TIMER2.0 (http://timer.cistrome.org) and UALCAN (http://ualcan.path.uab.edu/analysis-prot.html) databases were used to analyze the expression of WSB2 in normal and pan-cancer tissues. TIMER2.0 collected 10,897 samples of 32 types of cancer from TCGA (https://portal.gdc.cancer.gov), and the statistical significance of differential expression was evaluated using the Wilcoxon test (15). The data acquisition and analysis methods of UALCAN were as follows, TCGA-Assembler was used to download TCGA 3-level RNA-seq data associated with 33 types of cancer, Student's t-test was used to evaluate whether differences in expression levels between normal tumors and primary tumors were significant (16). P-values were downloaded from the two databases, types of cancer that expressed WSB2 in both two databases were identified and the expression P-values of 23 types of cancer were obtained. Finally, an expression heat map was drawn using Microsoft Excel (Microsoft). P<0.05 was considered to indicate a statistically significant difference.

The Human Protein Atlas (HPA; http://www.proteinatlas.org/) provides a comprehensive map of the expression and distribution of nearly all human proteins (~26,000) across human tissues and organs (17). Entering ‘WSB2’ into the database and clicking on the ‘CANCER’ module provides access to immunohistochemical images of cancerous tissues. Alternatively, clicking on the ‘Tissue’ module allows for the retrieval of immunohistochemical images of normal tissues. In this context, the antibody selected was HPA077139.

The Kaplan-Meier Plotter (18) was used to analyze the relationship between WSB2 expression levels and the OS rate and relapse-free survival (RFS) in patients with various types of cancer. After entering the website, mRNA was selected as the data type, ‘Start KM Plotter for pan-cancer module’ was clicked, the target gene ‘WSB2’ was input to generate the survival analysis visualization. This platform automatically stratifies patient samples into two cohorts based on predefined biomarker expression quantiles. Subsequently, it carries out a comparative survival analysis between these cohorts by generating a Kaplan-Meier survival curve. Statistical outputs include the log-rank test P-value, hazard ratio with 95% confidence intervals and corresponding significance estimates.

SKOV3 (ovarian adenocarcinoma) and U2OS (osteosarcoma) cells were cultured in McCoy's 5A medium (Procell Life Science & Technology Co., Ltd.). A2780 cells (ovarian endometroid adenocarcinoma) were cultured in 1640 medium (Gibco, Thermo Fisher Scientific, Inc.). SVCT cells (human breast epithelial cells), MDA-MB-231 cells (triple-negative breast cancer), MCF-7 cells (estrogen receptor positive breast cancer), T47D cells (infiltrating ductal carcinoma of the breast), MCF-10A cells (non-tumorigenic epithelial cells), HeLa cells (cervical cancer), U87 MG cells (glioblastoma of unknown origin) and 293T cells (embryonic kidney cells) were cultured in Dulbecco's modified Eagle's medium (Gibco, Thermo Fisher Scientific, Inc.).

Plasmids were transfected into cells according to the instructions of Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). MDA-MB-231 and MCF-7 cells were transfected with pCMV-HA-WSB2 or pCMV-HA (cat. no. HG-VPC0051, HonorGene) plasmids and assessed using reverse transcription-quantitative PCR (RT-qPCR), Cell Counting Kit 8 (CCK-8), 5-ethynyl-2′-deoxyuridine (EdU), cell proliferation, wound healing, Transwell and colony formation assays. MDA-MB-231, A2780, U87 MG, HeLa and MCF-7 cells were transfected with pCMV-SP1(human)-3×FLAG-Neo (cat. no. P39378; Wuhan Miaoling Biotechnology Co., Ltd.) or control pCMV-3×FLAG-Neo (cat. no. P1303; Wuhan Miaoling Biotechnology Co., Ltd.) plasmids for RT-qPCR.

Total RNA from SVCT, 293T, U87 MG, MCF-10A, MCF-7, A2780, T47D, MDA-MB-231, U2OS, SKOV3, and HeLa cells was extracted using the Trizol (cat. no. 15596018; Invitrogen; Thermo Fisher Scientific, Inc.) reagent, and the RNA was reverse-transcribed into cDNA using a FastKing cDNA first strand synthesis kit (cat. no. KR116; Tiangen Biotech Co., Ltd.). The cDNA was used as the template for the qPCR step of the RT-qPCR protocol, which used a SYBR Green kit (cat. no. MF013; Mei5 Biotechnology Co., Ltd.). The RT-qPCR thermocycling conditions were as follows: Initial denaturation at 95°C for 3 mins, 40 cycles of 95°C for 15 sec, 60°C for 15 sec, and 72°C for 30 sec WSB2, specificity protein 1 (SP1), NUPR1 (encoding nuclear protein 1, transcriptional regulator), LDLRAD4 (encoding low density lipoprotein receptor class A domain containing 4) and MDM2 (encoding double min 2 Protein) mRNA were quantified using the 2^−ΔΔCq method (19), with GAPDH mRNA (encoding glyceraldehyde-3-phosphate dehydrogenase) as the internal reference. The sequences of the primers used are shown in Table I.

Primers (Table II) were used to amplify the coding sequence region of WSB2 and inserted into the EcoRI and KpnI sites of the pCMV-HA vector. The −1624/-787 region of the WSB2 promoter was analyzed according to the TRANSFAC (http://www.gene-regµlation.com) and TESS (http://www.cbil.upenn.edu./cgi-bin/tess/tess) databases. The promoter region of WSB2 was also amplified using the primers shown in Table II, inserted into the KpnI and HindIII sites of the pGL3-basic vector and named as pWSB2-1.6k, pWSB2-0.8k, pWSB2-0.3k respectively.

MDA-MB-231 cells (7×10⁴/well) were cultured in 24-well plates until reaching >85% confluency. A total of 1 µg pWSB2-1.6k, pWSB2-0.8k, pWSB2-0.3k or pGL3-basic plasmids was transfected into the cells using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), followed by incubation at 37°C for 6 h. Then, the cells were treated with 150 or 200 nM plicamycin or DMSO (Sigma-Aldrich; Merck KgaA). At 24 h after treatment, a luciferase assay was conducted as previously described (20).

MDA-MB-231 cells were transfected with 24 µg of pCMV-SP1(human)-3×FLAG-Neo or control pCMV-3×FLAG-Neo per 10-cm dish, followed by incubation at 37°C for 48 h. Then, 1×10⁶ cells were lysed with 200 µl lysis buffer (P0013C, Beyotime), and 50 µl lysate was used per CHIP reaction. ChIP assays were carried out according to the manufacturer's protocol using a Millipore ChIP assay kit (cat. no. 17-295, Millipore Sigma). The following 10 µg primary antibodies were used: Anti-SP1 (cat. no. ab231778; Abcam) and anti-immunoglobulin G (IgG; cat. no. sc-2345; Santa Cruz Biotechnology, Inc.). The amount of each specific DNA fragment in the immunoprecipitants was determined using PCR and RT-qPCR as aforementioned, using the primers CHIP-SP1-F and CHIP-SP1-R, as shown in Table I.

At 24 h after transfection, the cell density was adjusted to 2×10⁴ cells /ml and 100 µl of MDA-MB-231 or MCF-7 cells were inoculated into 96-well plates. The cells were incubated for 24, 48 and 72 h, respectively, before being incubated with 10 µl CCK-8 (cat. no. CA1210; Beijing Solarbio Science & Technology Co., Ltd.) reagent to each well. Following incubation at 37°C for 2 h, the absorbance value of 450 nm was measured using a plate reader.

At 48 h after transfection, the MDA-MB-231 or MCF-7 cells were incubated with 50 µM EdU (cat. no. C10310-1; Guangzhou RiboBio Co., Ltd.) at 37°C for 1 h. The cells were fixed, stained, images were captured and the number of EdU-positive cells was determined as described previously (21).

After transfection, the cells were cultured for 2–3 weeks in DMEM, fixed with 4% paraformaldehyde (cat. no. BL539A; Biosharp Life Sciences) for 30 min at room temperature, stained with 850 µl of 0.5% crystal violet (cat. no. G1063; Beijing Solarbio Science & Technology Co., Ltd.) for 30 mins at room temperature, washed twice with PBS, dried and the cell clone count was determined manually.

Transfected MDA-MB-231 or MCF-7 cells were grown in serum-starved DMEM at 37°C for 6 h until a monolayer formed. The cell monolayer was scratched vertically using a 200 µl pipette tip, cultured in DMEM at 37°C and imaged using an inverted optical microscope at 0, 24 and 48 h, respectively. ImageJ (version 1.54; National Institutes of Health) was used to quantify the average degree of wound healing.

A Transwell chamber was purchased from Corning Inc. (cat. no. 3422) and was placed in a 24-well plate. MDA-MB-231 or MCF-7 cells were resuspended in serum-free medium. After counting, 1×10⁴ cells were taken and inoculated into the upper chamber of the Transwell plate. Subsequently, 750 µl of medium containing 10% serum was added to the lower chamber. The Transwell chambers were incubated in the culture dish for 12–48 h at 37°C and fixed with 4% paraformaldehyde for 30 min at room temperature. The cells were stained using 850 µl 0.5% crystal violet for 15 min at room temperature, washed with PBS and imaged using an inverted microscope. Each sample was imaged in five random fields and the number of cells that passed through the filter membrane were counted.

Standard technique was used to carry out western blotting (22) and the following specific primary antibodies were used: Anti-WSB2 (cat. no. ab127176; Abcam, 1:2,000), anti-GADPH (cat. no. CSB-MA000071Mom; Cusabio Technology, LLC, 1:10,000), anti-β-Actin (cat. no. AC026; ABclonal Biotech Co., Ltd. 1:10,000 dilution), anti-HA-tag (cat. no. B1021; Suzhou Botelon Immunotechnology Co., Ltd. 1:5,000), anti-E-cadherin (cat. no. BD-PT1454; Suzhou Botelon Immunotechnology Co., Ltd.; 1:500 dilution), anti-proliferating cell nuclear antigen (PCNA; cat. no. D220014-0025; Sangon Biotech Co., Ltd. 1:1,000 dilution), anti-Vimentin (cat. no. BD-PB4686; Suzhou Botelon Immunotechnology Co., Ltd. 1:500), anti-p53 antibody (p53) (cat. no. CSB-PA07889A0Rb; Cusabio Technology, LLC. 1:1,000 dilution), anti- regulated kinase 1/2 (ERK1/2) (cat. no. PTM-5850; Hangzhou Jingjie Biotechnology Co., Ltd.; 1:1,000), anti-Snail (cat. no. CY3066; Shanghai Aibei Biotechnology Co., Ltd.; 1:2,000). The labeled secondary antibodies were obtained from Sangon Biotech Co., Ltd. (cat. no. D110058-0025, 1:5,000 dilution) and Cusabio Technology, LLC (cat. no. CSB-PA573747, 1:5,000 dilution). Primary antibodies were incubated overnight at 4°C and secondary antibodies were incubated for 2 h at room temperature.

MDA-MB-231 cells were transfected with pCMV-HA-WSB2 and pCMV-HA. At 48 h after transfection, total RNA was extracted and purified as previously described (23). cDNA library construction and RNA-seq were performed by GeneChem (Shanghai, China) as previously described (24). A total of three samples were used for the transcriptome sequencing analysis. Differentially expressed mRNAs were identified, and Gene Ontology (geneontology.org/) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis were conducted as previously described (25,26). The data from the transcriptome sequencing analysis were deposited at the Sequence Read Archive database (ncbi.nlm.nih.gov/sra) and are accessible via the accession no. PRJNA1189658.

Statistical analysis was carried out using GraphPad Prism 8.2.1. (Dotmatics). The results are presented as the mean ± standard deviation from three independent experiments. Unpaired Student's t-test was used to compare the significant differences between the two groups of samples, and one-way ANOVA was employed for comparisons among multiple groups followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

Expression of WSB2 in pan-cancer was primarily analyzed. First, the mRNA expression levels of WSB2 in pan-cancer were evaluated using the UALCAN and TIMER2.0 databases. The results were consistent between UALCAN and TIMER2.0 (Fig. 1A). Online pan-cancer analysis revealed that the expression of WSB2 was increased in breast invasive carcinoma (BRCA), cervical and endocervical cancer types (CESC), cholangiocarcinoma (CHOL), liver HCC (LIHC), lung squamous cell carcinoma (LUSC), prostate adenocarcinoma (PRAD) and uterine corpus endometrial carcinoma (UCEC), but reduced in colon adenocarcinoma (COAD), kidney chromophobe (KICH), rectum adenocarcinoma (READ) and thyroid carcinoma (THCA), compared with that in their corresponding normal tissues according to TIMER2.0 and UALCAN (Fig. 1A). Subsequently, the HPA database was used to evaluate the protein expression of WSB2 in pan-cancer which revealed that WSB2 protein expression was different in tumor and normal tissues (Fig. 1B). WSB2 expression levels in 11 cell lines were examined using RT-qPCR. Analysis revealed that WSB2 was expressed at different levels in these 11 cell lines (Fig. 1C).

Transcription factors are proteins that can bind to the promoter of a gene, thereby regulating its expression (27). Transcription factors also regulate the expression of WSB2. In the present study, three different truncated WSB2 promoter-luciferase expression vectors, pWSB2-1.6k, pWSB2-0.8k and pWSB2-0.3k, were constructed (Fig. 2A). Analysis of the luciferase reporter assays revealed that pWSB2-1.6k had the highest activity (Fig. 2B), indicating that the −1624/-787 region of the WSB2 promoter may be important for WSB2 transcription. The −1624/-787 region was then analyzed according to the TRANSFAC and TESS databases, and several binding sites for the transcription factor SP1 were found in the −990/-798 region (Fig. 2A). Subsequently, analysis revealed that plicamycin, an SP1 specific inhibitor, dose-dependently reduced pWSB2-1.6k luciferase activity (Fig. 2C). In addition, the results of ChIP-qPCR analysis indicated significantly enhanced recruitment of SP1 to the promoter of endogenous WSB2 (Fig. 2D). Overexpression of SP1 upregulated the mRNA levels of WSB2 in MDA-MB-231, MCF-7, A2780 and U87 MG cells, but not in HeLa cells when compared with Flag-vector (Fig. 2E). Combined TIMER and UALCAN analysis revealed a strong association between WSB2 and SP1 expression in multiple types of cancer (R>0.3; Fig. 2F). These results indicate that SP1 carries out a key role in regulating WSB2 expression levels by binding to its promoter region.

UALCAN was used to evaluate the relationship between the WSB2 expression levels and the tumor pathological stage. The expression of WSB2 was positively associated with the pathological stage of BRCA, CESC, CHOL, LIHC, LUSC and UCEC; but was negatively associated with the pathological stages of COAD, KICH, READ and THCA (Fig. S1).

Kaplan-Meier analysis was used to assess the relationship between WSB2 expression levels and survival prognosis in patients with cancer. Analysis revealed that in BRCA, CESC, HNSC, LIHC, LUAD, LUSC, paraganglioma-pheochromocytoma syndromes and sarcoma), increased expression of WSB2 was associated with shorter OS (Fig. 3A). In addition, increased expression of WSB2 was associated with shorter RFS in BRCA and THCA, and decreased WSB2 expression was associated with poor prognosis of patients with TGCT (Fig. 3B)

Bioinformatic analysis revealed that WSB2 is highly expressed in BRCA (Fig. 1A) and is associated with its pathological stage (Fig. S1) and OS (Fig. 3A). Our previous research demonstrated that microRNA miR-28-5p inhibits the migration of breast cancer cells by regulating WSB2 expression levels (9). In addition, compared with that in the normal breast cell lines SVCT and MCF-10A, WSB2 is highly expressed in MCF-7 and MDA-MB-231 breast cancer cells (Fig. 1C). To investigate the biological function of WSB2 in breast cancer, MDA-MB-231 and MCF-7 cells were transiently transfected with pCMV-HA-WSB2. RT-qPCR and western blotting were used to determine the transfection efficiency (Fig. 4A and B). Overexpression of WSB2 in MDA-MB-231 and MCF-7 increased cell viability compared with that of the control group (Fig. 4C). Furthermore, analysis of the EdU proliferation assay revealed that overexpression of WSB2 increased the number of cells in the S phase (Fig. 4D and E). Next, the results of colony formation experiments revealed that overexpression of WSB2 increased the number of colonies (Fig. 4F and I). Wound healing and Transwell assays revealed that overexpression of WSB2 promoted the migration of MCF-7 and MDA-MB-231 cells (Fig. 4G-H and Fig. 4J). Western blotting was used to validate the possible proteins involved in WSB2-regulated cell proliferation and metastasis. The results revealed that WSB2 increased the expression levels of vimentin, Snail and ERK1/2, and inhibited the expression of p53 and E-cadherin in MDA-MB-231 and MCF-7 cells (Fig. 4K and L). In summary, these results suggest that WSB2 may promote the proliferation, migration and colony formation of breast cancer cells.

Transcriptome sequencing analysis was used to identify the differentially expressed genes (DEGs) between MDA-MB-231 cells overexpressing WSB2 and MDA-MB-231 cells transfected with the negative control. Analysis revealed that compared with the control group, 118 DEGs were identified, including 77 up- and 41 downregulated genes (Fig. 5A). The results of KEGG enrichment analysis revealed that the DEGs were mainly enriched in the ‘p53 signaling pathway’ and ‘transcriptional misregulation in cancer’ (Fig. 5B). Furthermore, NUPR1 and MDM2, which were enriched in the aforementioned two signaling pathways, and LDLRAD4, which was randomly selected, were used to validate the results of the transcriptome sequencing analysis using RT-qPCR. Overexpression of WSB2 significantly decreased the expression of LDLRAD4 and MDM2 in MCF-7 and A2780 cells, the expression of NUPR1 and MDM2 in MDA-MB-231 cells, and the expression of NUPR1, MDM2 and LDLRAD4 in U87 MG and HeLa cells Fig. 5C. Further screening and validation are needed to identify the target genes regulated by WSB2.