Human Bladder Cancer Tissues Research Articles

Circ_0071662, a novel tumor biomarker, suppresses bladder cancer cell proliferation and invasion by sponging miR-146b-3p.

Here, we characterized a new circRNA, named circ_0071662, which was downregulated in human bladder cancer tissues and cell lines, compared with matched adjacent normal tissues and normal bladder epithelial cells. Lower circ_0071662 level was observed in patients with advanced bladder cancer and was positively associated with poorer prognosis, including higher degrees of lymph node invasion and distal metastasis, and lower survival rate. Gain- and loss-of-functions showed that circ_0071662 suppressed cell proliferation and invasion in human bladder cell lines T-24 and J82. Bioinformatics analysis revealed that there are six binding sites of miR-146b-3p on circ_0071662 sequence, and pull-down assays demonstrated miR-146b-3p directly bound with circ_0071662. Moreover, circ_0071662 negatively regulated miR-146b-3p expression and positively regulated expression of miR-146b-3p target genes HPGD and NF2. Furthermore, miR-146b-3p could rescue the inhibition of cell proliferation and invasion caused by circ_0071662 overexpression. In conclusion, circ_0071662 suppresses bladder cancer cell proliferation and invasion by sponging miR-146b-3p.

XPC deficiency leads to centrosome amplification by inhibiting BRCA1 expression upon cisplatin-mediated DNA damage in human bladder cancer

Xeroderma pigmentosum group C (XPC) is a well-known DNA damage recognition protein. Defects in XPC lead to carcinogenesis and progression of many human cancers. In the current study, we defined a novel, important role of XPC in preventing centrosome amplification during cisplatin-mediated DNA damage response. From experiments with human bladder cancer tissue, urothelial tissue from Xpc knockout mice and XPC-silenced cell lines, we found that attenuated XPC expression was associated with increased centrosome amplification in human bladder cancer. A significant increase in centrosome amplification was observed in XPC-silenced cells upon cisplatin treatment. XPC deficiency leads to reduced BRCA1 expression via upregulating its transcriptional repressor, Pit-1. The BRCA1 downregulation results in more DNA double strand breaks accumulation and persistent activation of the ATM-Chk1/Chk2 signaling, resulting in a prolonged G2/M arrest during which centrosome can over-duplicate and lead to centrosome amplification. XPC complementation in silenced cells could reduce Pit-1 expression, increase BRCA1 expression and recover the status of centrosome amplification. Our study reveals a new function for XPC in preventing chromosomal instability, providing new information on cancer chemotherapy and potential clinical significance for cancer management.

Suppressed OGT expression inhibits cell proliferation while inducing cell apoptosis in bladder cancer

BackgroundThis study aimed to explore hyper-O-linked N-acetylglucosaminylation (O-GlcNAcylation) with an elevation of the expression of O-linked-β-N-acetylglucosamine transferase (OGT) in human bladder cancer.MethodsImmunohistochemical staining for OGT and O-GlcNAcylation was performed in 20 paired human bladder cancer and adjacent normal tissues, as well as in human bladder cancer tissue microarrays (N = 169). The expression level of OGT and O-GlcNAcylation in cell lines were detected using the Western blot analysis. The effects of O-GlcNAcylation on the cell proliferation of bladder cancer were detected using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and clone formation assays. Cell apoptosis and cell cycle analysis were detected using flow cytometry. The autophagy of bladder cancer cells was investigated using the Western blot analysis, and GFP–LC3 plasmid was used to detect the autophagic flux. MTT assay was performed to detect the sensitivity of bladder cancer cells to cisplatin after OGT knockdown.ResultsThe expression of OGT and the O-GlcNAcylation were upregulated in bladder cancer tissues and cell lines. O-GlcNAcylation and OGT were observed in nucleus and cytoplasm and found to be higher in muscle-invasive bladder cancer (MIBC) than in non-muscle-invasive bladder cancer (NMIBC). Reducing hyper-O-GlcNAcylation by OGT knockdown inhibited the proliferation of bladder cancer cells in vitro and xenograft tumor growth in vivo, triggered apoptosis, as well as led to cell cycle arrest. It also increased autophagy in bladder cancer cells. This study demonstrated increased autophagy pro-survival, but not pro-death. Reducing hyper-O-GlcNAcylation by OGT knockdown facilitated the chemosensitivity of bladder cancer cells to cis-platinum.ConclusionsThe data indicated that hyper-O-GlcNAcylation enhanced oncogenic phenotypes and was involved in DNA damage response in bladder cancer.

Protein arginine methyltransferase 5 promotes bladder cancer growth through inhibiting NF-kB dependent apoptosis.

Protein arginine methyltransferase 5 (PRMT5) has emerged as a key regulator of tumorigenesis. However, how PRMT5 functions in bladder cancer, the most common malignancy of the urological system, is unknown. We described here that PRMT5 is highly expressed in bladder cancer cell lines and primary human bladder cancer tissues. PRMT5 enhances the proliferation and colony formation of bladder cancer cells. PRMT5 knockdown induces bladder cancer cell apoptosis. Mechanistically, PRMT5 enhances NF-kB activation by targeting crucial anti-apoptotic genes such as BCLXL and c-IAP1, thereby inhibiting tumor cell apoptosis and sustaining proliferation. Importantly, PRMT5 inhibitor opposed tumor growth and BCLXL and c-IAP1 transcription in the bladder cancer xenograft model. Collectively, the current suggests the crucial role of PRMT5 as a promising therapeutic target in bladder cancers.

Hypermethylated in cancer 1 (HIC1) suppresses bladder cancer progression by targeting yes-associated protein (YAP) pathway.

Bladder cancer (BCa) is the most common malignant tumor in the urinary system. Growing evidence suggests that as a tumor suppressor gene, hypermethylated in cancer 1 (HIC1) is correlated with various malignancies in the modulation of tumor progression. This study aims to investigate the effect of HIC1 on regulating the proliferation, migration, and invasion of BCa. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot (WB) were used to evaluate the expression of HIC1 messenger RNA and protein in human BCa tissues and cells. Proliferation, migration and invasion assays, and flow cytometry assay were performed to assess the biological functional role of HIC1 in BCa. Co-immunoprecipitation (Co-IP) examined the protein-protein interaction. The signaling pathways involved in the mode of action of HIC1 in BCa were also investigated. HIC1 was found downregulated in tested samples. Cloning formation assay and cell-proliferation activity analysis showed that overexpression of HIC1 significantly inhibited the proliferation of BCa cells, while knockdown led to the opposite, namely the promotion of the proliferation. Flow cytometry assay confirmed the arrest of the cell cycle at the G1 phase with overexpression of HIC1 observed. Moreover, HIC1 inhibited migration and invasion of BCa. Co-IP showed the binding between YAP (yes-associated protein) and TEAD (TEA domain/transcription enhancer factor family members) as well as the cancerostatic activity of HIC1, partially manifested via its negative regulation of YAP signaling pathway. Our data unprecedently showed that HIC1 was responsible for the inhibition of proliferation, migration, and invasion of BCa via the YAP signaling pathway. These findings suggested that therapeutic strategies regulating HIC1 expression might provide effective treatments for BCa.

LIMK2 acts as an oncogene in bladder cancer and its functional SNP in the microRNA-135a binding site affects bladder cancer risk.

LIM kinases modulate multiple aspects of cancer development, including cell proliferation and survival. As the mechanisms of LIMK‐associated tumorigenesis are still unclear, we analyzed the tumorigenic functions of LIM kinase 2 (LIMK2) in human bladder cancer (BC) and explored whether the newly identified LIMK2 3´‐UTR SNP rs2073859 (G‐to‐A allele) is correlated with clinical features. Expression levels of LIMK2 in 38 human BC tissues and eight cell lines were examined using quantitative real‐time PCR and immunohistochemistry. LIMK2 was overexpressed in most BC tissues (27/38, 71%) and BC‐derived cell lines (6/8), and was more frequently overexpessed in high‐grade than low‐grade BC (80% vs. 47%). The effects of LIMK2 on BC cell proliferation, survival and migration, were studied by overexpression and RNA interference approaches in vitro and in vivo. LIMK2 overexpression promoted proliferation, migration and invasion of BC cells, while LIMK2 depletion inhibited cell invasion and viability and induced growth arrest in vitro and in vivo. PCR‐Restriction Fragment Length Polymorphism (RFLP) was used to genotype LIMK2 SNP rs2073859 and multivariate logistic regression applied to assess the relationship between allele frequency and clinical features in 139 BC patients. Functional analyses localized SNP rs2073859 within the microRNA‐135a seed‐binding region and revealed significantly lower LIMK2 G allele expression. The frequency of A genotypes (AG + AA) was higher in the BC group than normal controls and correlated with risks of high‐grade and high‐stage BC. In conclusion, LIMK2 may function as an oncogene in human BC, while allele‐specific regulation by microRNA‐135a may influence disease risk.

An ex vivo Tissue Culture Model for the Assessment of Individualized Drug Responses in Prostate and Bladder Cancer.

Urological malignancies, including prostate and bladder carcinoma, represent a major clinical problem due to the frequent occurrence of therapy resistance and the formation of incurable distant metastases. As a result, there is an urgent need for versatile and predictive disease models for the assessment of the individualized drug response in urological malignancies. Compound testing on ex vivo cultured patient-derived tumor tissues could represent a promising approach. In this study, we have optimized an ex vivo culture system of explanted human prostate and bladder tumors derived from clinical specimens and human cancer cell lines xenografted in mice. The explanted and cultured tumor slices remained viable and tissue architecture could be maintained for up to 10 days of culture. Treatment of ex vivo cultured human prostate and bladder cancer tissues with docetaxel and gemcitabine, respectively, resulted in a dose-dependent anti-tumor response. The dose-dependent decrease in tumor cells upon administration of the chemotherapeutic agents was preceded by an induction of apoptosis. The implementation and optimization of the tissue slice technology may facilitate the assessment of anti-tumor efficacies of existing and candidate pharmacological agents in the complex multicellular neoplastic tissues from prostate and bladder cancer patients. Our model represents a versatile “near-patient” tool to determine tumor-targeted and/or stroma-mediated anti-neoplastic responses, thus contributing to the field of personalized therapeutics.

High LINC01605 expression predicts poor prognosis and promotes tumor progression via up-regulation of MMP9 in bladder cancer.

The advent of high-throughput sequencing methods has facilitated identification of novel long non-coding RNAs (lncRNAs), which have been demonstrated to play an important role in multiple tumors. Moreover, with the assistance of bioinformatics analysis, LINC01605 has been found to be up-regulated in bladder cancer (BC) tissues compared with normal tissues. Hence, the present study was to explore its specific biological role and related mechanism in BC. The relative expression level of LINC01605 was measured in a cohort of BC tissues with matched normal tissues as well as human BC cell lines by quantitative real-time PCR (qRT-PCR). Survival analysis was performed to explore the relationship between LINC01605 expression and the prognosis of BC patients. The biological function of LINC01605 was studied in vitroand in vivo, by means of CCK-8 assay, colony formation assay, transwell assay, and tumor xenografts mice model. LINC01605 was found to be frequently highly expressed in both human BC cells and tissues. Survival analysis indicated that high LINC01605 expression was associated with higher histological grade and clinical stages. In addition, down-regulated LINC01605 in BC cells could significantly inhibit the abilities of proliferation, migration, and invasion in vitro and knockdown of LINC01605 in subcutaneous xenograft tumor model could impede tumorigenesis in vivo. Mechanistically, LINC01605 could activate epithelial–mesenchymal transition (EMT) signaling pathway and promote the expression of matrix metallopeptidase (MMP) 9 (MMP9). In summary, our results shed light on that LINC01605, as a new prognostic biomarker, could promote the proliferation, migration, and invasion of BC cells via activating EMT signaling pathway and up-regulating MMP9 expression.

Amyloid precursor protein is overexpressed in bladder cancer and contributes to the malignant bladder cancer cell behaviors.

To investigate the expression of amyloid precursor protein in bladder cancer, and to study its role in malignant bladder cancer cell behaviors. Immunohistochemistry and western blotting analysis were used to detect the amyloid precursor protein level in bladder cancer tissues and cell lines. The effect of amyloid precursor protein on bladder cancer cell proliferation, migration, invasion and cell cycle was evaluated by using small interfering ribonucleic acid. The levels of RAS, RAF, MEK, phosphorylated MEK, extracellular regulated protein kinases, phosphorylated extracellular regulated protein kinases, protein kinase B and phosphorylated protein kinase B were determined by western blot after amyloid precursor protein knockdown. The effect of amyloid precursor protein on the extracellular regulated protein kinases pathway was further evaluated using extracellular regulated protein kinases pathway agonist, ceramide C6. The expression of amyloid precursor protein was significantly increased in the human bladder cancer tissues compared with matched normal bladder tissues. The overexpression of amyloid precursor protein was significantly associated with tumor stage, tumor size, histological grade and lymph node metastasis. The proliferation, migration and invasion of human bladder cancer cells were significantly inhibited by the silencing of amyloid precursor protein. In addition, knockdown of amyloid precursor protein arrested the cell cycle progression of bladder cancer cells in the G2/M phase. Mechanistic analysis showed that knockdown of amyloid precursor protein significantly decreased the phosphorylation of extracellular regulated protein kinases. Ceramide C6 could rescue the malignant bladder cancer cell behaviors inhibited by the silencing of amyloid precursor protein. Amyloid precursor protein is upregulated in bladder cancer, and might be closely associated with bladder cancer cell growth and survival. Amyloid precursor protein could potentially be used as a therapeutic target for bladder cancer treatment.

High-Performance Identification of Human Bladder Cancer Using a Signal Self-Amplifiable Photoacoustic Nanoprobe.

Cancer diagnostics has been an important research field, and identification of small lesions that are less noticeable plays a vital role in thoroughly removing the tumor, thereby reducing the recurrence rate of cancer. Herein, we synthesized a signal self-amplifiable photoacoustic (PA) liposomal nanoprobe composed by ammonium hydrogen carbonate (AHC) payload and aggregated purpurin-18 (P18) within the bilayer. Under PA laser irradiation, P18 aggregates efficiently generated local heat, leading to the launch of wide-band ultrasonic emission. In parallel, the heat also triggered the decomposition of AHC and production of CO2 bubbles, which consequently dramatically amplified the acoustic signal. For clinical translation, by decorating bladder cancer (BC) specific CD44v6 antibody onto nanoprobe, we were capable of utilizing this high sensitive and specific PA probe for human BC tissue imaging. The results indicated that small tumor lesion (<5 mm) was identified and the tumor-to-normal tissue ratio was ∼18 folds enhancement by using this PA probe, which rendered the tumor boundary distinct. All together, we developed a new strategy for exploring high-performance imaging probes, which might potentially benefit for the imaging-guided surgery in clinics.

ISO, via Upregulating MiR-137 Transcription, Inhibits GSK3β-HSP70-MMP-2 Axis, Resulting in Attenuating Urothelial Cancer Invasion

Our most recent studies demonstrate that miR-137 is downregulated in human bladder cancer (BC) tissues, while treatment of human BC cells with isorhapontigenin (ISO) elevates miR-137 abundance. Since ISO showed a strong inhibition of invasive BC formation in the N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN)-induced invasive BC mouse model, the elucidation of a potential biological effect of miR-137 on antagonizing BC invasion and molecular mechanisms underlying ISO upregulation of miR-137 are very important. Here we discovered that ectopic expression of miR-137 led to specific inhibition of BC invasion in human high-grade BC T24T and UMUC3 cells, while miR-137 deletion promoted the invasion of both cells, indicating the inhibitory effect of miR-137 on human BC invasion. Mechanistic studies revealed that ISO treatment induced miR-137 transcription by promoting c-Jun phosphorylation and, in turn, abolishing matrix metalloproteinase-2 (MMP-2) abundance and invasion in BC cells. Moreover, miR-137 was able to directly bind to the 3′ UTR of Glycogen synthase kinase-3β (GSK3β) mRNA and inhibit GSK3β protein translation, consequently leading to a reduction of heat shock protein-70 (HSP70) translation via targeting the mTOR/S6 axis. Collectively, our studies discover an unknown function of miR-137, directly targeting the 3′ UTR of GSK3β mRNA and, thereby, inhibiting GSK3β protein translation, mTOR/S6 activation, and HSP70 protein translation and, consequently, attenuating HSP70-mediated MMP-2 expression and invasion in human BC cells. These novel discoveries provide a deep insight into understanding the biomedical significance of miR-137 downregulation in invasive human BCs and the anti-cancer effect of ISO treatment on mouse invasive BC formation.

Abstract 499: MicroRNA-411 downregulation promotes tumorigenesis and inhibits chemotherapy sensitivity by upregulating MLLT11 expression in human bladder cancer

Abstract Although several previous studies have reported the implication of various microRNAs (miRNAs) in regulation of human bladder cancer (BC) development, alterations and function of many miRNAs in BC carcinogenesis and anti-cancer therapy are not explored yet at present. Here, we screened 1900 known miRNAs and first discovered that miR-411 was one of major miRNAs that was downregulated in a n-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN)-induced BC mouse model. This miR-411 downregulation was also observed in human BC tissues and cell lines. The results from evaluating the relationship between miR-411 and patient survival in BC using the TCGA database also indicated that miR-411 was positively correlated with survival (&amp;gt;3 years). Our studies also showed that miR-411 inhibited tumorigenesis and increased chemotherapy sensitivity in human BC. Mechanistic studies revealed that overexpression of miR-411 repressed expression of ALL1-fused gene from the chromosome 1q (MLLT11) by binding to the 3′-untranslated region of MLLT11 mRNA, induced p21 expression, and caused cell cycle arrest at the G2/M phase, in turn inhibiting BC tumorigenic growth. Ectopic expression of miR-411 also increased the sensitivity of BC cells to the chemotherapeutic agents, whereas MLLT11 restoration in miR-411-overexpressing cells increased resistance to the chemotherapeutic agents. Citation Format: Honglei Jin, Haishan Huang, Chuanshu Huang, Xiaohui Hua, Junlan Zhu. MicroRNA-411 downregulation promotes tumorigenesis and inhibits chemotherapy sensitivity by upregulating MLLT11 expression in human bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 499.

MiR-192-5p suppresses the growth of bladder cancer cells via targeting Yin Yang 1.

Bladder cancer has been identified as one of the most malignant cancers with high incidence and mortality. The underlying mechanisms by which regulate the tumorigenesis of bladder cancer deserve further investigation. Here, we found that miR-192-5p was downregulated in human bladder cancer cell lines and tissues. Overexpression of miR-192-5p significantly inhibited the growth of bladder cancer cells, while depletion of miR-192-5p exerted opposite effect. Bioinformatics analysis and molecular mechanism study identified that miR-192-5p targeted the transcription factor Yin Yang 1 (YY1) and decreased the expression level of YY1. Highly expressed YY1 attenuated the potential tumor suppressive function of miR-192-5p. The expression of miR-192-5p was negatively correlated with that of YY1 in bladder cancer tissues. These results indicated that miR-192-5p might serve as a promising target in bladder cancer diagnosis and therapy.

LINE-1 ORF1 Protein Is Up-regulated by Reactive Oxygen Species and Associated with Bladder Urothelial Carcinoma Progression.

Reactivation of long interspersed nuclear element-1 (LINE-1) and oxidative stress are suggested to have oncogenic potential to drive tumorigenesis and cancer progression. We previously demonstrated that reactive oxygen species (ROS) caused hypomethylation of LINE-1 elements in bladder cancer cells. In this study, we investigated the expression of LINE-1-encoded protein (ORF1p) and oxidative stress marker 4-hydroxynonenal (4-HNE) in human bladder cancer tissues, as well as induction of ORF1p expression by ROS in bladder cancer cell lines. Thirty-six cancerous and 15 non-cancerous adjacent tissues were immunohistochemically stained for ORF1p and 4-HNE. ORF1p expression and cell migration were determined in bladder cancer cells exposed to H2O2 Results: ORF1p and 4-HNE expression was higher in cancerous than non-cancerous tissues. Elevated ORF1p expression was associated with increased 4-HNE expression and with advanced tumors. H2O2 provoked oxidative stress and up-regulated ORF1p expression in VM-CUB-1 compared to the untreated control, and to a lesser degree in TCCSUP. H2O2 exposure enhanced cell migration in UM-UC-3, TCCSUP and VM-CUB-1. Elevated ORF1p expression is associated with tumor progression. ROS experimentally induce ORF1p expression and promote migration in bladder cancer cells.

MicroRNA-411 Downregulation Enhances Tumor Growth by Upregulating MLLT11 Expression in Human Bladder Cancer

Although several previous studies have reported the implication of various microRNAs (miRNAs) in regulation of human bladder cancer (BC) development, alterations and function of many miRNAs in bladder cancer growth are not explored yet at present. Here, we screened 1,900 known miRNAs and first discovered that miR-411 was one of the major miRNAs, which was down-regulated in n-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN)-induced BCs. This miR-411 down-regulation was also observed in human BC tissues and cell lines. The results from evaluating the relationship between miR-411 and patient survival in BC using the TCGA (The Cancer Genome Atlas) database indicated that miR-411 was positively correlated with DFS (disease-free survival). Our studies also showed that miR-411 inhibited tumor growth of human BC cells in a xenograft animal model. Mechanistic studies revealed that overexpression of miR-411 repressed the expression of ALL1-fused gene from the chromosome 1q (AF1q) (MLLT11) by binding to the 3′ untranslated region (UTR) of mllt11 mRNA and in turn induced p21 expression and caused cell cycle arrest at the G2/M phase, further inhibiting BC tumor growth. Collectively, our results improve our understanding of the role of miR-411 in BC tumor growth and suggest miR-411 and MLLT11 as potential new targets for the treatment of BC patients.

BTG2 is a tumor suppressor gene upregulated by p53 and PTEN in human bladder carcinoma cells.

Although widely deemed as a tumor suppressor gene, the role of B‐cell translocation gene 2 (BTG2) in bladder cancer is still inconclusive. We investigated the role and regulatory mechanism of BTG2 in bladder cancer. BTG2 expression in human bladder tissues was determined by RT‐qPCR and immunoblotting assays. Expressions of BTG2 and PTEN in bladder carcinoma cells were determined by immunoblotting, RT‐qPCR, or reporter assays. The 3H‐thymidine incorporation assay, flow cytometry, and the xenograft animal model were used to determine the cell growth. BTG2 expression was lower in human bladder cancer tissues than normal bladder tissues. Highly differentiated bladder cancer cells, RT4, expressed higher BTG2 than the less‐differentiated bladder cancer cells, HT1376 and T24. Overexpression of BTG2 in T24 cells inhibited cell growth in vitro and in vivo. Camptothecin and doxorubicin treatments in RT‐4 cells or transient overexpression of p53 into p53‐mutant HT1376 cells induced p53 and BTG2 expression. Further reporter assays with site‐mutation of p53 response element from GGGAAAGTCC to GGAGTCC within BTG2 promoter area showed that p53‐induced BTG2 gene expression was dependent on the p53 response element. Ectopic PTEN overexpression in T24 cells blocked the Akt signal pathway which attenuated cell growth via upregualtion of BTG2 gene expression, while reverse effect was found in PTEN‐knockdown RT‐4 cells. PTEN activity inhibitor (VO‐OHpic) treatment decreased BTG2 expression in RT‐4 and PTEN‐overexpressed T24 cells. Our results suggested that BTG2 functioned as a bladder cancer tumor suppressor gene, and was induced by p53 and PTEN. Modulation of BTG2 expression seems a promising way to treat human bladder cancer.

TM4SF1 regulates apoptosis, cell cycle and ROS metabolism via the PPARγ-SIRT1 feedback loop in human bladder cancer cells

Transmembrane-4-L-Six-Family-1 (TM4SF1) is a member of the L6 family and functions as a signal transducer to regulate cell development, growth and motility. Here we show that TM4SF1 is strongly upregulated in human muscle invasive bladder cancer (MIBC) tissues, corroborating the bioinformatical results of transcriptome analysis. Moreover, tissue microarray (TMA) shows significant correlations (p < 0.05) between high expression of TM4SF1 and T stage, TNM stage, lymph node metastasis status and survival rate of MIBC patients, indicating a positive association between TM4SF1 expression and poorer prognosis. Furthermore, in vitro and in vivo studies indicate that the proliferation of human bladder cancer (BCa) cells is significantly suppressed by knockdown of TM4SF1 (p < 0.05). Functionally, the reduction of TM4SF1 could induce cell cycle arrest and apoptosis possibly via the upregulation of reactive oxygen species (ROS) in BCa cells. In addition, these observations could be recovered by treatment with GW9662 (antagonist of PPARγ) and resveratrol (activator of SIRT1). Taken together, our results suggest that high expression of TM4SF1 predicts poor prognosis of MIBC.

Anti-oncogenic activities of cyclin D1b siRNA on human bladder cancer cells via induction of apoptosis and suppression of cancer cell stemness and invasiveness.

The human cyclinD1 gene generates two major isoforms, cyclinD1a and cyclinD1b, by alternative splicing. Although cyclin D1b mRNA is hardly expressed in normal human tissues, it is detected in approximately 60% of human bladder cancer tissues and cell lines. In the present study, to assess the therapeutic ability of cyclinD1b siRNA, we investigated the anti-oncogenic effects of cyclinD1b siRNA on human bladder cancer cell lines, SBT31A and T24, which express cyclinD1b mRNA. Knockdown of cyclinD1b by specific siRNA significantly suppressed cell proliferation, invitro cell invasiveness and three-dimensional (3D) spheroid formation in these cell lines. Cell cycle analyses revealed that cyclinD1b siRNA inhibited G1-S transition in T24 cells. The increase in the sub-G1 fraction, morphological aberrant nuclei with nuclear fragmentation and caspase-3 activity in SBA31A cells treated with cyclinD1b siRNA showed that cyclinD1b siRNA induced apoptosis. In T24 cells, knockdown of cyclinD1b suppressed the expression of the stem cell marker CD44. Knockdown of cyclinD1b or CD44 suppressed the invasiveness under 3D spheroid culture conditions and expression of N-cadherin. Tumor growth of SBT31A cells in nude mice was significantly inhibited by cyclinD1b siRNA. Taken together, these results indicate that knockdown of cyclinD1b suppresses the malignant phenotypes of human bladder cancer cells via induction of apoptosis and suppression of cancer cell stemness and epithelial-mesenchymal transition. Applying cyclinD1b siRNA will be a novel therapy for cyclinD1b-expressing bladder cancers.

MicroRNA-154 as a prognostic factor in bladder cancer inhibits cellular malignancy by targeting RSF1 and RUNX2.

Recent studies have demonstrated that microRNA-154 (miR-154) is involved in tumorigenesis, progression, invasion and metastasis in several types of human cancer. However, whether it plays a role in bladder cancer (BC) is unclear. The aim of the present study was to determine miR-154 levels in human BC tissues and investigate the correlation between miR-154 levels and clinicopathological characteristics as well as patient outcome. Using RT-qPCR, we found that the expression levels of miR-154 were significantly lower in BC tissues compared to adjacent normal tissues. We also demonstrated that downregulation of miR-154 was associated with advanced clinicopathological features and worse prognoses for patients with BC. Using a variety of integrated approaches, we demonstrated that both runt-related transcription factor 2 (RUNX2) and remodeling and spacing factor 1 (RSF1) were miR-154 targets. Notably, there was an inverse correlation between RSF1, RUNX2 and miR-154 expression in BC tissues. The biological functions of miR-154 were examined in vitro using Cell Counting Kit-8 (CCK-8), wound healing, and Transwell assays with T24 human bladder carcinoma cells transfected with miR-154 mimics or negative controls. These assays demonstrated that miR-154 significantly suppressed proliferation, migration and invasion of T24 cells (P<0.05). Furthermore, overexpression of RSF1 and RUNX2 rescued miR-154-induced inhibition of these aggressive behaviors. Our results indicated that miR-154, and its downstream targets RSF1 and RUNX2, are promising options for future BC therapies.

CircHIPK3 sponges miR-558 to suppress heparanase expression in bladder cancer cells.

Increasing evidences suggest that circular RNAs (circRNAs) exert crucial functions in regulating gene expression. In this study, we perform RNA-seq and identify 6,154 distinct circRNAs from human bladder cancer and normal bladder tissues. We find that hundreds of circRNAs are significantly dysregulated in human bladder cancer tissues. We further show that circHIPK3, also named bladder cancer-related circular RNA-2 (BCRC-2), is significantly down-regulated in bladder cancer tissues and cell lines, and negatively correlates with bladder cancer grade, invasion as well as lymph node metastasis, respectively. Over-expression of circHIPK3 effectively inhibits migration, invasion, and angiogenesis of bladder cancer cells invitro and suppresses bladder cancer growth and metastasis invivo Mechanistic studies reveal that circHIPK3 contains two critical binding sites for the microRNA miR-558 and can abundantly sponge miR-558 to suppress the expression of heparanase (HPSE). Taken together, our findings provide evidence that circRNAs act as "microRNA sponges", and suggest a new therapeutic target for the treatment of bladder cancer.