Epithelial-mesenchymal Transition In Bladder Cancer Research Articles

CircFSCN1 induces tumor progression and triggers epithelial–mesenchymal transition in bladder cancer through augmentation of MDM2-mediated p53 silencing

BackgroundCompelling evidences indicated that circular RNA (circRNA) was a novel class of non-coding RNA that played critical and distinct roles in various human cancers. Their roles and underlying mechanisms, however, in bladder cancer (BC) remained largely unknown. MethodsA novel circRNA derived from oncogene FSCN1, namely circFSCN1, was selected from a microarray analysis. The phenotypic alterations were assessed with functional experiments in vitro and in vivo. RNA immunoprecipitation, RNA pull-down, luciferase reporter assay, and rescue experiments were sequentially proceeded to clarify the interactions among circFSCN1, miR-145-5p, MDM2, and p53. ResultsWe observed that the expression of circFSCN1 was elevated in BC cell lines and tissues. Next, we validated the fundamental properties of circFSCN1. In the meanwhile, we noticed that elevated circFSCN1 level, pathological T stage, and tumor grade were identified as independent factors associated with cancer-specific survivals of patients with BC，as determined by univariate and multivariable COX regression analyses. Phenotype studies demonstrated the promoting effects of circFSCN1 on the proliferation, migration, invasion, and epithelial–mesenchymal transition (EMT) of BC cells. Mechanistically, we elucidated that circFSCN1, primarily localized in the cytoplasm, upregulated the expression of MDM2, a well-known inhibitor of p53, by directly binding to miR-145-5p. ConclusionsElevated circFSCN1 induces tumor progression and EMT in BC via enhancing MDM2-mediated silencing of p53 by sponging miR-145-5p. Targeting circFSCN1, a novel identified target, may be conducive in impeding BC progression and providing survival benefits for patients with BC.

RAB14 promotes epithelial-mesenchymal transition in bladder cancer through autophagy‑dependent AKT signaling pathway

Bladder cancer (BLCA) is the 9th most common cancer of mortality. Autophagy and epithelial to mesenchymal transition (EMT) have an essential role in cancer invasion and metastasis. However, the relationship between autophagy and EMT is still poorly understood in BLCA. Functional enrichment and pathway network analysis were carried out. Comprehensive protein-protein interactions (PPI) networks were proposed to prioritize candidate autophagy-related genes. Furthermore, an autophagy-related signature and a nomogram model were established by integrating clinical information and this signature risk score to evaluate candidate autophagy-related genes. RAB14 expression and its association with pathological information and survival were evaluated in samples from TCGA dataset. Knocking down RAB14 in T24 cells was constructed, and immunofluorescence staining, transmission electron microscopy, immunohistochemistry and western blotting and a series of functional assays were performed to evaluate the migration, invasion, EMT and autophagy abilities of BLCA cells. The autophagy-related gene RAB14 was the only candidate gene identified by three kinds of analytic approaches. RAB14 was highly upregulated in BLCA and correlated with clinical outcomes based on TCGA BLCA datasets. Knocking down RAB14 was found to inhibit EMT and autophagy in T24 cells. RAB14 levels were positively related to those of LC3B and Beclin1, two genes with critical roles in the autophagy process, and the correlation was further confirmed in clinical tissue specimens by IHC and western blot analysis. In addition, RAB14-promoted EMT, migration, and invasion in T24 cells could be partially reversed by autophagy activator, rapamycin. The effects of RAB14 on autophagy was associated with level of p-Akt, indicating that they were possibly mediated via PI3K/AKT signaling. These findings indicated that autophagy-related gene RAB14-promoted EMT, migration and invasion of bladder cancer via the Akt-associated autophagic pathway.

The Impact of Cancer-Associated Fibroblasts on Drug Resistance, Stemness, and Epithelial-Mesenchymal Transition in Bladder Cancer: A Comparison between Recurrent and Non-Recurrent Patient-Derived CAFs

This study comparatively evaluated the possible effects of recurrent and non-recurrent patient-derived Cancer-Associated Fibroblasts (CAFs-R and -NR) on the bladder cancer cell line, EJ138. Both groups of CAFs increased cisplatin resistance and altered cell cycle distribution alongside induced resistance to apoptosis. Later, the scratch assay confirmed the cell migration-inducing effects of CAFs on cells. Nonetheless, only CAFs-R managed to increase sphere-formation and clonogenic levels in EJ138 cells, which were later validated by upregulating pluripotency transcription factors. Besides, CAFs-R also affected the expression levels of some of the EMT markers. Our study suggests that CAFs-R had stronger pro-tumorigenic effects on EJ138 cells.

TGF-β1 dominates stromal fibroblast-mediated EMT via the FAP/VCAN axis in bladder cancer cells

BackgroundBladder cancer is one of the most common malignant tumors of the urinary system and is associated with a poor prognosis once invasion and distant metastases occur. Epithelial-mesenchymal transition (EMT) drives metastasis and invasion in bladder cancer. Transforming growth factor β1 (TGF-β1) and stromal fibroblasts, especially cancer-associated fibroblasts (CAFs), are positive regulators of EMT in bladder cancer. However, it remains unclear how TGF-β1 mediates crosstalk between bladder cancer cells and CAFs and how it induces stromal fibroblast-mediated EMT in bladder cancer. We aimed to investigate the mechanism of TGF-β1 regulation of stromal fibroblast-mediated EMT in bladder cancer cells.MethodsPrimary CAFs with high expression of fibroblast activation protein (FAP) were isolated from bladder cancer tissue samples. Subsequently, different conditioned media were used to stimulate the bladder cancer cell line T24 in a co-culture system. Gene set enrichment analysis, a human cytokine antibody array, and cytological assays were performed to investigate the mechanism of TGF-β1 regulation of stromal fibroblast-mediated EMT in bladder cancer cells.ResultsAmong the TGF-β family, TGF-β1 was the most highly expressed factor in bladder cancer tissue and primary stromal fibroblast supernatant. In the tumor microenvironment, TGF-β1 was mainly derived from stromal fibroblasts, especially CAFs. In stimulated bladder cells, stromal fibroblast-derived TGF-β1 promoted bladder cancer cell migration, invasion, and EMT. Furthermore, TGF-β1 promoted the activation of stromal fibroblasts, inducing CAF-like features, by upregulating FAP in primary normal fibroblasts and a normal fibroblast cell line. Stromal fibroblast-mediated EMT was induced in bladder cancer cells by TGF-β1/FAP. Versican (VCAN), a downstream molecule of FAP, plays an essential role in TGF-β1/FAP axis-induced EMT in bladder cancer cells. VCAN may also function through the PI3K/AKT1 signaling pathway.ConclusionsTGF-β1 is a critical mediator of crosstalk between stromal fibroblasts and bladder cancer cells. We revealed a new mechanism whereby TGF-β1 dominated stromal fibroblast-mediated EMT of bladder cancer cells via the FAP/VCAN axis and identified potential biomarkers (FAP, VCAN, N-cadherin, and Vimentin) of bladder cancer. These results enhance our understanding of bladder cancer invasion and metastasis and provide potential strategies for diagnosis, treatment, and prognosis.

β-asarone inhibits the migration, invasion, and EMT of bladder cancer through activating ER stress.

β-asarone (β-as), a compound extracted from Acorus calamus, has been found to have anticancer effects on a variety of human cancers. However, the potential effect of β-as on bladder cancer (BCa) remains unknown. After exposure to β-as, migration, invasion, and epithelial-mesenchymal transition (EMT) of BCa were determined by wound healing, transwell, and Western blot assays. Expression of proteins involved in the EMT and ER stress were explored by Western blot assays. Nude mouse xenograft model was served as the model system in vivo. The migration, invasion, and EMT of BCa were significantly inhibited after β-as treatment. Further experiments revealed that endoplasmic reticulum (ER) stress is involved in β-as-mediated metastasis inhibition. In addition, β-as significantly up-regulated activating transcription factor 6 (ATF6), a branch of ER stress, and promoted its Golgi cleavage and nuclear localization. ATF6 silencing attenuated β-as-mediated metastasis and EMT inhibition in BCa cells. Our data suggests that β-as inhibits migration, invasion, and EMT of BCa by activating the ATF6 branch of ER stress. Thus, β-as represents a potential candidate for BCa treatment.

Tumor microenvironment and epithelial-mesenchymal transition in bladder cancer: Cytokines in the game?

Bladder cancer (BlCa) is a highly immunogenic cancer. Bacillus Calmette-Guérin (BCG) is the standard treatment for non-muscle invasive bladder cancer (NMIBC) patients and, recently, second-line immunotherapies have arisen to treat metastatic BlCa patients. Understanding the interactions between tumor cells, immune cells and soluble factors in bladder tumor microenvironment (TME) is crucial. Cytokines and chemokines released in the TME have a dual role, since they can exhibit both a pro-inflammatory and anti-inflammatory potential, driving infiltration and inflammation, and also promoting evasion of immune system and pro-tumoral effects. In BlCa disease, 70-80% are non-muscle invasive bladder cancer, while 20-30% are muscle-invasive bladder cancer (MIBC) at the time of diagnosis. However, during the follow up, about half of treated NMIBC patients recur once or more, with 5-25% progressing to muscle-invasive bladder cancer, which represents a significant concern to the clinic. Epithelial-mesenchymal transition (EMT) is one biological process associated with tumor progression. Specific cytokines present in bladder TME have been related with signaling pathways activation and EMT-related molecules regulation. In this review, we summarized the immune landscape in BlCa TME, along with the most relevant cytokines and their putative role in driving EMT processes, tumor progression, invasion, migration and metastasis formation.

ADAM12 promotes gemcitabine resistance by activating EGFR signaling pathway and induces EMT in bladder cancer.

Gemcitabine (GEM)-based chemotherapy regimens is widely used in bladder cancer (BC) patients. However, GEM resistance may occur and result in treatment failure and disease progression. A disintegrin and metalloprotease 12 (ADAM12) plays a critical role in many cancers. However, the role of ADAM12 in GEM resistance of BC remains unclear. We analyzed the relationship between ADAM12 expression and tumor characteristics using the data downloaded from The Cancer Genome Atlas (TCGA) database and Gene Expression Omnibus (GEO) database. Then, we established GEM resistant BC cell lines and used quantitative real-time PCR, western blot, cell counting kit-8, immunohistochemistry, and xenograft mouse model to investigate the role of ADAM12 in GEM resistance. In general, ADAM12 was found to be upregulated in GEM resistant BC cells. ADAM12 knockdown increased the chemosensitivity of BC cells. We further proved that ADAM12 could promote GEM resistance by activating the epidermal growth factor receptor (EGFR) signaling pathway in BC. Furthermore, the epithelial-mesenchymal transition (EMT) phenotype was observed in GEM resistant BC cells. ADAM12 induced EMT process and promotes tumor progression in BC. Our findings suggested that ADAM12 was a key gene for GEM resistance and positively correlated with malignancy of BC. It might serve as a novel and valuable therapeutic target for BC.

Melatonin Inhibits EMT in Bladder Cancer by Targeting Autophagy.

Melatonin, a naturally biosynthesized molecule secreted by the pineal gland, exhibits antitumor activities against several different types of cancer. The mechanisms of action of melatonin against tumor progression involve cellular apoptosis, antimetastatic activity, antioxidant and mutagenic effects, antiangiogenic activity, and the restoration of cancer immune surveillance. Melatonin has anticancer activity when administered alone or in combination with standard chemotherapeutic agents, with measurable improvements seen in the clinical endpoints of tumor regression and patient survival. However, scant clinical evidence supports the use of melatonin in bladder cancer treatment. Our study has found that melatonin treatment suppresses the bladder cancer cell migratory ability by inhibiting the epithelial-mesenchymal transition (EMT) process, which appears to be linked to melatonin-induced decreases in bladder cancer cell autophagy. Finally, an evaluation of in vivo melatonin-induced antitumor effects in an orthotopic animal model of bladder cancer indicated that melatonin treatment slightly prolonged the survival of tumor-bearing mice. Our study offers novel insights into the use of melatonin in bladder cancer treatment.

Daurisoline Inhibiting Tumor Angiogenesis and Epithelial-Mesenchymal Transition in Bladder Cancer by Mediating HAKAI Protein Stability.

Daurisoline can suppress the development of liver and lung cancers, but its effect on bladder cancer has not been investigated. This study probed into the mechanism underlying the effects of daurisoline on angiogenesis and epithelial-mesenchymal transition (EMT) in bladder cancer. Tissue samples were taken from 40 patients with bladder cancer to analyze the expression of HAKAI and the relationship between HAKAI expression and patient survival. After the gain of function of HAKAI and/or treatment with daurisoline or heat shock protein 90 (HSP90) inhibitor geldanamycin, bladder cancer cells were collected for western blot detection of EMT-related proteins and transwell invasion assay. Tube formation assay assessed the angiogenesis of human umbilical vein endothelial cells (HUVECs) cultured in a conditioned medium of bladder cancer cells. The relationships between daurisoline, HSP90, HAKAI, and E-cadherin (E-cad) were analyzed using drug affinity responsive target stability (DARTS) assay and co-immunoprecipitation (co-IP) method. The effect and action mechanism of daurisoline were validated in nude mice. HAKAI was up-regulated 1.26-fold in bladder cancer tissues (P = 0.004) and correlated with poor prognosis. Daurisoline or geldanamycin inhibited EMT of bladder cancer cells and HUVEC angiogenesis. HAKAI overexpression reversed the suppression by daurisoline or geldanamycin. HAKAI was a client protein of HSP90, which could be directly targeted by daurisoline. HAKAI could target E-cad. Daurisoline also counteracted the promotive effects of overexpressed HAKAI on bladder carcinoma growth in nude mice. Daurisoline suppresses EMT and angiogenesis in bladder cancer by targeting HSP90 and disrupting the stability of HAKAI protein to up-regulate the expression of E-cad.

<p>α-E-Catenin (CTNNA1) Inhibits Cell Proliferation, Invasion and EMT of Bladder Cancer</p>

AimBladder cancer (BLCA) is an urogenital system tumor with a high morbidity. We aimed to explore the function and potential mechanism of α-E-catenin (CTNNA1) in BLCA.MethodsThe CTNNA1 expression in BLCA tissues was detected using qRT-PCR and immunohistochemistry. QRT-PCR and Western blot were performed to measure the CTNNA1 expression in BLCA cell lines. CTNNA1 expression was up-regulated in T24 and UMUC-2 cells by CTNNA1 overexpression plasmid transfection. Cell proliferation, apoptosis, migration and invasion were respectively assessed by CCK-8 assay, flow cytometry, wound healing assay and transwell assay. The expression levels of epithelial–mesenchymal transition (EMT)-related factors were tested by qRT-PCR and Western blot. BLCA nude mice models were constructed to explore the effects of CTNNA1 on BLCA in vivo. Gene set enrichment analysis (GSEA) was proceeded to identify the CTNNA1-related pathways in BLCA.ResultsThe expressions of CTNNA1 were down-regulated in BLCA tissues and cell lines, and its low expression indicated poor prognosis of BLCA patients. CTNNA1 inhibited cell proliferation, migration, invasion and EMT and promoted cell apoptosis in BLCA cells. CTNNA1 enhanced E-cadherin expression and suppressed N-cadherin, snail, MMP2 and MMP9 expressions in BLCA cells, which suggested that CTNNA1 repressed EMT in BLCA cells. Moreover, CTNNA1 could inhibit tumor growth in vivo. CTNNA1 was positively associated with P53 and apoptosis pathways in BLCA cells.ConclusionCTNNA1 inhibited cell proliferation, migration, invasion and EMT and promoted cell apoptosis in BLCA via activating P53 and apoptosis pathways. CTNNA1 might be a novel target in BLCA therapy.

LncRNA CASC9 sponges miR‑758‑3p to promote proliferation and EMT in bladder cancer by upregulating TGF‑β2.

The long noncoding RNA cancer susceptibility candidate 9 (CASC9) has been revealed to be an oncogenic gene in several types of cancer, and high CASC9 expression is related to tumorigenesis and cancer progression. However, the role of CASC9 in bladder cancer (BC), particularly during epithelial-mesenchymal transition (EMT), has not been characterized. RT-qPCR, EdU, CCK-8, wound scratch, Transwell and flow cytometric assays were performed to detect CASC9 expression, miR-758-3p expression and their functions in BC. RNA FISH was used to detect CASC9 subcellular localization. Luciferase reporter assay, RT-qPCR assay and western blotting were used to explore the relationship of CASC9, miR-758-3p and TGF-β2. In the present study, it was revealed that CASC9 regulated EMT in BC. CASC9 expression was significantly upregulated in BC cell lines and specimens compared to that in adjacent normal bladder tissues. Upregulated CASC9 was associated with increased invasion ability and poor prognosis of BC. CASC9 knockdown inhibited BC cell proliferation, migration and invasion. Furthermore, a bioinformatics study and luciferase reporter assays revealed that CASC9 functioned as a ceRNA for miR-758-3p. CASC9 inhibited microRNA (miR)-758-3p activity and resulted in the de-suppression of its target transforming growth factor (TGF)-β2. TGF-β signaling driven by TGF-β2 was crucial for CASC9 to promote EMT in BC. Collectively, these results indicated that CASC9 sponged miR-758-3p to regulate the expression of TGF-β2, which activated the TGF-β signaling pathway and promoted proliferation and EMT in BC.

Transcription factor YY1 mediates epithelial-mesenchymal transition through the TGFβ signaling pathway in bladder cancer.

Bladder cancer is one of the most aggressive urothelial tumors. Previous studies have suggested that epithelial-mesenchymal transition (EMT) contributes to bladder cancer progression. However, the regulatory network of EMT in bladder cancer remains elusive. In this study, we found Yin Yang 1 (YY1) is a critical regulator of EMT in bladder cancer. First, we showed that YY1 was upregulated in bladder cancer tissues than that in adjacent normal tissues. Then, we proved that YY1 promoted EMT of bladder cancer cells. Further experiments indicated that YY1 affected the EMT of bladder cancer through transforming growth factor-β (TGFβ) signaling pathway. Taken together, our study identifies YY1 as a key EMT driver in bladder cancer, suggesting it as a potential therapeutic target.

HMGN5 promotes IL-6-induced epithelial-mesenchymal transition of bladder cancer by interacting with Hsp27.

Bladder cancer (BC) is one of the most common cancers worldwide, with a high rate of recurrence and poor outcomes. High-mobility group nucleosome-binding domain 5 (HMGN5) is overexpressed in many cancers and could cause carcinogenesis in BC. By protein-protein-interaction (PPI) analysis, we found that heat shock protein 27 (Hsp27), also a crucial functional factor in BC carcinogenesis, is significantly related to HMGN5. Hsp27 is required for IL-6-mediated EMT via STAT3/Twist signaling in prostate cancer. Here, we hypothesize that HMGN5 may interact with Hsp27 to affect IL-6-induced EMT and invasion in BC via STAT3 signaling. In the present study, we found that HMGN5 and Hsp27 are highly expressed in BC tissues and positively correlated with each other. HMGN5 interacts with Hsp27 in vitro, to modulate the cell invasion and EMT in BC. Moreover, HMGN5 could modulate IL-6-Hsp27-induced EMT and invasion in BC cells by regulating STAT3 phosphorylation and STAT3 targeting of the Twist promoter. HMGN5 interacts with Hsp27 to promote tumor growth in a human BC xenograft model in nude mice. In summary, HMGN5 interacts with Hsp27 to promote IL-6-induced EMT, therefore promoting invasion in BC and contributing to the progression of BC.

HnRNP-F regulates EMT in bladder cancer by mediating the stabilization of Snail1 mRNA by binding to its 3' UTR.

BackgroundHeterogeneous nuclear ribonucleoprotein F (hnRNP-F) has been implicated in multiple cancers, suggesting its role in tumourigenesis, but the potential oncogenic role and mechanism of hnRNP-F in bladder cancer (BC) remain incompletely understood.MethodsHnRNP-F was identified by proteomic methods. A correlation of hnRNP-F expression with prognosis was analysed in 103 BC patients. Then, we applied in vitro and in vivo methods to reveal the behaviours of hnRNP-F in BC tumourigenesis. Furthermore, the interaction between hnRNP-F and Snail1 mRNA was examined by RNA immunoprecipitation (RIP), and Snail1 mRNA stability was measured after treatment with actinomycin D. Finally, the binding domain between hnRNP-F and Snail1 mRNA was verified by constructing Snail1 mRNA truncations and mutants.FindingHnRNP-F is significantly upregulated in BC tissue, and its increased expression is associated with a poor prognosis in BC patients. HnRNP-F is necessary for tumour growth, inducing epithelial-mesenchymal transition (EMT) and metastasis in BC. The changes in Snail1 expression were positively correlated with hnRNP-F at both the mRNA and protein levels when hnRNP-F was silenced or enhanced, suggesting that Snail1 is likely a downstream target of hnRNP-F that mediates its effects on enhancing invasion, metastasis and EMT in BC. The overexpression of hnRNP-F caused an increase in the stability of Snail1 mRNA. Our RNA chip analysis revealed that hnRNP-F could combine with Snail1 mRNA, and we further demonstrated that hnRNP-F could directly bind to the 3′ untranslated region (3′ UTR) of Snail1 mRNA to enhance its stability.InterpretationOur findings suggest that hnRNP-F mediates the stabilization of Snail1 mRNA by binding to its 3′ UTR, subsequently regulating EMT.

Upregulation of NDRG1 predicts poor outcome and facilitates disease progression by influencing the EMT process in bladder cancer

N-myc downstream regulated gene 1 (NDRG1) is an intracellular protein involved in cell differentiation and was recently reported to exert various effects in several cancers. However, its expression and role in bladder cancer remain unclear. Our study enrolled 100 bladder cancer patients to detect NDRG1 expression in tumour tissues by immunohistochemistry. Correlations between NDRG1 expression and clinical factors were analysed. An NDRG1 overexpression plasmid and NDRG1 siRNAs were transfected into bladder cancer cell lines. Cell biological behaviours were assessed by CCK-8, flow cytometry, wound healing and Transwell assays. Additionally, the influence of NDRG1 on epithelial-mesenchymal transition (EMT) was investigated by western blotting and real-time PCR. NDRG1 expression in urine from bladder cancer patients was examined by ELISA. NDRG1 protein levels were significantly increased in bladder cancer patients and correlated with tumour stage (p = 0.025), lymph node metastasis (p = 0.034) and overall survival (p = 0.016). Patients with high NDRG1 expression had poorer outcomes than those with low NDRG1 expression. NDRG1 overexpression was associated with increased cell proliferation, migration, and invasion and decreased apoptotic cell numbers; NDRG1 knockdown resulted in the inverse effects. Moreover, upregulated NDRG1 expression was associated with downregulated Cytokeratin 7 and Claudin-1 expression and upregulated N-cad, β-catenin and slug expression. Downregulated NDRG1 expression was associated with the inverse effects. Urine protein levels could distinguish bladder cancer patients from healthy controls, with an area under the curve of 0.909. NDRG1 promoted EMT in bladder cancer and could be an effective diagnostic and prognostic biomarker in bladder cancer patients.

MicroRNA-338-3p inhibits the progression of bladder cancer through regulating ETS1 expression.

MicroRNA-338-3p (miR-338-3p) was reported to influence the metastasis and development of several human cancers. However, in bladder cancer (BC), the special function of miR-338-3p remains unknown. Here, we aimed at exploring the miR-338-3p function in the progression of BC. miR-338-3p and ETS1 expressions were examined by quantitative Real-time polymerase chain reaction (qRT-PCR) in BC samples. Following that, transwell assays for cell migration and invasion were performed. And MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay for cell proliferation was conducted as well. Western blot was employed to examine the epithelial-mesenchymal transition (EMT) marker expressions. Finally, the relationship between miR-338-3p and E26 transformation specific-1 (ETS1) was verified by luciferase reporter assay. The decreased miR-338-3p expression was examined in BC cells. Moreover, miR-338-3p upregulation repressed cell proliferation ability in BC. Next, miR-338-3p upregulation also depressed cell metastasis and EMT in BC cells. Furthermore, ETS1 was a direct target of miR-338-3p and inversely associated with its expression. And upregulation of ETS1 partially rescued the suppression of miR-338-3p for cell proliferation and metastasis in BC. Upregulation of miR-338-3p inhibited the proliferation, metastasis and EMT in BC by suppressing ETS, showing that miR-338-3p might block the development of BC through regulating ETS1 expression.

HnRNP-F Regulates EMT in Bladder Cancer by Mediating the Stabilization of Snail mRNA Through Binding to Its 3'UTR

Background: Heterogeneous nuclear ribonucleoprotein F (HnRNP-F) has previously been implicated in multiple cancers, suggesting its roles in tumorigenesis, but the potential oncogenic role and mechanism of hnRNP-F in bladder cancer (BC) remains incompletely understood. Methods: HnRNP-F was identified by proteomic methods. A correlation of hnRNP-F expression with the prognosis was analyzed in103 BC patients. Then, we applied in vitro and in vivo methods to reveal the behaviors of hnRNP-F in BC tumorigenesis. Furthermore, the interaction between hnRNP-F and Snail mRNA was examined by RNA immunopreciptiation (RIP), and Snail mRNA stability was measured after treatment with actinomycin D. Finally, the binding domain between hnRNP-F and Snail mRNA was tested through Snail mRNA truncations and mutants. Finding: HnRNP-F is significantly up-regulated in BC tissue, and its increased expression is associated with poor prognosis in BC patients. HnRNP-F was necessary for tumor growth, inducing Epithelial-mesenchymal transition (EMT) and metastasis in BC. The changes of Snail expression were positively consistent with hnRNP-F in both mRNA and protein levels when hnRNP-F was silenced or enhanced, suggesting that Snail is likely to be the downstream target of hnRNP-F that mediates its effects on enhancing invasion, metastasis and EMT in BC. Overexpression of hnRNP-F caused an increase in the stability of the Snail mRNA was enhanced. Our RNA chip analysis revealed that hnRNP-F could combine with the Snail mRNA, and we further demonstrated that hnRNP-F could directly bind to the 3'untranslated region (3'UTR) of Snail mRNA to enhance its stability. Interpretation: Our findings suggest that hnRNP-F mediates the stabilization of Snail mRNA via binding to its 3'UTR, subsequently regulating EMT. Funding Statement: This study was supported by Natural Science Foundation Committee of China (NSFC 81802941) (F.L.), and the Natural Science Foundation of Guangdong Province of China (2018A0303130287) (F.L.). Declaration of Interests: The authors declare no conflicts of interest. Ethics Approval Statement: All patients were given written informed consent and all procedures were approved by bioethics committee of the authors' hospital.

Effect of microRNA-497-5p on epithelial-to-mesenchymal transition in bladder cancer

Objective To observe the expression of microRNA-497 (miR-497) in bladder cancer cells and investigate its possible functions in epithelial-mesenchymal transition (EMT) of bladder cancer. Methods The expression of miR-497 in bladder cells and human normal bladder tissue were measured by real-time quantitative polymerase chain reaction (RT-qPCR). MiR-497 mimics and miR-497 inhibitor were transfected into bladder cancer EJ cells and RT-qPCR was used to analyze the effect of transfection. Then, morphological changes of cells were observed under the inverted microscope and the wound healing assay, transwell invasion assay were used to detect the cell migration and invasion of bladder cancer EJ cells transfected by miR-497 mimics and miR-497 inhibitor. cytometry. Finally, Western blotting was applied to detect the levels of EMT-related proteins. Results MiR-497 levels were obviously down-regulated in bladder cancer cells (5637, EJ, BIU-87, TCCSUP and T24) in comparison to human bladder pillon biochemical cell (SV-HUC-1), the difference was statistically significant (P=0.000). Compared with negative control, overexpression of miR-497 significantly suppressed EJ cells migration [ (74.00±3.61)% vs. (25.33±6.11)%] and invasion [ (219±17) cells vs. (109±3) cells], and EJ cells turned into epithelioid cells morphology, when compared with the control group. Moreover, opposite results were obtained when miR-497 inhibitor was transfected into EJ cells. Compared with mimics NC, the protein expression of E-cadherin was upregulated (P=0.000), while N-cadherin and vimentin downregulated (P=0.001, P=0.000)in miR-497 mimics transfection group. Conclusion MiR-497 may play an important role of EMT in bladder cancer. Key words: MicroRNA-497; Bladder cancer; Migration; Invasion; Epithelial-to-mesenchymal transition

Epithelial–mesenchymal transition promotes SOX2 and NANOG expression in bladder cancer

Bladder cancer is the most common malignant tumor of the urothelium and is classified into non-muscle-invasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC). Stemness markers such as SOX2 and NANOG are frequently overexpressed in various aggressive cancers, including MIBC; epithelial–mesenchymal transition (EMT) has been proposed as a potential trigger of stemness in cancers. To determine whether cancer stemness is acquired via EMT in bladder cancer, we studied the effect of EMT on the expression of SOX2 and NANOG in bladder cancer cell lines. We also analyzed their expression in clinical tissue samples. Our results revealed that a potent EMT inducer (transforming growth factor β1) reduced the expression of the epithelial marker E-cadherin and increased expression of both SOX2 and NANOG in epithelial-type bladder cancer cells. As for clinical bladder cancer samples, in NMIBC, E-cadherin expression was slightly diminished, and the expression of both SOX2 and NANOG was negligible. In contrast, in MIBC, E-cadherin expression was highly and heterogeneously diminished, while the expression of both SOX2 and NANOG was increased. We also noticed that either E-cadherin or SOX2 (or NANOG) was expressed (ie, in a manner exclusive of each other). In addition, the concentration of E-cadherin showed a significant negative correlation with tumor grade and stage, while expression of SOX2 and NANOG positively correlated with those clinicopathological parameters. These findings suggest that EMT promotes stemness of bladder cancer cells, contributing to tumor aggressiveness. This EMT–cancer stemness axis may also play an important role in the pathogenesis of NMIBC and MIBC.

Downregulation of pigment epithelium-derived factor is associated with increased epithelial-mesenchymal transition in bladder cancer.

Pigment epithelium-derived factor (PEDF) has been reported to inhibit angiogenesis in bladder cancer, but whether it modulates epithelial-mesenchymal transition (EMT) remains unknown. We have included 68 samples of non-muscle invasive bladder cancer in this study. Immunohistochemistry was performed in all sample sections for expressions of PEDF, SNAI2, Vimentin and E-cadherin, which were quantified and The EMT Index (EMTi) was calculated by Vimentin/E-cadherin. Levels of expression were studied for correlations with clinicopathological parameters. Patients were grouped with high (EMTi≤1) and low (EMTi>1) EMT level for comparisons of clinicopathological parameters. In the univariate analysis, EMT level was shown to be significantly associated with tumor onset and PEDF level, but not with gender, occurrence, stage, grade, or SNAI2 level. Further multivariate analysis revealed that PEDF level was significantly negatively correlated with EMTi independent of other clinicopathological parameters. Our results indicate that PEDF may serve as a promising novel modality for the treatment of bladder cancer, as it can inhibit not only angiogenesis but also EMT process of bladder cancer.