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RNF19A inhibits bladder cancer progression by regulating ILK ubiquitination and inactivating the AKT/mTOR signalling pathway

Biology Direct volume 19, Article number: 102 (2024) Cite this article

Abstract

Background

The role of the RING finger protein superfamily in carcinogenesis has been widely studied, but one member of this family, RNF19A, has not yet been thoroughly explored in bladder cancer (BCa).

Methods

The expression levels of RNF19A in BCa samples and cell lines were analysed through data mining of public resources and further experiments. BCa cells in which RNF19A was stably overexpressed or knocked down were generated through lentivirus infection. The effects of RNF19A on cell proliferation, migration, and invasion were explored by performing a series of in vitro experiments, including CCK-8, colony formation, wound healing, and Transwell invasion assays. Using bioinformatics methods and multiple experiments, including western blot, qRT‒PCR, immunoprecipitation, cycloheximide, ubiquitination, and rescue assays, the mechanism underlying the effect of RNF19A on the progression of BCa was investigated.

Results

Here, we found that RNF19A expression was reduced in BCa samples and cell lines and that lower RNF19A expression predicted shorter overall survival of BCa patients. Functionally, forced expression of RNF19A suppressed BCa cell proliferation, migration, and invasion by inactivating the AKT/mTOR signalling pathway, whereas silencing RNF19A had the opposite effects. Mechanistically, RNF19A could directly interact with ILK and promote its ubiquitination and degradation. Rescue experiments revealed that forced ILK expression partially rescued the decreased phosphorylation of AKT, mTOR, and S6K1 caused by RNF19A overexpression and that the increased levels of the p-AKT, p-mTOR, and p-S6K1 proteins induced by RNF19A knockdown were eliminated after silencing ILK. Similarly, the effects of RNF19A overexpression or knockdown on the phenotypes of cell proliferation, migration, and invasion could also be restored by forced or decreased ILK expression.

Conclusions

RNF19A suppressed the proliferation, migration, and invasion abilities of BCa cells by regulating ILK ubiquitination and inactivating the AKT/mTOR signalling pathway. RNF19A might be a potential prognostic biomarker and promising therapeutic target for BCa.

Introduction

Bladder cancer (BCa), one of the most prevalent malignancies of the genitourinary system, ranks ninth in incidence and thirteenth in mortality among all cancers [1]. Globally, approximately 573,000 new cases of BCa are estimated to occur annually, and more than 213,000 people die from BCa each year [2, 3], imposing a considerable societal burden on humans. Recent studies have revealed that genomic factors combined with environmental factors such as smoking account for the tumorigenesis of BCa [4]. Histologically, BCa can typically be classified into non-muscle-invasive bladder cancer (NMIBC), muscle-invasive bladder cancer (MIBC), or a metastatic form [5]. Nearly 70% of BCa cases are NMIBC when first diagnosed, and unfortunately, approximately 15% of NMIBC cases eventually develop into high-risk MIBC or advanced-stage disease [6, 7]. Currently, the standard interventions for BCa patients include surgical resection, chemotherapy, and immunotherapy [8]. Although great advancements have been made in these strategies in the last few decades, the prognoses for patients with MIBC and advanced tumours are still extremely poor, with 5-year disease-free survival rates of less than 30% [9]. In addition, the side effects of chemotherapy drugs and postoperative complications impede the efficacy of these therapies and lead to decreased quality of life. To date, the molecular mechanisms related to BCa pathogenesis and progression are largely unknown. Novel insights into the driver genes of BCa aggressiveness could provide better therapeutic modalities.

Ring finger protein 19A (RNF19A), also known as Dorfin, belongs to the RING finger protein superfamily, the members of which were previously reported to possess E3 ligase activity [10]. The RNF19A structure contains three highly conserved domains in its N-terminus, including a RING (IBR) domain between two RING finger motifs, which confer the E3 ligase activity of RNF19A [11, 12]. Although the role of RNF19A is poorly understood, the literature has confirmed the dysregulation or dysfunction of RNF19A in various cancers. In non-small cell lung cancer (NSCLC), Cheng et al. [13] reported that RNF19A was highly expressed in tumour samples and predicted increased carcinogenesis and poor outcomes in patients with NSCLC. Further experiments indicated that RNF19A promoted cell proliferation and inhibited apoptosis in NCSCL by inducing the ubiquitination-mediated degradation of p53. Zhu et al. [14] reported that RNF19A promoted the ubiquitination of BARD1, leading to the inhibition of homologous recombination (HR)-mediated DNA repair and thereby increasing the therapeutic efficiency of poly-(ADP‒ribose) polymerase inhibitors (PARPis) in breast cancer. Despite these findings, the biological role and associated mechanism of RNF19A in bladder cancer remain largely unclear.

Here, we identified a previously unreported tumour suppressor function of RNF19A in BCa. We revealed that RNF19A was expressed at low levels in BCa samples and cell lines and was positively correlated with patients’ clinical outcomes. Gain- and loss-of-function experiments suggested that the overexpression of RNF19A inactivated the AKT/mTOR signalling pathway and subsequently impeded cell proliferation, migration, and invasion in BCa. Mechanistically, RNF19A directly interacted with ILK and promoted its ubiquitination and degradation, thus inactivating the AKT/mTOR signalling pathway. Rescue experiments revealed that the pharmacological inhibition of AKT/mTOR signalling or silencing of ILK could partly attenuate the effect of RNF19A knockdown on promoting cell proliferation, migration, and invasion. Thus, our findings showed that RNF19A inhibited bladder cancer progression by regulating ILK ubiquitination and inactivating the AKT/mTOR signalling pathway, indicating its potential as a therapeutic target for BCa treatment.

Materials and methods

Cell culture

BCa cell lines, including UM-UC-3, T24, and 5637 cells, and the human embryonic kidney epithelial cell line HEK-293T were obtained from the Cell Bank of the Shanghai Chinese Academy of Sciences (Shanghai, China). The immortal ureteral epithelial cell line SV-HUC-1 was purchased from Pricella Biotechnology (Wuhan, China). UM-UC-3, HEK-293T, and SV-HUC-1 cells were maintained in DMEM (Gibco, USA). T24 and 5637 cells were cultured in McCoy’s 5a (Gibco, USA) and RPMI 1640 media (Gibco, USA), respectively. All the complete media were supplemented with 10% foetal bovine serum (FBS; Gibco, USA) and a 1% penicillin/streptomycin solution (Servicebio, Wuhan, China). The cells were maintained at 37 °C with 5% CO2 and appropriate humidity. All the cell lines assayed in the present study were identified by STR profiling.

Sample collection

54 Paired BCa samples and adjacent normal tissues were collected from Renmin Hospital of Wuhan University. The resulting samples were immediately frozen and stored in liquid nitrogen after being washed with cold PBS. None of the patients received any prior radiotherapy or chemotherapy before surgery, and informed consent was obtained from each patient. This study was approved by the Ethics Committee of Renmin Hospital of Wuhan University (Ethics number: WDRY2019-K035).

Lentiviral infection

The lentiviruses used for RNF19A overexpression (LV-RNF19A) or knockdown (shRNF19A) and the corresponding control lentivirus (LV-Ctrl, shNeg) in the present study were all purchased from Sangon Biotech (Shanghai, China). For the generation of stable cell lines, BCa cells were plated in 6-well plates and cultured to reach a confluence of approximately 40–50%. Then, suitable amounts of the lentivirus mixture were added to the plate and incubated for 12 h. The supernatants were subsequently removed, and the cells were further cultured for 36 h in media supplemented with 20% PBS. The stably infected cells were selected with puromycin (3 µg/ml, Sigma), and the knockdown or overexpression efficiency was confirmed through qRT‒PCR and western blot assays.

Plasmid transfection

A Flag-tagged human RNF19A expression plasmid (Flag-RNF19A), an HA-tagged ILK expression plasmid (HA-ILK), and a His-tagged ubiquitin (Ub) expression plasmid (His-Ub) were obtained from GeneCreate Biotech (Wuhan, China). The plasmid transfection experiments were conducted using HighGene reagent (ABclonal, China) according to the instructions provided. Forty-eight hours after transfection, the cells were collected for further experiments.

Cell proliferation assay

Cell proliferation was assessed by performing cell counting kit-8 (CCK-8) and colony formation assays. Briefly, 5 × 103 BCa cells in 100 µl of complete media were seeded into each well of 96-well plates in triplicate. The cells were cultured for 0, 24, 48, or 72 h, and 10 µl of CCK-8 solution (Beyotime Biotechnology, Shanghai, China) was added to each well. After an incubation for 2 h at 37 °C in the dark, the optical density (OD) at 450 nm was determined using a microplate reader (Bio-Rad Laboratories, USA). For the colony formation assay, 5 × 102 BCa cells from each group were seeded into 6-well plates. The cells were cultured for 12 days. The supernatants were discarded every four days, and fresh media were added to the plates. At the end of the experiments, the cells were washed with PBS and fixed with 4% paraformaldehyde (Servicebio, Wuhan, China) for 20 min, after which they were stained with 0.1% crystal violet (Solarbio, China). The clones were observed and photographed using a camera, and the number of clones was counted using ImageJ software.

Wound healing and transwell invasion assays

Cell migration and invasion abilities were evaluated by wound healing and Transwell invasion assays, respectively. Briefly, a total of 5 × 105 BCa cells in 2 ml of complete media were seeded in 6-well plates and grown until they reached confluence. A straight scratch was created in the bottom of the plate using a sterilized pipette tip (10 µl). Then, the supernatants were removed, and the cells were washed with PBS and cultured in serum-free media for 36 h. The wound areas of each group were observed and photographed under an inverted microscope (Olympus, Japan). The wound healing rate was determined using the following formula: wound healing rate = (wound area at 0 h – wound area at 36 h)/wound area at 0 h×%. For the Transwell invasion assay, 1 × 104 BCa cells in 200 µl of serum-free media were added to the upper chamber of Transwell (8-µm pore size, Corning, USA), which were precoated with a thin layer of Matrigel (BD Biosciences). The bottom chamber was filled with 600 µl of media containing 20% FBS. After an incubation for 36 h, the cells in the upper chamber were removed with a cotton swab, and the invading cells at the bottom of the chambers were fixed with 4% paraformaldehyde for 20 min at room temperature, followed by staining with 0.1% crystal violet for 10 min. After two washes with PBS, the cells that crossed the Transwell chamber were observed and photographed using an inverted microscope. The average number of invaded cells in five random fields was determined using ImageJ software. All the experiments were repeated at least in triplicate.

Quantitative real-time PCR (qRT‒PCR)

Briefly, total RNA was isolated from cells and tissue samples using an RNA extraction kit (Vazyme, Nanjing, China) according to the manufacturer’s instructions and reverse transcribed into cDNA using a PrimeScript kit (Takara, Beijing, China). The qRT‒PCR assay was conducted on a LightCycler96 using SYBR qPCR SuperMix Plus (Novo Protein: Shanghai, China). The expression levels of the target genes were determined using the comparative Ct method (ΔΔCT) and normalized to the expression level of the GAPDH gene. The sequences of primers used for qRT‒PCR were as follows: GAPDH, forward primer, 5’-CCAGAACATCATCCCTGCCT-3’; reverse primer, 5’-CCTGCTTCACCACCTTCTTG-3’; RNF19A, forward primer, 5’-CCATCCGAGACAACCTGAGT-3’; reverse primer, 5’-ACTGTTCCCAAGCTGACTGT-3’; and ILK, forward primer, 5’-GTGAAGGTGCTGAAGGTTCG-3’; reverse primer, 5’-AAGCAAACTTCACAGCCTGG-3’.

Western blot and immunoprecipitation (IP) assays

Total proteins were prepared from cells and tissue samples using RIPA buffer containing 1% protease inhibitor (PMSF) and a cocktail. The protein concentration was estimated via the BCA method. A total of 20 µg of protein in 10 µl of solution was subjected to SDS-PAGE to separate proteins with different molecular weights, which were subsequently transferred to a PVDF membrane. After the membranes were blocked with non-fat milk for 1 h and washed twice with TBST, different primary antibodies were incubated with the membranes at 4 °C overnight to probe specific targets. The next day, after three washes with TBST, the membranes were incubated with the corresponding secondary antibodies at room temperature for 2 h. Finally, the protein bands were visualized using a chemiluminescence kit (Beyotime Biotechnology, Shanghai, China). The relative protein expression levels were measured using ImageJ software and normalized to the internal control GAPDH. For the IP assay, total proteins were extracted from the indicated cells using IP lysis buffer according to previously described methods. 10% of the cell extracts were added to a new tube and were regarded as input samples. The remaining cell extracts were precleared with 20 µl of Protein A/G magnetic beads for 4 h. After centrifugation at 4 °C for 10 min, the supernatants were transferred to a new tube and incubated with the target antibody or anti-rabbit IgG with gentle rotation at 4 °C overnight for protein pull-down. The next day, 50 µl of Protein A/G magnetic beads was added to each tube and incubated for 2 h with gentle rotation at 4 °C. The magnetic beads were subsequently washed with IP lysis buffer three times. After the supernatants were discarded, the magnetic beads were boiled in 50 µl of SDS loading buffer, and the samples were finally subjected to immunoblot analysis.

Cycloheximide (CHX) assay

A CHX assay was conducted to analyse the protein half-life and protein stability. Briefly, cells from different groups were treated with CHX (20 µg/ml) for the indicated durations (0, 2, 4, and 8 h). The cells were subsequently collected, and total proteins were isolated for immunoblot analysis to measure protein abundance.

Ubiquitination assay

An in vivo ubiquitination assay was performed in HEK-293T cells transfected with the required plasmids, such as Flag-RNF19A, HA-ILK, and His-Ub. Thirty-six hours after transfection, the cells in the different groups were treated with MG132 (10 µM, Sigma, USA) for 6 h. The cells were collected and lysed with IP lysis buffer. Subsequently, the lysates were immunoprecipitated with HA- or Flag-tag magnetic beads. Next, the beads were washed with IP lysis buffer, followed by boiling in 50 µl of SDS loading buffer. Finally, the samples were subjected to immunoblotting to detect ubiquitin.

Subcutaneous tumour formation in nude mice

The in vivo study was approved by the Ethics Committee of Renmin Hospital of Wuhan University (Ethics number: 20220806B). Four-week-old male nude mice (BALB/c-nu) were purchased from Hunan SJA Laboratory Animal Co., Ltd. (Hunan, China) and were housed in an SPF chamber at standard temperature and humidity. The mice were randomly divided into two groups: LV-Ctrl and LV-RNF19A. Equal amounts (1 × 106) of T24 cells stably overexpressing RNF19A or control cells were subcutaneously injected into the flanks of nude mice. The xenograft tumour volumes were monitored every week using callipers. The tumour volume was calculated using the following formula: tumour volume = a×b2/2 (a: tumour length; b: tumour width). The mice were euthanized five weeks after the injection, and the tumours were resected and photographed using a camera. The resected samples were frozen and stored at -80 °C for further experiments.

Immunohistochemistry (IHC)

The IHC analysis was conducted as previously described [15, 16]. The following primary antibodies were used: anti-RNF19A (1:200; Cat: NBP1-87989; Novus Biologicals), anti-ILK (1:100; Cat: sc-20019; Santa Cruz Biotechnology), anti-Ki-67 (1:200; Cat: #9027; Cell Signaling Technology), anti-p-AKT (1:200; Cat: #4060; Cell Signaling Technology), and anti-p-mTOR (1:200; Cat: #2976; Cell Signaling Technology).

Statistical analysis

All the data were statistically analysed using GraphPad Prism 9 and are presented as the means ± SDs. Differences between groups were determined by two-tailed paired or unpaired Student’s t tests or one-way ANOVA. If the P value was less than 0.05, the data were considered significantly different.

Results

RNF19A was aberrantly downregulated in BCa and positively correlated with the clinical outcomes of BCa patients

First, we explored the expression of RNF19A across cancers by analysing sequencing data from various tumours in the TIMER 2.0 database. Compared with that in corresponding normal tissues, RNF19A was highly expressed in Cholangiocarcinoma (CHOL), Esophageal Carcinoma (ESCA), Head and Neck squamous cell carcinoma (HNSC), Liver hepatocellular carcinoma (LIHC), and Stomach adenocarcinoma (STAD), whereas its expression was downregulated in Bladder Urothelial Carcinoma (BLCA), Colon adenocarcinoma (COAD), Kidney Chromophobe (KICH), Kidney renal papillary cell carcinoma (KIRP), Lung squamous cell carcinoma (LUSC), Rectum adenocarcinoma (READ), Thyroid carcinoma (THCA), and Uterine Corpus Endometrial Carcinoma (UCEC) (Fig. 1A). We also analysed RNF19A expression in bladder cancer using the UALCAN database and found that RNF19A was aberrantly downregulated in bladder cancer (Fig. 1B). Moreover, RNF19A expression was even lower in BCa patients with increased lymph node metastasis (Fig. 1C). We investigated RNF19A mRNA and protein levels in BCa samples by performing qRT–PCR, western blot, and IHC assays to determine the profile of RNF19A expression in BCa. The results suggested that RNF19A expression was significantly decreased in BCa tissues compared to adjacent normal tissues (Fig. 1D-G), which was consistent with findings from the public databases. In addition, we measured RNF19A expression in BCa cell lines (UM-UC-3, J82, SW780, T24, and 5637) and immortalized ureteral epithelial cells (SV-HUC-1), and the results revealed slightly lower RNF19A mRNA and protein levels in BCa cells than in normal SV-HUC-1 cells (Fig. 1H-I). Additionally, we investigated the prognostic predictive performance of RNF19A in BCa. The Kaplan–Meier survival analysis revealed that BCa patients with high RNF19A expression experienced longer overall survival (OS) than those with low RNF19A expression (Fig. 1J). Taken together, these findings strongly support the hypothesis that RNF19A is aberrantly downregulated in BCa tissues and cells and might serve as a valuable prognostic predictor for BCa.

Fig. 1
figure 1

RNF19A was expressed at low levels in BCa tissues and cell lines and was positively correlated with the clinical outcomes of patients with BCa. (A) RNF19A mRNA expression was analysed in various cancers using the TIMER database. (B) The RNF19A mRNA level was compared in normal and BCa tissues using the UALCAN database. (C) RNF19A mRNA expression in BCa with different lymphatic metastasis statuses, as determined from the UALCAN database. (D) qRT‒PCR analysis of RNF19A mRNA levels in normal and BCa tissues. (E-F) western blot analysis of RNF19A protein levels in normal and BCa tissues and quantitative analysis. (G) IHC analysis of RNF19A expression in normal and BCa tissues. Scale bar, 200 μm. (H-I) RNF19A mRNA and protein levels were detected in BCa cell lines (UM-UC-3, J82, SW780, T24, and 5637) and immortal ureteral epithelial cells (SV-HUC-1). (J) Kaplan‒Meier analysis of the correlation between overall survival and RNF19A expression levels. *P < 0.05, **P < 0.01, and ***P < 0.001

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Overexpression of RNF19A suppressed cell proliferation, migration, invasion and the EMT in BCa cells

T24 and 5637 cells were stably infected with a lentivirus containing an RNF19A overexpression plasmid to clarify the function of RNF19A in BCa cells. The overexpression efficiency of RNF19A was validated through qRT‒PCR and western blot assays. Compared with those in the LV-Ctrl group, the RNF19A mRNA and protein levels in BCa cells in the LV-RNF19A group were dramatically increased (Fig. 2A-C). The results of the CCK-8 and colony formation assays suggested that forced expression of RNF19A resulted in reduced cell proliferation, as evidenced by decreased OD values and colony numbers in the LV-RNF19A group (Fig. 2D-E). In the wound healing assay, the relative migration rates were lower in the LV-RNF19A group than in the LV-Ctrl group, indicating that the overexpression of RNF19A significantly inhibited the motility of BCa cells (Fig. 2F-G). Similarly, in the transwell invasion assay, the invasion capacity of BCa cells in the LV-RNF19A group was impaired (Fig. 2H-I). The epithelial‒mesenchymal transition (EMT) is a reversible process that is tightly associated with tumour cell proliferation, migration, and invasion [17]. We subsequently examined the levels of marker proteins (E-cad, N-cad, and Vim) of the EMT. The results of the western blot assay revealed that E-cad expression was notably increased, whereas N-cad and Vim levels were decreased after the overexpression of RNF19A (Fig. 2J-L). Our data suggested that the overexpression of RNF19A suppressed cell proliferation, migration, and invasion and inhibited the EMT in BCa cells.

Fig. 2
figure 2

The overexpression of RNF19A suppressed cell proliferation, migration, invasion and the EMT process in BCa cells. (A) qRT‒PCR analysis of RNF19A mRNA expression in T24 and 5637 cells stably overexpressing RNF19A. (B-C) western blot analysis of RNF19A protein levels in T24 and 5637 cells overexpressing RNF19A and a quantitative analysis. (D-G) After RNF19A was overexpressed, cell proliferation was assessed via CCK-8 and colony formation assays. (H-K) After RNF19A was overexpressed, cell migration and invasion abilities were evaluated via wound healing and transwell invasion assays. (L-O) Protein levels of EMT-related markers (E-cad, N-cad, and Vim) in BCa cells stably overexpressing RNF19A. *P < 0.05, **P < 0.01, and ***P < 0.001

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Knockdown of RNF19A promoted cell proliferation, migration, invasion, and the EMT in BCa cells

Stable RNF19A-knockdown BCa cell lines (T24 and 5637) were constructed via infection with lentiviral vectors carrying an shRNA targeting RNF19A (shRNF19A) to further confirm the effect of RNF19A on the malignant behaviours of BCa cells. qRT‒PCR and western blot experiments demonstrated that the RNF19A mRNA and protein levels were markedly lower in the shRNF19A group than in the shNeg group (Fig. 3A‒C). Through in vitro CCK-8 and colony formation assays, we found that RNF19A knockdown increased the growth rate of BCa cells, as evidenced by increases in optical density values and colony numbers (Fig. 3D-E). Wound healing assays revealed that the relative migration rates were greater in the shRNF19A group than in the shNeg group, suggesting that the migration ability of BCa cells increased after RNF19A was knocked down (Fig. 3F-G). The results of the transwell invasion assay revealed that RNF19A knockdown led to an enhanced cell invasion capacity (Fig. 3H-I). Next, we examined the protein expression levels of EMT markers in BCa cells. The results of the western blot assay suggested that the knockdown of RNF19A dramatically increased N-cad and Vim protein expression but resulted in a reduction in the E-cad protein level. Overall, our data revealed that RNF19A was a key negative regulator of cell proliferation, migration, and invasion in BCa cells.

Fig. 3
figure 3

Knockdown of RNF19A promoted cell proliferation, migration, invasion, and the EMT in BCa cells. (A) qRT‒PCR analysis of RNF19A mRNA expression in T24 and 5637 cells with stable RNF19A knockdown. (B-C) western blot analysis of RNF19A protein levels in T24 and 5637 cells after the knockdown of RNF19A and quantitative analysis. (D-G) After RNF19A was knocked down, cell proliferation was assessed via CCK-8 and colony formation assays. (H-K) After RNF19A was knocked down, cell migration and invasion were evaluated via wound healing and transwell invasion assays. (L-O) Protein levels of EMT-related markers (E-cad, N-cad, and Vim) in BCa cells with stable RNF19A knockdown. *P < 0.05, **P < 0.01, and ***P < 0.001

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RNF19A negatively regulated the AKT/mTOR signalling pathway in BCa

We explored the biological mechanism of RNF19A by stratifying TCGA bladder cancer dataset into RNF19AHigh and RNF19ALow groups and identified differentially expressed genes (DEGs) between the two groups; these genes were regarded as RNF19A-related DEGs. A total of 1466 genes were altered, with 523 upregulated and 943 downregulated genes in the RNF19AHigh group compared with those in the RNF19ALow group (Fig. 4A). The expression patterns of these genes in BCa samples with high or low RNF19A expression are displayed in Supplementary Fig. 1A. These DEGs related to RNF19A were subsequently subjected to functional enrichment analyses. The results of the GO analysis suggested that molecular transducer activity, receptor activity, and ion transmembrane transporter activity were the three most enriched terms with respect to molecular functions. In terms of biological processes, these genes were particularly enriched in single-organism, multicellular organismal, and single-multicellular organism processes. In terms of cellular component, these DEGs were enriched mainly in intrinsic components of the membrane, extracellular region, and extracellular region part (Supplementary Fig. 1B). According to the KEGG enrichment analysis, these DEGs related to RNF19A were significantly enriched in neuroactive ligand‒receptor interaction, the mTOR signalling pathway, the oestrogen signalling pathway, the cAMP signalling pathway, and the calcium signalling pathway (Fig. 4B). Moreover, the GSEA results revealed a close association between the RNF19A expression level and pathways, including ubiquitin-mediated proteolysis and mTOR signalling (Fig. 4C-D). Additionally, the correlation analysis revealed that RNF19A expression was negatively correlated with the expression of multiple genes (EIF4EBP1, PGF, RPS6KB2, MLST8, PIK3CD, VEGFB, PIK3R5, and EIF4E2) involved in the mTOR signalling pathway (Fig. 4E-L). Overall, these analyses prompted us to infer that RNF19A might participate in regulating the mTOR signalling pathway. Therefore, we examined the phosphorylation levels of AKT (p-AKT T473), mTOR (p-mTOR S2448), and S6K1 (p-S6K1 T389), which indicate the activation of the mTOR signalling pathway. Our results revealed that the levels of phosphorylated AKT, mTOR, and S6K1 were also significantly reduced in RNF19A-overexpressing BCa cells compared with control cells (Fig. 4G-H). Conversely, RNF19A knockdown led to increased protein levels of p-AKT, p-mTOR, and p-S6K1 (Fig. 4I-J). We treated BCa cells in which RNF19A was stably knocked down with LY294002 (a small-molecule inhibitor of AKT/mTOR signalling) to further investigate whether RNF19A functions by regulating the activation of AKT/mTOR signalling (Supplementary Fig. 2A-F). Our results confirmed that the effects of RNF19A knockdown on promoting cell proliferation, migration and invasion were inhibited by the addition of LY294002. Overall, the above results suggested that RNF19A negatively regulated the AKT/mTOR signalling pathway to influence the biological behaviours of BCa cells.

Fig. 4
figure 4

RNF19A negatively regulated the AKT/mTOR signalling pathway in BCa. (A) Volcano plot showing the differentially expressed genes between the RNF19Ahigh and RNF19Alow groups. (B) KEGG enrichment analysis of RNF19A-related DEGs. (C) GSEA plots depicting the enrichment of ubiquitin-mediated proteolysis in samples with high RNF19A expression. (D) GSEA plots depicting the enrichment of the mTOR signalling pathway in samples with low RNF19A expression. (E-L) Correlation analyses of RNF19A expression and the expression levels of multiple genes in the mTOR signalling pathway, including EIF4EBP1, PGF, RPS6KB2, MLST8, PIK3CD, VEGFB, PIK3R5, and EIF4E2. (M-O) Expression of proteins involved in mTOR signalling in T24 and 5637 cells stably overexpressing RNF19A. (P-R) Protein levels of AKT, p-AKT T473, mTOR, p-mTOR S2448, S6K1, and p-S6K1 T389 in T24 and 5637 cells after RNF19A was knocked down. *P < 0.05, **P < 0.01, and ***P < 0.001

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RNF19A targeted ILK as a ubiquitination substrate

Moreover, we wanted to explore the detailed mechanism by which RNF19A regulated the activation of AKT/mTOR signalling in BCa. Previous studies have shown that RNF19A can function as an E3 ligase to regulate the ubiquitination and stability of downstream targets [13, 18]. Therefore, we speculated that RNF19A might promote the ubiquitination of some kinases to influence AKT/mTOR signalling. We subsequently used the BioGRID portal to identify potential direct targets of RNF19A. ILK, an integrin-linked kinase that promotes the growth of various tumours, attracted our attention since it is aberrantly expressed in bladder cancer and likely participates in regulating the AKT/mTOR pathway [19,20,21]. We then performed protein IP assays to detect the interaction between RNF19A and ILK. Our results confirmed that endogenous RNF19A and ILK physically interacted in BCa cells (Fig. 5A). Then, we overexpressed HA-ILK and Flag-RNF19A in HEK-293T cells and performed co-IP experiments. As shown in Fig. 5B, the results indicated that RNF19A specifically interacted with ILK. Next, we asked whether RNF19A influenced the expression levels of ILK. qRT‒PCR revealed that the overexpression or knockdown of RNF19A had no effect on the ILK mRNA level (Fig. 5C‒D). Surprisingly, the results of the western blot assay showed that forced expression of RNF19A led to a reduction in the ILK protein level, whereas silencing RNF19A increased the ILK protein level (Fig. 5E-H). Moreover, we analysed RNF19A and ILK expression in human BCa samples, and found that RNF19A and ILK were negatively corrected (Supplementary Fig. 3A-B). In the cycloheximide (CHX) chase assay, we found that the overexpression of RNF19A shortened the half-life of the ILK protein, whereas the knockdown of RNF19A slowed its degradation (Fig. 5I-L). Additionally, ubiquitination assays revealed that the level of ubiquitinated ILK dramatically increased after ectopic RNF19A expression (Fig. 5M). In contrast, the downregulation of RNF19A led to reduced ILK polyubiquitylation (Fig. 5N). Based on these findings, we concluded that RNF19A could directly interact with ILK and target ILK as a ubiquitination substrate.

Fig. 5
figure 5

RNF19A targeted ILK as a ubiquitination substrate. (A) Cell lysates from T24 and 5637 cells were immunoprecipitated with IgG, anti-RNF19A, or anti-ILK antibodies, and the precipitates were then subjected to western blot analysis. (B) HEK-293T cells transfected with the indicated plasmids were lysed and then immunoprecipitated with anti-HA or anti-Flag antibodies. The precipitates were detected by western blotting. (C-D) qRT‒PCR was performed to detect ILK mRNA levels after RNF19A was overexpressed or knocked down. (E-H) western blot analyses of ILK protein levels in BCa cells with stable overexpression or knockdown of RNF19A and quantitative analyses. (I-L) The protein levels of ILK in T24 and 5637 cells with stable overexpression or knockdown of RNF19A after CHX treatment were measured via quantitative analyses. (M-N) An in vivo ubiquitination assay using ILK pull-down confirmed that the overexpression of RNF19A promoted the ubiquitination of ILK, whereas the knockdown of RNF19A had the opposite effect. *P < 0.05, **P < 0.01, and ***P < 0.001

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RNF19A suppressed cellular malignancy via the ILK-mediated inactivation of AKT/mTOR signalling

We first explored the effect of ILK on the activation of the AKT/mTOR signalling pathway to determine whether the tumour-suppressive function of RNF19A in BCa cells relied on ILK. Western blot assays revealed that silencing ILK led to reduced levels of p-AKT, p-mTOR, and p-S6K1 (Fig. 6A-C), whereas forced expression of ILK resulted in increased protein levels of p-AKT, p-mTOR, and p-S6K1 (Fig. 6D-F). We subsequently manipulated the ILK expression level in RNF19A-deficient or RNF19A-overexpressing 5637 and T24 cells. Western blot assays revealed that forced ILK expression partially rescued the decreased levels of p-AKT, p-mTOR, and p-S6K1 caused by RNF19A overexpression (Fig. 6G-I). Consistently, the increased phosphorylation levels of proteins in the AKT/mTOR signalling pathway induced by RNF19A knockdown were eliminated after ILK expression was silenced (Fig. 6J-L). In vitro functional assays suggested that the overexpression of ILK restored the phenotypes of cell proliferation, migration, and invasion that were inhibited by RNF19A overexpression (Supplementary Fig. 4A-B,E,G). Moreover, the enhanced malignant behaviours induced by RNF19A knockdown were attenuated after downregulating ILK, as confirmed by CCK-8, wound healing, and transwell invasion assays (Supplementary Fig. 4C-D,F,H). Therefore, we concluded that RNF19A affected the activity of the AKT/mTOR signalling pathway through the regulation of ILK and suppressed the proliferation, migration, and invasion of BCa cells.

Fig. 6
figure 6

RNF19A regulated the activation of AKT/mTOR signalling via ILK. (A-C) western blot analysis of the levels of phosphorylated AKT, mTOR, and S6K1 in T24 and 5637 cells transfected with an siRNA targeting ILK and quantitative analyses. (D-F) western blot analysis of phosphorylated AKT, mTOR, and S6K1 levels in BCa cells overexpressing ILK and quantitative analyses. (G-I) Protein levels of AKT, p-AKT, mTOR, p-mTOR, S6K1, and p-S6K1 in RNF19A-overexpressing BCa cells after transfection with the ILK overexpression plasmid or empty vector and quantitative analyses. (J-L) western blot results showing that the increased levels of phosphorylated AKT, mTOR, and S6K1 induced by RNF19A knockdown were eliminated after silencing ILK. *P < 0.05, **P < 0.01, and ***P < 0.001

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RNF19A inhibited BCa growth in vivo

Finally, we created a subcutaneous tumour formation model in BALB/c-nu nude mice and monitored tumour growth every week to further evaluate the effect of RNF19A on BCa cell growth in vivo. As shown in Fig. 7A-C, the tumour volume, size, and weight were smaller in the LV-RNF19A group than in the LV-Ctrl group, indicating that tumour growth was suppressed in vivo after RNF19A was overexpressed. Western blot and IHC assays revealed increased RNF19A protein levels in tumour samples from the LV-RNF19A group compared with those from the LV-Ctrl group. Moreover, the protein level of ILK and the levels of phosphorylated AKT, mTOR, and S6K1 were markedly lower in the LV-RNF19A group than in the LV-Ctrl group (Fig. 7D-E and G). qRT‒PCR experiments revealed that the RNF19A mRNA level was significantly higher in tumour samples from the LV-RNF19A group than in those from the LV-Ctrl group, whereas ILK mRNA expression remained unaltered after the forced expression of RNF19A (Fig. 7F). Collectively, these data indicated that RNF19A inhibited BCa growth in vivo, which was associated with decreased ILK protein levels and inactivation of the AKT/mTOR signalling pathway.

Fig. 7
figure 7

RNF19A inhibited BCa growth in vivo. (A) Tumour growth curves of the LV-Control and LV-RNF19A groups. (B) Size of the tumours in the LV-Control and LV-RNF19A groups. (C) Comparison of tumour weight between the LV-Control and LV-RNF19A groups. (D-E) western blot analysis of the levels of the RNF19A, ILK, and proteins involved in mTOR signalling in tumour samples from the LV-Control and LV-RNF19A groups. (F) qRT‒PCR analysis of RNF19A and ILK mRNA levels in tumour samples from the LV-Control and LV-RNF19A groups. (G) IHC analysis of RNF19A, Ki-67, ILK, p-AKT, and p-mTOR levels in tumour samples from the LV-Control and LV-RNF19A groups. *P < 0.05, **P < 0.01, and ***P < 0.001

Full size image

Discussion

Ubiquitination, one of the most important posttranslational modifications (PTMs) in eukaryotic cells, is a reversible process that plays a multifunctional role in regulating cellular behaviours under physiological and pathological conditions [22, 23]. Ubiquitin (Ub), a 76-amino acid protein, acts as a linkage site for multiple polyubiquitin chain modifications, which ultimately determine the intracellular function or fate of target proteins [24]. In the ubiquitination cascade, three enzymes, Ub-activating enzymes (E1s), Ub-conjugating enzymes (E2s), and Ub ligases (E3s), play specific roles. Briefly, E1s can activate and transfer Ub to E2s. Thereafter, the E3s transmit the activated Ub molecules to the substrates with the cooperation of E2s [25]. Ubiquitinated proteins exhibit changes in their localization, stability, trafficking, or intracellular function and thus participate in various biological processes, such as the cell cycle, cell growth, apoptosis, differentiation, and senescence [26, 27]. Recently, the role of abnormal ubiquitination activity in the pathogenesis of various cancers has attracted increasing attention [28]. Multiple E3 ligases are aberrantly expressed in BCa and function as tumour-promoting or tumour-inhibiting factors by mediating the ubiquitination and degradation of downstream proteins and could also serve as prognostic biomarkers [29,30,31]. For example, RNF126 is upregulated in BCa and functions as an oncoprotein in BCa by directly binding to and promoting the ubiquitination and degradation of PTEN [32]. The downregulation of RNF128 was significantly associated with an advanced tumour grade, stage, nodal metastasis, vascular invasion and a high mitotic rate in BCa. Furthermore, lower expression of RNF128 predicts shorter disease-specific survival and metastasis-free survival in patients with BCa [33]. Despite these previous reports, the expression, functional role and associated mechanism of E3 ligases in BCa remain largely unknown.

In this work, we revealed a previously unreported role of RNF19A in BCa. We showed that RNF19A expression was reduced in BCa tissues and cell lines through data mining of public resources and further experiments. Moreover, lower expression of RNF19A was correlated with nodal metastasis and shorter overall survival of BCa patients, indicating the potential of RNF19A as a prognostic predictor in BCa patients. Further assessment of the predictive performance of RNF19A in multiple central and real-world cohorts of BCa patients is warranted. Furthermore, we discovered that the overexpression of RNF19A suppressed cell proliferation, migration, invasion and the EMT in vitro, whereas silencing RNF19A had the opposite effects. In addition, the subcutaneous tumour model revealed that overexpressing RNF19A impeded tumour growth in vivo. Therefore, our findings support the tumour inhibitory role of RNF19A in BCa and suggest that RNF19A might be a prognostic biomarker for patients with BCa.

We revealed the downstream pathways associated with RNF19A by conducting bioinformatic analyses using datasets from TCGA database, as previously described [34]. We found that the RNF19A-related DEGs were enriched mainly in the mTOR signalling pathway via a KEGG enrichment analysis. Moreover, GSEA revealed that lower expression of RNF19A was significantly related to the mTOR signalling pathway. In addition, RNF19A expression was negatively correlated with the expression of multiple genes related to mTOR signalling. These findings provided clues for the regulatory role of RNF19A in mTOR signalling in BCa. Mammalian or mechanistic target of rapamycin (mTOR), a serine/threonine protein kinase with a molecular weight of 289 kDa, is the catalytic subunit of two structurally and functionally distinct protein complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) [35]. mTOR is a central signalling hub that integrates multiple intracellular and environmental signals in eukaryotic cells. mTOR kinase is often overactivated in human malignancies, which drives tumorigenesis and progression [36]. mTOR signalling is often activated by the upstream kinase AKT, resulting in the nuclear translocation of phosphorylated mTOR and ultimately activating downstream effectors such as S6K1 and 4EBP1 to regulate cell proliferation, survival, metabolism and autophagy [37]. Therefore, we examined the effect of RNF19A on the activity of mTOR signalling via western blotting, and the results revealed decreased phosphorylation levels of AKT, mTOR, and S6K1 in RNF19A-overexpressing BCa cells. Conversely, the silencing of RNF19A resulted in the upregulation of p-AKT, p-mTOR, and p-S6K1, suggesting that RNF19A negatively regulates AKT/mTOR signalling in BCa. Additionally, the effects of RNF19A knockdown on promoting cell proliferation, migration and invasion were inhibited by the addition of LY294002, a small-molecule inhibitor of AKT/mTOR signalling. Collectively, these findings suggest that RNF19A negatively regulates the AKT/mTOR signalling pathway to influence the biological behaviours of BCa cells.

Considering that RNF19A functioned as an E3 ligase rather than as a protein kinase in a previous study, we explored the potential substrates of RNF19A using the BioGRID portal. Integrin-linked kinase (ILK), an ankyrin repeat-containing serine‒threonine protein kinase, attracted our attention for the following reasons: (1) ILK was previously identified as an interacting protein of RNF19A [38]; and (2) ILK can phosphorylate multiple downstream proteins, such as GSK-3 and AKT [39]. We first confirmed whether ILK was a direct target of RNF19A by measuring the physical interaction between RNF19A and ILK via IP experiments. The results of the qRT‒PCR and western blot assays revealed that RNF19A negatively regulated the ILK protein level but had no effect on its mRNA expression, suggesting that RNF19A regulated ILK expression through a posttranscriptional mechanism. In the CHX chase assay, the overexpression of RNF19A shortened the half-life of the ILK protein, whereas knockdown of RNF19A slowed its degradation, suggesting that RNF19A decreased the stability of the ILK protein. In addition, an in vivo ubiquitination assay revealed that upregulation of RNF19A promoted ILK polyubiquitylation. Thus, our findings revealed that RNF19A could interact with ILK and promote its ubiquitination and degradation. We manipulated ILK expression in RNF19A-deficient or RNF19A-overexpressing cells to assess whether RNF19A inhibits BCa by destabilizing ILK. Our data revealed that forced ILK expression partially rescued the decreased phosphorylation levels of AKT, mTOR, and S6K1 caused by RNF19A overexpression and that the increased protein levels of p-AKT, p-mTOR, and p-S6KA induced by RNF19A knockdown were eliminated after silencing ILK. Similarly, the effects of RNF19A overexpression or knockdown on the phenotypes of cell proliferation, migration, and invasion could also be restored by forced or decreased ILK expression. Therefore, we concluded that RNF19A suppresses the proliferation, migration, and invasion of BCa cells by regulating ILK ubiquitination and inactivating the AKT/mTOR signalling pathway.

Our study had several limitations. First, the driving factor that results in decreased RNF19A expression in BCa should be further investigated. Second, the effects of RNF19A on the cell cycle distribution, stemness, apoptosis, angiogenesis, and chemosensitivity of BCa deserve further exploration. Third, the domains of RNF19A and ILK responsible for their interaction need to be investigated. In addition, analysing the associations among RNF19A, ILK, and the AKT/mTOR signalling pathway in human BCa samples would be more useful.

In conclusion, we showed that RNF19A expression was reduced in BCa tissues and cell lines and that lower RNF19A expression was correlated with an advanced tumour stage and worse clinical outcomes. RNF19A functions as a tumour suppressor in BCa by suppressing cell proliferation, migration, and invasion in vitro and impeding tumour growth in vivo. Furthermore, we revealed that RNF19A could directly interact with ILK, promote ILK ubiquitination and inactivate the AKT/mTOR signalling pathway, thereby impeding cell proliferation, migration, and invasion in BCa. Therefore, our findings strongly suggest that RNF19A is a promising therapeutic target for BCa.

Data availability

No datasets were generated or analysed during the current study.

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Acknowledgements

We sincerely acknowledge all the authors of this study.

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Authors and Affiliations

  1. Department of Urology, The First Affiliated Hospital of Yangtze University, The First people’s Hospital of Jingzhou, Jingzhou, 434000, China

    Hao Deng, Guanghai Ji & Shaoping Cheng

  2. Department of Urology, Renmin Hospital of Wuhan University, Wuhan, 430060, China

    Hao Deng & Fan Cheng

  3. Department of Urology, Shanghai Public Health Clinical Center, Shanghai, 200083, China

    Jun Ma

  4. Department of Oncology, The First Affiliated Hospital of Yangtze University, The First people’s Hospital of Jingzhou, Jingzhou, 434000, China

    Jun Cai

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Contributions

HD, GHJ, JM designed the study. HD performed the experiments. JC, FC and SPC reviewed and revised the manuscript. All authors reviewed the manuscript.

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Correspondence to Jun Cai, Shaoping Cheng or Fan Cheng.

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Deng, H., Ji, G., Ma, J. et al. RNF19A inhibits bladder cancer progression by regulating ILK ubiquitination and inactivating the AKT/mTOR signalling pathway. Biol Direct 19, 102 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13062-024-00562-2

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