USP13 functions as a tumor suppressor by blocking the NF-kB-mediated PTEN downregulation in human bladder cancer

For the use of clinical materials for research purposes, prior patients’ written consent and approval were obtained from the China Medical University and The First Affiliated Hospital of China Medical University. A total of 50 patients with bladder urothelial cell carcinoma underwent partial cystectomies or radical cystectomies from 2014 to 2016 at the Department of Urology of the First Affiliated Hospital of China Medical University in China (Table 1). The tissue specimens were harvested and then immediately frozen in liquid nitrogen and stored at − 80 °C. Histologically, the tumors were classified according to the 2004 World Health Organization histologic classification of urinary tract tumors, and were staged using the 2002 American Joint Committee on Cancer system.

The human bladder carcinoma cell lines, 5637 and UM-UC-3 cells were purchased from the cell bank of Chinese Academy of Sciences (Shanghai, China). These cells are maintained in RPMI 1640 (HyClone, Logan, UT, USA) supplemented with 10% FBS (HyClone) and 1% penicillin-streptomycin (HyClone) at 37 °C, and regularly tested to ensure that they are mycoplasma-free.

Total RNA, including micro-RNA from cultured cells and fresh surgical bladder tissues, was extracted using a miRNeasy™ Mini Kit (Qiagen), according to the manufacturer’s instructions. cDNA synthesis and quantitative real-time PCR were performed using a mercury LNA™ Universal RT microRNA PCR kit (Exiqon, Skelstedet, Vedbaek, Denmark). The hsa-miR-130b-3p, has-miR-301b-3p and U6 LNA™ PCR primer sets were purchased from Exiqon. qRT-PCR was performed using SYBR® Premix Ex Taq™ (Tli RNaseH Plus; Takara Biotechnology CO. LTD., Dalian, China) and LightCyclerTM 480 II system (Roche, Basel, Switzerland). β-actin and U6 snRNA were used as endogenous controls for mRNA and miRNA, respectively. The primers used to amplify the target genes are listed in Additional file 5: Table S1. The relative levels of expression were quantified and analyzed using LightCyclerTM 480 software 1.5.1.6.2 (Roche, Basel, Switzerland). The 2-ΔΔCT method was performed to calculate the relative expression, and expression levels of negative controls were used for calibration. Three independent experiments were performed to analyze the relative gene expression.

Luciferase reporter assay was performed using a Dual Luciferase Reporter Assay Kit (Promega) according to the manufacturer’s protocol. The luciferase reporters, psiCHECK2-USP13 wild type and psiCHECK2-USP13 mutant type were synergized by purchased from GenePharma (Shanghai, China). The luciferase reporter construct was co-transfected with agomir of miR-130b-3p and miR-301b-3p into the 5637 cells by Lipofectamine 3000 (Invitrogen) according to the manufacturer’s guidelines. The relative luciferase activity was measured by Synergy HTX multi-mode microplate reader (BioTek).

Antibodies to USP13 (ab99421), NF-kB p65 (ab16502), NF-kB phosph-p65 (ab86299), pan-AKT (ab8805), phosph-AKT (Ser473) (ab8932), phosph-AKT (Thr308) (ab8933), PTEN (ab170941) and GAPDH (ab181602) (all from abcam) were used according to the manufacturers’ protocols. The western blotting analysis was performed as described before. Briefly, equal amounts of protein extracts were separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were blocked with Tris-buffered saline plus Tween-20 (TBS-T; 0.1% Tween-20) with 5% (w/v) non-fat dry milk and were then incubated with primary antibodies in TBS-T at 4 °C overnight. After three washes with TBS-T for 15 min each, the membranes were incubated with the appropriate HRP-labeled secondary antibodies for 1 h at 37 °C. The immunobands were visualized using the ECL reagents (Transgen Biotechnology, Beijing, China) on a MicroChemi Chemiluminescent Imaging System (DNR Bio-Imaging Systems, Mahale HaHamisha, Jerusalem, Israel). The densitometric values for each band were calculated by Image J 1.46r software (Wayne Rasband, National institutes of Health, Bethesda, MA, USA), and the statistical analysis were conducted based on the ratios of target protein/GAPDH.

The Flag-vector, Flag-USP13, pLvx-USP13, pLvx-p65, pLvx-PTEN and relative pLvx-vector were constructed by GenePharma (shanghai, China). shRNAs were constructed using pLKO.1 vector according to Addgene TRC Cloning Protocol. The micro RNA agomir and antagomir were synergized by and purchased from GenePharma (Shanghai, China). Transfections were performed using the Lipofectamine 3000 Reagent (Invitrogen) following the manufacturer’s protocol. Final concentrations for miRNA agomir or plasmids were 50 nM and 0.75 μg/ml. Cells were cultured in a six-well plate with 2 ml culture medium. The concentration for lentivirus transduction was 5 × 10⁶ transducing units of lentivirus. The stable cell lines were constructed using puromycin (200 μgml− 1).

The cell proliferation capacity was evaluated by a Cell Counting Kit-8 (CCK-8) (Dojindo, Tokyo, Japan) and a cell colony formation assay, according to the manufacturer’s protocol. The absorbance value was measured at 450 nm to determine cell viability using a 96-well plate reader (model 680, Bio-Rad, Hertfordshire, UK). For cell colony formation assay, the cells were plated in 6-well plates (300 cells per well), and after 24 h, cells were subject to indicated treatments, and followed by 10- days incubation in complete medium. Colonies were fixed with 10% formaldehyde for 10 min and stained with 1.0% crystal violet for 5 min, and images were captured. The number of colonies, defined as > 50 cells/colony, was counted.

The transwell assay was performed to evaluated cell invasive and migrative capacities using the transwell (Corning) with or without matrigel (BD Biosciences), respectively, according to the manufacturer’s instructions. Cells were re-suspended in RPMI 1640 containing 1% FBS, and 0.2 ml cell suspension (1 × 10⁴/ml) was seeded into the top chamber, whereas 0.6 ml of RPMI 1640 containing 10% FBS was filled in the lower chamber as the chemoattractant. After 24 h of incubation at 37 °C with 5% CO2, the number of cells invading or migrating to the lower chamber was counted in 10 randomly selected visual fields, and the images were captured by a Leica DM3000 microscope (Leica).

miR-130b/301b overexpressing or USP13 knockdown 5637 cells (3 × 104 per well) were seeded into 24-well culture plates and cultured for 24 h. Then, the cells were harvested, washed three times in phosphate buffered saline (PBS), and resuspended in 0.4 ml of ice-cold PBS. The resuspended cells were incubated with propidium iodide (PI) and a fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody specific for Annexin V (BD, San Diego, CA, USA). The results were measured by flow cytometry (Becton Dickinson Biosciences, San Jose, CA), and the data was analyzed by the ModFit LT software package. The experiments were performed independently in triplicate.

Clinical pathological sections of bladder cancer tissue specimens from thirty bladder cancer patients were provided by Department of Pathology at the First hospital of China Medical University. The expression of USP13 and PTEN was detected using an UltraSensitive™ SP (Mouse/Rabbit) IHC kit (Maxin-Bio, Fuzhou, Fujian, China) according to the manufacturer’s instructions. Briefly, sections were firstly dewaxed in xylene and ethanol, and antigen retrieval was performed using a microwave for 10 min at 100 °C. The sections were then incubated with antibodies for 1 h, followed by biotinylated anti-IgG antibody and streptavidin-biotinylated-complex horseradish peroxidase. DAB and hematoxylin were used for nuclear staining. The images were then captured by upright metallurgical microscope (Olympus, Tokyo, Japan) under an original magnification of 200×. Scoring was done by two pathologists who counted the intensity of positive cells in a defined area. To quantify NF-kB p65, USP13 or PTEN staining, staining intensity was classified as: 0 (no staining), 1 (low staining), 2 (medium staining) and 3 (high staining). 0 and 1 were defined as low expression, and 2 and 3 were defined as high expression. USP13 and PTEN expression was classified as USP13-low or USP13-high and PTEN-low or PTEN-high, respectively. Fisher’s exact test was used to correlateUSP13 and PTEN staining.

For immunoprecipitation, cells were harvested in lysis buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% NP40, 1 mM EDTA, 1 mM sodium orthovanadate, and a 1× protease inhibitor cocktail. To immunoprecipitate Flag-tagged proteins, lysates were firstly incubated with anti-Flag M2 beads (Sigma) overnight, beads were then washed three times, and precipitated proteins were eluted with SDS loading buffer.

UM-UC-3 cells were transduced with a lentiviral control vector or a construct harboring human NF-kB p65. Simultaneously, NF-kB p65-expressing cells were transduced with either a lentiviral vector encoding USP13 (NF-kB p65 + USP13) or PTEN (NF-kB p65 + PTEN). Four types of UM-UC-3 cells (vector, NF-kB p65, NF-kB p65 + USP13, NF-kB p65 + PTEN) were used for the orthotopic tumor model and the metastasis model.

BALB/c nude mice (4–6 weeks old, 14–16 g) were purchased from Beijing Vital River Experimental Animal Technology Co. Ltd. and housed in department of laboratory animal science of China Medical University. The study was approved by Medical Laboratory Animal Welfare and Ethics Committee of China Medical University and the methods were carried out in accordance with the approved guidelines. The mice were randomly divided to groups (n = 5 for each group). The cells were separately injected into the flanks of athymic nude mice to establish a xenograft tumor. Tumors were examined twice weekly; the length, width, and thickness were measured with calipers, and tumor volumes were calculated using the equation (Length × Width²)/2. 50 days after injection, the animals were euthanized, and the tumors were excised, weighed, paraffin-embedded and subjected to staining assays.

We previously reported that miR-130b acts as an oncogene in BC, and is directly regulated by NF-kB via binding with the gene promoter. And miR-130b is capable of maintaining activation of NF-kB by decreasing the expression of CYLD, which is a deubiquitylation enzyme and functions as a NF-kB inhibitor [24]. MiR-130b belongs to the miR-130 family, which is composed of four miRNA precursor genes: miR-130a (at chr11), miR-301a (at chr17), miR-130b and miR-301b (at chr22). The stem-loop precursors of miR-130b and miR-301b are coded 327 bp apart at chr22 (Additional file 1: Figure S1a), and they constitute as clustered miRNAs. The mature RNAs generated from miR-130b~301b cluster are hsa-miR-130b-3p and hsa-miR-301b-3p (hereafter referred as miR-130b and miR-301b, respectively), which share a common seed sequence. The miR-130b/301b cluster belongs to a pan-cancer oncogenic miRNA superfamily, and has been identified to target critical tumor suppressor genes [25]. Since we have discovered the tumorigenic role of miR-130b in BC, we further sought to explore the regulatory functions of miR-130b/301b cluster.

Using bioinformatic analysis software (Targetscan [26] and miRDB [27]), we predicted several potential target genes of miR-130 family which display as tumor suppressor genes or crucial mediators in cancer-related pathways. Among them, we focused our attention on USP13 because of its potential role as a tumor suppressor by sustaining PTEN protein stability. Moreover, PTEN loss has been reported in human cancers previously and its loss is associated with constitutive NF-kB activation [28]. Thus, we sought to find if USP13 could be targeted by NF-kB/miR-130b~301b signaling and further facilitate the loss of PTEN. To investigate the regulatory role of USP13 in BC, we detected mRNA expression of USP13 in a cohort of 50 pairs of bladder cancer tissues and matched normal bladder mucosa tissues. The expression of miR-130b/301b was detected respectively as well. Results showed that expression of USP13 was significantly decreased in BC tissues compared with matched non-tumorous tissues (Fig. 1a). In contrast, the presence of miR-130b/301b was notably elevated in BC tissues (Fig. 1b and c). We next analyzed expression of USP13 gene and miR-130b/301b based on the data from The Cancer Genome Atlas (TCGA) database by starBase [29] and UALCAN [30] software. The evidence collected from TCGA-BLCA further supported our findings (Additional file 1: Figure S1b~S1e). In attempt to analyze the correlation between the expression of USP13 and miR-130b/301b in BC tissues, we performed Spearman’s correlation coefficient analysis based on the experimental results from our clinical cohort. Interestingly, we found USP13 expression was inversely correlated with the expression of miR-130b and miR-301b, respectively (Fig. 1d and e).

Next, we respectively overexpressed or knocked down miR-130b/301b in two BC cell lines, 5637 and UM-UC-3. 5637 (Grade I) and UM-UC-3 (Grade III) are two cell lines from primary urinary bladder cancer, and are commonly used as non-muscle invasive or muscle invasive bladder cancer models, respectively [31]. qRT-PCR (Fig. 1f and g) and western blot (Fig. 1h) analysis were performed to measure the alteration of USP13 expression. Results showed that overexpression of miR-130b/301b significantly decreased USP13 expression in transcription and translational levels, and knockdown of miR-130b/301b revealed an opposite effect on USP13 expression. The dual-luciferase reporter assay was then performed to assess the binding of miR-130b/301b with the 3’UTR region of USP13 mRNA. The results showed overexpression of miR-130b/301b notably inhibited the transcription of reporter construct containing wild type 3’UTR sequence of USP13 gene, whereas did not significantly affect the transcription of the control construct bearing the mutant 3’UTR of USP13 gene (Fig. 1i and j). We then employed the miR-130b/301b “mutant” agomir with the mutant seed sequence as a negative control, and respectively co-transfected the test cells with reporter plasmid bearing wild type USP13 3’UTR containing miR-130b/301b agomir or miR-130b/301b mutant agomir (Fig. 1i). Results revealed that the mutant of miR-130b/301b seed sequence blocked the binding of miR-130b/301b with the 3’UTR of USP13 gene, therefore abolished the inhibition of miR-130b/301b for the transcription of reporter gene (Fig. 1k). Taken together, USP13 is a target of miR-130b/301b and its expression decreased in BC tissues. Furthermore, we found decreased expression level of USP13 is associated with an increased risk of progression to BC, suggesting a tumor suppressor role of USP13 in bladder cancer.

The in vitro phenotypic impact of miR-130b or miR-301b was assessed in BC cell lines, 5637 and UM-UC-3. A remarkable increase in cell proliferation was observed by cell counting kit-8 (CCK-8) (Fig. 2a and b) and colony formation assays (Fig. 2c) in miR-130b/301b overexpressed BC cells, whereas knockdown of miR-130b/301b significantly suppressed cell proliferation (Additional file 2: Figure S2a, S2b, S2c and S2d). Moreover, results of transwell assay displayed a significant enhancement in cell invasive and migrative capacities in response to miR-130b/301b overexpression (Fig. 2d and e), in contrast, miR-130b/301b knockdown remarkably reduced the number of invaded and migrated cells (Additional file 2: Figure S2e and S2f). To study the biological function of USP13 in bladder cancer, we overexpressed USP13 by transducing the BC cells with lentivirus vector encoding USP13 (Additional file 2: Figure S2 g). However, colony formation assay and transwell assay revealed no significant changes in cell proliferation, invasion and migration (Additional file 2: Figure S2 h and S2i). In contrast, USP13 knockdown by shRNA dramatically improved proliferation along with invasion and migration of the BC cells (Fig. 2f, g, h, i and j). We also tested the impact of miR-130b/301b or USP13 on cell apoptosis, however no significant difference in cell apoptosis was observed in response to miR-130b/301b overexpression or USP13 knockdown (Additional file 2: Figure S2j and S2 k). These findings suggested that USP13 might exert its anti-tumor function by interacting with tumor suppressors and sustain its function. This is in consistent with previously reported findings that USP13 deubiquitinates and stabilizes PTEN protein thus suppresses tumorigenesis in human cancers. To test this hypothesis, we then re-overexpressed PTEN expression in USP13 knocked down BC cells (Additional file 2: Figure S2 l). Results suggested that cell proliferative, invasive and migrative capacities were rescued in response to PTEN re-introduction (Fig. 2k, l, m, n). This supported our idea that USP13 might play as a tumor suppressor by blocking PTEN downregulation.

To investigate the functional role of USP13 in BC, USP13 expression was knocked down by two independent USP13 shRNAs (Additional file 3: Figure S3a and S3b) in 5637 and UM-UC-3 cells. Next, we analyzed the expression of PTEN, AKT, along with phospho-AKT (Ser473) and phospho-AKT (Thr308) proteins by western blot analysis. Results indicated that USP13 knockdown notably decreased PTEN protein expression and increased phosphor-AKT levels in BC cells (Fig. 3a and b). We then sought to explore whether miR-130b/301b could reduce PTEN expression via targeting USP13. Loss-of-function analysis was performed in 5637 and UM-UC-3 cells stably expressing USP13 and PTEN protein. Results showed that overexpression of miR-130b/301b dramatically decreased protein expression of USP13 and PTEN, while restoration of USP13 completely reversed the effect (Fig. 3c and d). In addition, no alteration of PTEN expression was observed in USP13-overexpression cells (Fig. 3e and f). This helps explain our previous finding that USP13 overexpression exerts no significant influence on BC cells phenotype.

We have demonstrated in our last study that NF-kB enhances transcription of miR-130b by binding to the promoter, and we have identified the Kappa-B binding site within the promoter region of miR-130b precursor by chromatin-immunoprecipitation (Chip) assay (Additional file 3: Figure S3c). The genomic structure of miR-130b and miR-301b suggested that the cluster could be driven by the same upstream promoter. Hence, we predicted that NF-kB drove the transcription of miR-130b/301b cluster and enhanced the presence/expression of both miR-130b-3p and miR-301b-3p. To assess the possibility, we endogenously activated NF-kB signaling by stimulating the test cells with tumor necrosis factor (TNF)-alpha. Transcriptional levels of NF-kB downstream targets, namely, NFKB inhibitor alpha (NFKBIA), TNF alpha induced protein 3 (TNFALP3), X-linked inhibitor of apoptosis (XIAP), C-C motif chemokine ligand 5 (CCL5), C-X-C motif chemokine ligand 8 (CXCL8), were detected to confirm the activation of NF-kB signaling (Additional file 3: Figure S3d and S3e). In consistent with our hypothesis, expression of miR-130b/301b is significantly increased following TNF-alpha stimulation (Additional file 3: Figure S3f and S3 g). Furthermore, we overexpressed NF-kB by transducing the cells with lentivirus vector encoding NF-kB subunit p65 (pLVX-p65) (Additional file 3: Figure S3 h). As our prediction, miR-130b/301b was upregulated in response to p65 overexpression (Additional file 3: Figure S3i and S3j). Thus, miR-130b/301b cluster is a transcriptional target of NF-kB.

Given the function of miR-130b/301b-USP13 in regulating PTEN expression and the established role of miR-130b/301b as a downstream target of NF-kB signaling, we then asked whether NF-kB regulate PTEN protein expression through miR-130b/301b-USP13 axis. Loss-of-function analysis was performed to assess our hypothesis. We transfected the cells with NF-kB subunit p65 and measured NF-kB activation by detecting the nucleus p65 protein expression level. Results indicated that NF-kB p65 overexpression dramatically decreased both USP13 and PTEN expression, while depletion of miR-130b/301b partially rescued expression of USP13 and PTEN. In addition, restoration of USP13 completely reversed the effect of NF-kB p65 overexpression on reducing USP13 and PTEN expression (Fig. 3g and h). In addition, co-immunoprecipitation (IP) was then performed to verify the direct interaction between USP13 and PTEN proteins. We ectopically expressed flag-tagged USP13 in 5637 cells (Additional file 3: Figure S3 k) and detected PTEN protein in the anti-flag antibody pull-down protein products in western blot assay. Of note, TNF-alpha stimulation in flag-tagged USP13 expressed 5637 cells with reduced the PTEN protein level was detected in the pull-down proteins (Fig. 3i, j and Additional file 4: Figure S4). These results supported a role for USP13 in the NF-kB-driven PTEN downregulation and tumorigenesis of BC. More importantly, NF-kB induced PTEN loss is most likely to be controlled by USP13 protein to a large extent.

Having found that USP13 is capable of blocking NF-kB induced PTEN downregulation, we further attempted to verify whether USP13 functions as a tumor suppressor in NF-kB-driven tumorigenesis of BC. The in vitro phenotypic impact of NF-kB p65 overexpression was assessed in BC cell lines. CCK-8 (Fig. 4a and b) and colony formation assay (Fig. 4c, d and e) revealed the overexpression of NF-kB p65 dramatically increased cell proliferation and survival, while depletion of miR-130b/301b or restoration of USP13 partially rescued the enhancement of cell viability induced by NF-kB p65 overexpression. Transwell assay was then performed to assess cell invasive and migrative capacities. Results suggested that overexpression of NF-kB p65 greatly increased the number of migrated/invaded cells, however knockdown of miR-130b/301b or restoration of USP13 rescued the increased cell invasion and migration caused by NF-kB activation (Fig. 4f, g and h). These data supported our notion that USP13 suppresses tumorigenesis by blocking the NF-kB-driven PTEN downregulation in BC. Nonetheless, USP13 was not able to fully rescue the oncogenic function of NF-kB on BC cells, suggesting the possible involvement of other pathways in the process.

To evaluate the biological function of USP13 in vivo, UM-UC-3 cells were subcutaneously injected into nude mice. There were four groups based on the pretreatment of the cells: group 1 (vector) was injected with UM-UC-3 cells transduced with control lentivirus vector; group 2 (NF-kB p65) was injected with UM-UC-3 cells transduced with lentivirus construct harboring NF-kB p65; group 3 (NF-kB p65 + USP13) and group 4 (NF-kB p65 + PTEN) were respectively injected with UM-UC-3 cells transduced with lentivirus vector containing NF-kB p65 and either USP13 or PTEN. We found that tumor lumps in the NF-kB p65 overexpressing group were significantly larger and heavier than that in the control group. Furthermore, restoration of either USP13 or PTEN could partially reduce the tumor growth induced by NF-kB. On the 50th day, the mice were sacrificed and tumor lumps size and weight in each group were measured (Fig. 5a, b and c). The tumors were then subjected to staining assay (Fig. 5d). Expression of NF-kB p65, USP13 or PTEN proteins was measured by IHC staining assay and was quantified by classifying the staining intensity as 0 (no staining), 1 (low staining), 2 (medium staining) and 3 (high staining). Results suggested that the overexpression of NF-kB p65 significantly decreased the expression levels of USP13 and PTEN, and re-introduction of USP13 rescued PTEN expression (Fig. 5e, f and g).

To further examine the effect of USP13 on metastasis in vivo, the same four groups of engineered cells were injected into the tested mice via the tail vein. After 50 days of injection, the mice were sacrificed, and the lungs were removed and examined. We observed that the metastatic lesions at the surface of the lungs were more plentiful in the NF-kB overexpressing group than in the control group. Lung metastasis could also be observed in USP13 or PTEN restoration group, but the number was significantly decreased comparing with that of the NF-kB overexpressing group (Fig. 5h). The lungs were then subjected to hematoxylin-eosin staining, and the numbers of metastatic nodules were counted under the microscope. Consistent with the morphological characteristics, there were more metastatic lesions in the lungs of mice injected with NF-kB overexpression cells compared with the control group, and USP13 or PTEN re-introduction decreased the number of lung metastatic nodules comparing with that of the NF-kB overexpressing group (Fig. 5i).

To further demonstrate the correlation between expression of USP13 and PTEN, we performed IHC staining in 30 human BC specimens (Fig. 6a). Based on staining intensity, we defined USP13 samples as either USP13-low or -high. Likewise, PTEN staining was defined as either PTEN-low or -high. Analysis of USP13 and PTEN expression (Fig. 6b) suggested that USP13-high was positively correlated with PTEN-high (p < 0.05, Fisher’s exact test). The above data again supported the potential association of USP13 and PTEN in human bladder tumors.