhanced apoptosis. However, USP21 overexpression enhanced cell proliferation and malignant behavior.
Several recent studies have shown that USP21 is involved in cell cycle control [12,22,23]. In patients with breast cancer, USP21 stabilizes FoxM1, a major cell cycle regulator that controls mitotic gene dynamics, them to enter and progress through mitosis [24]. Importantly, USP21 knockdown delayed breast cancer cell cycle progression in mouse xenograft tumors and in vitro cell proliferation [24]. Similarly, we found that the downregulation of USP21 expression in DU145 cells blocked the cell cycle transition from the G0/G1 to the S phase, whereas the upregulation of USP21 accelerated cell cycle progression. Moreover, the data from our gene enrichment analysis revealed that the molecules positively correlated with USP21 were mostly enriched in the cell cycle.
MEK2, along with other proteins such as GATA3 and Gli1, is deubiquitinated and stabilized by USP21, which aids tumor growth [19,21,25]. Notably, certain USPs, such as USP7, regulate the cellular turnover of various tumor suppressor proteins, including p53 [19]. Additionally, USP7 controls Hdm2 and Hdmx, which are known p53 antagonistic regulators. EZH2, which aids in metastasis and epithelial-to-mesenchymal transition, is deubiquitinated and stabilized by USP21, which increases cell proliferation and metastasis in patients with bladder cancer [20]. Further, knockdown of USP17 in prostate cancer cells blocked NF-κB signaling, which restrains prostate cancer proliferation [3]. In addition, USP14 overexpression, which promotes prostate cancer cell proliferation, depends in part on the function of ATF2 [1]. By inhibiting the expression of ATF2, subcutaneous tumor cell growth induced by USP14 overexpression in nude mice may also be reduced [1]. Although the present study showed that USP21 has the potential to regulate cell adhesion molecules, its role (and that of its downstream substrates) in prostate cancer requires further investigation. In addition, prostate cancer immune infiltration is associated with USP21 expression levels, but the detailed molecular mechanism still needs to be further explored, which is a limitation of this study.
In conclusion, USP21 mRNA was upregulated in patients with PRAD, indicating a poor prognosis. The downregulation of USP21 hindered the ability of DU145 cells to proliferate, migrate, and invade, and encouraged apoptosis. Overexpression of USP21 improved the proliferative, migratory, and invasive capacities of DU145 cells.
Therefore, the present study suggests that USP21 is crucial for the growth and spread of prostate cancer cells, providing a novel target for prostate cancer therapy. Additionally, USP21 may control the occurrence and progression of prostate cancer by regulating the cell cycle, cell adhesion, and immunological infiltration. Inhibition of USP21 expression in tumor tissues may represent a useful treatment option since patients with consistently high levels of USP21 expression have a poor prognosis. These findings imply that USP21 may be a candidate target for the targeted therapy for PRAD. Competing of interest: All authors declare that they have no competing interests. Informed consent: Not applicable. Ethical approval: Not applicable. Availability of data and materials: The data and materials are available from the corresponding author upon reasonable request.