Ability Of Bladder Cancer Cells Research Articles

Mesenchymal stem cells in tumor microenvironment: drivers of bladder cancer progression through mitochondrial dynamics and energy production

Mesenchymal stem cells (MSCs) in tumor microenvironment (TME) are crucial for the initiation, development, and metastasis of cancer. The impact and mechanism of MSCs on bladder cancer are uncertain. Here we analyzed 205 patient samples to explore the relationships between tumor-stroma ratio and clinicopathological features. A co-culture model and nude mouse transplantation were used to explore the biological roles and molecular mechanisms of MSCs on bladder cancer cells. We found that a high tumor-stroma ratio was significantly associated with a larger tumor size and higher T stage, pathological grade, number of vascular invasions, and poor overall survival. MSCs in TME promoted the ability of bladder cancer cells to proliferate, migrate, and invade in vitro and in vivo. Next, we demonstrated that MSCs enhance mitochondrial autophagy and mitochondrial biogenesis of bladder cancer cells, and increase energy production, thereby promoting bladder cancer cell progression. Kynurenine (Kyn) produced by MSCs could enhance mitochondrial function by activating the AMPK pathway. IDO1 inhibitor could reverse the tumor‑promoting effects of MSCs in vitro and in vivo. Our results demonstrated that tryptophan metabolites Kyn of MSCs in TME could enhance mitochondrial function by activating the AMPK pathway, thereby promoting bladder cancer cell progression.

Knockdown of AMIGO2 suppresses proliferation and migration through regulating PPAR-γ in bladder cancer

PurposeThis study aims to reveal the relationship between AMIGO2 and proliferation, migration and tumorigenicity of bladder cancer, and explore the potential molecular mechanisms.MethodsThe expression level of AMIGO2 is measured by qRT-PCR and immunohistochemistry (IHC). Stable AMIGO2 knockdown cell lines T24 and 5637 were established by lentivirus transfection. Cell Counting Kit (CCK-8 assay) was produced to determine cell proliferation, flow cytometry analysis was utilized to detect cell cycle, and wound healing assay was proceeded to test migration ability of bladder cancer cells. Xenograft mouse model was established for investigating the effect of AMIGO2 on tumor formation in vivo. The RNA Sequencing technology was applied to explore the underlying mechanisms. The expression level of PPAR-γ was measured by Western Blot.ResultsAMIGO2 was upregulated in bladder cancer cells and tissues. Inhibited expression of AMIGO2 suppresses cell proliferation and migration. Low AMIGO2 expression inhibited tumorigenicity of 5637 in nude mice. According to RNA-Seq and bioinformatics analysis, 917 DEGs were identified. The DEGs were mainly enriched in cell–cell adhesion, peroxisome proliferators-activated receptors (PPARs) signaling pathway and some other pathways. PPAR-γ is highly expressed in bladder cancer cell lines T24 and 5637, but when AMIGO2 is knocked down in T24 and 5637, the expression level of PPAR-γ is also decreased, and overexpression of PPAR-γ could reverse the suppression effect of cell proliferation and migration caused by the inhibition of AMIGO2.ConclusionAMIGO2 is overexpressed in bladder cancer cells and tissues. Knockdown of AMIGO2 suppresses bladder cancer cell proliferation and migration. These processes might be regulated by PPAR-γ signaling pathway.

Comparative transcriptomic analysis and mechanistic characterization revealed the use of formononetin for bladder cancer treatment.

Bladder cancer is one of the most common cancers worldwide, with 213 000 deaths reported in 2020. Patients with a progression from non-muscle-invasive bladder cancer to muscle-invasive disease have poorer prognosis and survival rates. Therefore, there is an urgent need to identify novel drugs to prevent the recurrence and metastasis of bladder cancer. Formononetin is an active compound extracted from the herb Astragalus membranaceus that possesses anticancer properties. Few studies have demonstrated the anti-bladder cancer effects of formononetin; however, the detailed mechanism remains unknown. In this study, we used two bladder cancer cell lines, TM4 and 5637, to investigate the potential role of formononetin in bladder cancer treatment. Comparative transcriptomic analysis was conducted to delineate the molecular mechanisms underlying the anti-bladder cancer effects of formononetin. Our results showed that formononetin treatment inhibited the proliferation and colony-forming abilities of bladder cancer cells. Additionally, formononetin reduced the migration and invasion of bladder cancer cells. Transcriptomic analysis further highlighted the involvement of formononetin-mediated two clusters of genes involved in endothelial cell migration (FGFBP1, LCN2, and STC1) and angiogenesis (SERPINB2, STC1, TNFRSF11B, and THBS2). Taken together, our results suggest the potential use of formononetin to inhibit the recurrence and metastasis of bladder cancer through the regulation of different oncogenes.

Scabertopin Derived from Elephantopus scaber L. Mediates Necroptosis by Inducing Reactive Oxygen Species Production in Bladder Cancer In Vitro

Bladder cancer remains one of the most common malignant tumors that threatens human health worldwide. It imposes a heavy burden on patients and society due to the high medical costs associated with its easy metastasis and recurrence. Although several treatment options for bladder cancer are available, their clinical efficacy remains unsatisfactory. Therefore, actively exploring new drugs and their mechanisms of action for the clinical treatment of bladder cancer is very important. Scabertopin is one of the major sesquiterpene lactones found in Elephantopus scaber L. Sesquiterpene lactones are thought to have fairly strong anti-cancer efficacy. However, the anticancer effect of sesquiterpenoid scabertopin on bladder cancer and its mechanism are still unclear. The aim of this study is to evaluate the antitumor activity of scabertopin in bladder cancer and its potential molecular mechanism in vitro. Our results suggest that scabertopin can induce RIP1/RIP3-dependent necroptosis in bladder cancer cells by promoting the production of mitochondrial reactive oxygen species (ROS), inhibit the expression of MMP-9 by inhibiting the FAK/PI3K/Akt signaling pathway, and ultimately inhibit the migration and invasion ability of bladder cancer cells. At the same time, we also demonstrated that the half-inhibition concentration (IC50) of scabertopin on various bladder cancer cell lines (J82, T24, RT4 and 5637) is much lower than that on human ureteral epithelial immortalized cells (SV-HUC-1). The above observations indicate that scabertopin is a potential therapeutic agent for bladder cancer that acts by inducing necroptosis and inhibiting metastasis.

Synergistic effect of metformin and doxorubicin on the metastatic potential of T24 cells

BackgroundThe motor ability of cancer cells to cross the basement membrane contributes to their implantation in a new location. Metastasis is a significant factor that worsens the prognosis of cancer patients. Thus, reducing cell invasiveness is an important aspect of anticancer therapy, also in bladder cancer treatment. MaterialThe study material was the T24 cell line of human urinary bladder cancer. The migratory potential of the cells and the effect of the treatment with individually doses and synergistic combination of doxorubicin and metformin in the 500:1 ratio for 24 h were analyzed. ResultsThe results obtained show a compound-initiated decrease in the motor abilities of bladder cancer cells compared to controls. A decrease in the rate of colony formation was observed, as well as inhibition of migration through inserts. The visualized reorganization of the vimentin and actin networks confirms the drug-initiated limitation of the metastatic potential of T24 cells. ConclusionAccording to our knowledge, we are the first to show, that combination of doxorubicin and metformin also worth considering in the treatment of bladder cancer. We showed that simultaneous administration of these cytostatic enhances the antiproliferative effect of drugs, but also limits cells' migratory potential.

Biological function and regulation mechanism of MTA2 expression in bladder cancer

Bladder cancer that occurs on the bladder mucosa is the most common malignant tumor in the urinary system. Exposure to aromatic amine chemicals and cigarette smoking are the two risk factors which causes bladder cancer. The mechanism of complex molecular signaling during its progress is still not understood clearly. High expression of metastasis associated gene 2 (MTA2) in malignant tumors such as ovarian cancer and hepatocellular carcinoma indicates its possible relationship with migration and invasion. However, expression level and biological function of MTA2 in bladder cancer tissues have not been studied. Here, we investigated the biological function, regulation mechanism and clinical application of metastasisrelated genes 2 (MTA2). The expression level of MTA2 in various bladder tumor cells was determined by Western Blot. T24 and EJ cells were selected for in vitro study. T24 cells and EJ cells were transfected with viral vector and SiMTA2, respectively, to induce MTA2 over- or low- expression. MTS colorimetric assay was used to explore the proliferative ability of bladder cancer cells in vitro under MTA2 over- or low-expression. Matrigel invasion assay and Transwell migration assay were used to detect the invasion and migration of cancer cells in vitro under different MTA2 expression conditions. The results of Western Blot assay on bladder cancer cell lines have shown that the MTA2expression level to be significantly lower in T24 cells compared to that of EJ and J82 cells. MTA2 level was significantly higher in MTA2 overexpressed T24 cells than those in the non-transfected and CD511B transfected bladder cancer cells, while the level of MTA2 was much lower in the MTA2-knockdown EJ cells than that in the non-transfected and si-NC transfected. The MTS colorimetric assay significant increase in proliferation of T24 cells with MTA2 overexpression, while the proliferation of EJ cells with MTA2 knockdown was decreased. Results of Transwell migration assay showed that MTA2 overexpression could significantly increase the migration ability of T24 cells, and MTA2 knockdown could significantly reduce the migration ability of EJ cells. Matrigel invasion assay showed that MTA2 overexpression could enhance the invasive ability of T24 cells significantly, while MTA2 knockdown weaken the invasive ability of EJ cells. The expression level of E-cadherin was lower in the T24 cells with MTA2 overexpression than that in the non-transfected group and CD511B transfected group, but the expression level of N-cadherin protein was significantly higher in the T24 cells with MTA2 overexpression compared to the other two groups. The expression level of E-cadherin protein was significantly higher in the EJ cells with MTA2 knockdown than that of the non-transfected group and si-NC transfected group, while the expression level of N-cadherin protein was significantly lower than that in the non-transfected group and si-NC transfected group.

Oncogenic E3 ubiquitin ligase NEDD4 binds to KLF8 and regulates the microRNA-132/NRF2 axis in bladder cancer

The neuronally expressed developmentally downregulated 4 (NEDD4) gene encodes a ubiquitin ligase that targets the epithelial sodium channel for degradation and has been implicated in tumor growth in various cancers. Hence, in this study, we intended to characterize the functional relevance of the NEDD4-mediated Kruppel-like factor 8/microRNA-132/nuclear factor E2-related factor 2 (KLF8/miR-132/NRF2) axis in the development of bladder cancer. NEDD4 and KLF8 were overexpressed in bladder cancer tissues and were associated with poorer patient survival rates. In bladder cancer cells, NEDD4 intensified the stability and transcriptional activity of KLF8 through ubiquitination to augment cell viability and migratory ability. Our investigations revealed that NEDD4 promotes the binding of KLF8 to the miR-132 promoter region and inhibits the expression of miR-132. KLF8 inhibited the expression of miR-132 to augment the viability and migratory ability of bladder cancer cells. Furthermore, miR-132 downregulated the expression of NRF2 to restrict the viability and migratory ability of bladder cancer cells. In addition, in vivo findings verified that NEDD4 regulates the KLF8/miR-132/NRF2 axis by accelerating tumor growth and lung metastasis. In conclusion, this study highlights NEDD4 as a potential therapeutic target against tumor recurrence and metastasis in bladder cancer.

Targeting S1PR1 May Result in Enhanced Migration of Cancer Cells in Bladder Carcinoma.

Simple SummaryMetastasis is critical to the prognosis of patients with bladder cancer, and it is important to understand the mechanism of its occurrence. S1PR1 expression is thought to be associated with poor prognosis, but it is unknown whether it is associated with tumor metastasis. Analysis of clinical gene expression data suggests that endothelial or immune cells in tumor tissue may be the source of S1PR1 expression. Comparative analysis of clinical tumor tissues with bladder cancer cells suggests that S1PR1 expression is associated with cellular adhesion. In vitro experiments demonstrated that S1PR1 expression was negatively correlated with cancer cell motility, and that S1PR1 inhibition by FTY-720 may cause an increase in cancer cell motility, suggesting that the use of S1PR1 inhibition as a synergistic therapy requires additional observations and considerations.Clinical bladder tumor histological analysis shows that high expression of S1PR1 is associated with poor patient prognosis. However, there are no studies that describe the underlying mechanism. To investigate the relative distribution and actual function of S1PR1 in bladder tumors, we analyzed multiple clinical databases in combination with tumor purity and immune cell infiltration simulations, as well as databases of well-defined histological phenotypes of bladder cancer, and single-cell sequencing of adjacent normal tissues and bladder tumors, and further compared them with bladder cancer cell lines. The results showed that S1PR1 expression was generally higher in normal tissues than in bladder cancer tissues, and its distribution was mainly in endothelial cells or immune cells. The association between high S1PR1 expression and poor prognosis may be due to tumor invasion of adjacent normal tissues, where highly expressed S1PR1 may affect prognostic interpretation. The effect of S1PR1 itself on cancer cells was associated with cell adhesion, and in bladder cancer cells, S1PR1 expression was negatively correlated with cell motility. Moreover, the use of FTY-720 will cause an increased metastatic ability of bladder cancer cells. In conclusion, we suggest that the use of S1PR1-specific inhibition as a synergistic treatment requires more observation and consideration.

Long Noncoding RNA LINC00707 Accelerates Tumorigenesis and Progression of Bladder Cancer via Targeting miR-145/CDCA3 Regulatory Loop

Introduction: Growing studies reveal that long noncoding RNA is involved in oncogenesis and progression. Previous studies have demonstrated that long intergenic noncoding RNA 00707 (LINC00707) stimulated tumor progress in numerous neoplasm types; however, the function of LINC00707 in bladder cancer (BC) was not yet clear. Our researches aimed to determine whether LINC00707 was dysregulated in BC and further study its biological functions. Methods: LINC00707 levels in BC tissues and cells were measured using reverse transcription-PCR (RT-PCR), and the associations between the levels of LINC00707 and clinicopathological features and the months of survival were also examined. Then, Cell Counting Kit-8 assays, flow cytometry, colony formation assays, and Transwell assays were applied for the assessment of the impact of LINC00707 on the abilities of BC cells. The interaction between LINC00707 and miR-145 or cell division cycle associated 3 was determined by luciferase reporter system and RT-PCR. Protein expressions of Wnt/β-catenin signaling were examined using RT-PCR and Western blot. Results: We found that LINC00707 expressions were notably upregulated in BC samples and cells. Higher expressions of LINC00707 were associated with T stage, grade, and shorter overall survival in BC patients. LINC00707 was also an independent prognostic factor for BC. In vitro assays confirmed that silencing LINC00707 expressions suppressed cell proliferation, colony formation, and metastasis. Mechanistic studies elucidated that LINC00707 was directly targeted to miR-145/CDCA3. Western blot assays revealed that Wnt/β-catenin signaling was inactivated by LINC00707 knockdown. Conclusion: Our work offers new insight into the function of LINC00707 in the tumorigenesis of BC.

LncRNA DANCR regulates lymphatic metastasis of bladder cancer via the miR-335/VEGF-C axis.

BackgroundSubstantial evidence indicate that long non-coding RNA (lncRNA) and microRNA (miRNA) act as key role in bladder cancer. Differentiation antagonistic ncRNA (DANCR) could be used as a biomarker in the occurrence and development of cancer. This study aims to explore the mechanism of DANCR/miR-335/VEGF-C axis affecting lymphatic metastasis of bladder cancer.MethodsqRT-PCR detects the expression of DANCR in bladder cancer cell lines (SW780, 5637, T24, UM-UC-3) and normal bladder cell lines (SV-HUC-1), and selects T24 cell lines for subsequent experiments. The expression levels of DANCR, miR-335 and VEGF were measured by qRT-PCR, and the dual luciferase reporter gene verified the targeted regulation of DANCR on miR-335 and miR-335 on VEGF. CCK-8, Transwell and Wound healing assay detect the proliferation, invasion and migration ability of bladder cancer cells, Endothelial cell adhesion assay and Western blot further prove the lymphatic metastasis of bladder cancer.ResultsIn this study, DANCR was highly expressed in bladder cancer cell lines. Transfection of si-DANCR significantly inhibits the proliferation, migration, invasion and lymphatic metastasis of bladder cancer cells. Dual luciferase assay confirmed that DANCR targets miR-335/VEGF-C. Transfection of miR-335 mimic promotes the proliferation, migration, invasion and lymphatic metastasis of bladder cancer cells, overexpression of DANCR eliminates the promotion of miR-335 mimic on bladder cancer cells. Further experiments proved that inhibition of miR-335 and overexpression of VEGF-C can reverse the inhibitory effect of silencing DANCR on bladder cancer cells.ConclusionsIn bladder cancer, DARCR plays an important role, which regulates the proliferation, migration, invasion and lymphatic metastasis of bladder cancer cells through the miR-335/VEGF-C molecular axis.

LncRNA LINC00355 promotes EMT and metastasis of bladder cancer cells through the miR-424-5p/HMGA2 axis.

Bladder cancer is a common malignant tumor with a high recurrence rate and mortality, while the detailed mechanisms for bladder cancer progression and metastasis are unknown. Recently, long non-coding RNAs (lncRNAs) have been reported to be involved in the development of cancers. In this study, we aim to investigate the role of lncRNA LINC00355 in bladder cancer progression and metastasis. The association between LINC00355 and the prognosis of bladder cancer patients was determined by Kaplan-Meier survival analysis. Cell migration and invasion ability were detected using the Transwell migration and invasion assay. The relationships of LINC00355, miR-424-5p, and High Mobility Group AT-Hook 2 (HMGA2) were verified through the luciferase assay and RNA pull-down assay. Xenograft tumor was established to evaluate tumor lung metastasis in vivo. qRT-PCR and western blot were used to detect gene expression. LINC00355 was upregulated in bladder cancer patients, especially in patients with higher TNM stage. Elevated LINC00355 was correlated with the poor prognosis of bladder cancer patients. Besides, overexpressed LINC00355 promoted migration, invasion, and epithelial-mesenchymal transition (EMT) ability of bladder cancer cells. Contrarily, decreased LINC00355 suppressed migration, invasion, and EMT ability of bladder cancer cells, and lung metastasis of xenograft tumors. Furthermore, LINC00355 could regulate HMGA2 expression by acting as a sponge for miR-424-5p. Overexpression of HMGA2 induced EMT of bladder cancer cells. Additionally, LINC00355 regulated the migration, invasion, and EMT ability of bladder cancer cells through modulating HMGA2 expression via sponging miR-424-5p. LINC00355 promoted migration, invasion, and EMT ability of bladder cancer through elevating HMGA2 expression via acting as a sponge for miR-424-5p.

USP24-GSDMB complex promotes bladder cancer proliferation via activation of the STAT3 pathway.

Background: Bladder cancer is the fourth and tenth most common malignancy in men and women worldwide, respectively. One of the main reasons for the unsatisfactory therapeutic control of bladder cancer is that the molecular biological mechanism of bladder cancer is complex. Gasdermin B (GSDMB) is one member of the gasdermin family and participates in the regulation of cell pyroptosis. The role of GSDMB in bladder cancer has not been studied to date.Methods: TCGA database was used to exam the clinical relevance of GSDMB. Functional assays such as MTT assay, Celigo fluorescent cell-counting assay, Annexin V-APC assay and xenografts were used to evaluate the biological role of GSDMB in bladder cancer. Mass spectrometry and immunoprecipitation were used to detect the protein interaction between GSDMB and STAT3, or GSDMB and USP24. Western blot and immunohistochemistry were used to study the relationship between USP24, GSDMB and STAT3.Results: In this study, bioinformatics analysis indicated that the mRNA expression level of GSDMB in bladder cancer tissues was higher than that in adjacent normal tissues. Then, we showed that GSDMB promoted bladder cancer progression. Furthermore, we demonstrated that GSDMB interacted with STAT3 to increase the phosphorylation of STAT3 and modulate the glucose metabolism and promote tumor growth in bladder cancer cells. Besides, we also showed that USP24 stabilized GSDMB to activate STAT3 signaling, which was blocked by the USP24 inhibitor.Conclusions: We suggested that aberrantly up-regulated GSDMB was responsible for enhancing the growth and invasion ability of bladder cancer cells. Then, we showed that GSDMB could bind to STAT3 and activate STAT3 signaling in bladder cancer. Furthermore, we also demonstrated that USP24 interacted with GSDMB and prevented GSDMB from degradation in bladder cancer cells. Therefore, the USP24/GSDMB/STAT3 axis may be a new targetable signaling pathway for bladder cancer treatment.

Phosphorylated MAPK14 Promotes the Proliferation and Migration of Bladder Cancer Cells by Maintaining RUNX2 Protein Abundance.

BackgroundMitogen-activated protein kinase 14 (MAPK14) acts as an integration point for multiple biochemical signal pathways. High expressions of MAPK14 have been found in a variety of tumors. Runt‑related transcription factor 2 (RUNX2) is related to many tumors, especially in tumor invasion and metastasis. However, the mechanism of these two genes in bladder cancer remains unclear.MethodsTCGA database and Western blot were used to analyze the mRNA and protein levels of the target gene in bladder cancer tissues and adjacent tissues. The proliferation ability of bladder cancer cells was tested by colony forming and EdU assay. The migration ability of cells was detected by transwell assay. Immunoprecipitation was utilized to detect protein–protein interaction. Cycloheximide chase assay was used to measure the half-life of RUNX2 protein.ResultsPhosphorylated mitogen-activated protein kinase 14 (P-MAPK14, Thr180/Tyr182) was highly expressed in bladder cancer tissues and bladder cancer cell lines. Accordingly, P-MAPK14 could be combined with RUNX2 and maintain its protein stability and promote the proliferation and migration of bladder cancer cells. In addition, the functional degradation caused by the downregulation of MAPK14 and P-MAPK14 could be partially compensated by the overexpression of RUNX2.ConclusionThese results suggest that P-MAPK14 might play an important role in the development of bladder cancer and in the regulation of RUNX2 protein expression. P-MAPK14 might become a potential target for the treatment of bladder cancer.

An 18-gene signature based on glucose metabolism and DNA methylation improves prognostic prediction for urinary bladder cancer

BackgroundGlucose metabolism and DNA methylation play important roles in cancers. We aimed to identify glucose metabolism-related genes that were DNA methylation associated to establish a prognostic signature of bladder cancer (BLCA). MethodsWith BLCA sample transcriptome data from The Cancer Genome Atlas (TCGA) and methylation data from TCGA 450 K microarray, glucose metabolism-related genes associated to prognosis and DNA methylation were identified and a prognostic signature was established. GSEA and WGCNA analysis were performed and two genes, UCHL1 and PYCR1, were selected for functional validations. Results18 target genes were identified and the signature based on them was considered an effective and independent prognostic factor. Several pathways were enriched in the high-risk group by GSEA and three modules of genes were identified by WGCNA. UCHL1 and PYCR1 proliferated proliferation, migration and invasion ability of bladder cancer cells. ConclusionsThe 18-gene signature is an independent prognostic factor for bladder cancer patients.

LncRNA AFAP1-AS1 promotes proliferation ability and invasiveness of bladder cancer cells.

It was the aim of this study to explore the role and mechanism of long non-coding RNA (lncRNA) AFAP1-AS1 in the progression of bladder cancer (BCa) by in vitro experiments. AFAP1-AS1 levels in 40 pairs of clinical BCa tissue samples and normal ones collected from BCa patients were determined, and paired sample t-test was applied to compare the differences between groups. The prognosis data of patients with BCa were collected, and survival analysis and t-test were performed to specify the interplay between AFAP1-AS1 and the prognosis of BCa patients. Subsequently, AFAP1-AS1 expression level in BCa and normal cells were further confirmed by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR), and Cell Counting Kit-8 (CCK-8), 5-Ethynyl-2'-deoxyuridine (EdU), and transwell assays were performed to figure out the influence of this lncRNA on the proliferation ability and invasiveness of BCa cells. Meanwhile, the interaction between AFAP1-AS1 and its sense mRNA was analyzed. We used co-transfection technology to simultaneously transfect si-AFAP1-AS1 and pcDNA3.1-AFAP1 or their corresponding negative controls into BCa cells, and cell proliferation and invasion ability in different subgroups were determined to explore the underlying mechanism through which AFAP1-AS1 plays a role in BCa progression. No matter in BCa tissues or in cell samples, compared to the corresponding normal controls, AFAP1-AS1 was found highly expressed; at the same time, in invasive bladder cancer tissues, the expression level of AFAP1-AS1 was also higher than that in non-invasive tissues. Meanwhile, survival analysis revealed that patients with BCa with high expression of AFAP1-AS1 owned a shorter overall survival rate than those with low expression, indicating a negative interplay between AFAP1-AS1 expression and patients' prognosis. In addition, in BCa cell lines, according to the results of CCK-8, EDU, and transwell assays, the proliferative capacity, as well as the invasive ability of BCa cells, were found weakened after downregulation of AFAP1-AS1. Meanwhile, a negative interplay was discovered between AFAP1-AS1 and its sense mRNA. Finally, the results of cell reversal experiment using co-transfection technique revealed that overexpression of AFAP1 can reverse the inhibitory impact of lncRNAAFAP1-AS1 on the malignant ability of BCa cells. AFAP1-AS1 may enhance the proliferation ability as well as the invasiveness of BCa cells so as to aggravate the degree of BCa malignancy.

Long non-coding RNA LINC00675 is associated with bladder cancer metastasis and patient survival.

Muscle-invasive bladder cancer (MIBC), a bladder cancer that spreads into the detrusor muscle of the bladder, leads to a poor outcome. Long noncoding RNA LINC00675 has been reported to play import roles in several cancer types, although its biological function and underlying mechanism in MIBC remain largely unclear. Eighty-nine patients with MIBC were enrolled in the present study. RNA expression was measured using a quantitative reverse transcription-polymerase chain reaction. Protein expression was detected using western blotting. Transwell assays were performed to analyze the abilities of bladder cancer cells to migrate and invade. An RNA microarray was carried out to analyze LINC00675-regulated mRNAs in bladder cancer cells. We found that LINC00675 expression was decreased in MIBC tissues compared to matched normal tissues, and was correlated with lymph node-metastatic MIBC. MIBC patients with low expression of LINC00675 had worse survival rates than those with high expression. Functional investigation showed that ectopic expression of LINC00675 inhibited bladder cancer cell migration, invasion and proliferation, whereas LINC00675 knockdown presented an opposite effect. Mechanistically, we found that LINC00675 inhibited β-catenin and its downstream gene expression. The findings of the present study suggest that LINC00675 regulates β-catenin expression, and is associated with bladder cancer metastasis and patient survival.

Inhibition of Heparanase Expression Results in Suppression of Invasion, Migration and Adhesion Abilities of Bladder Cancer Cells.

Heparan sulfate proteoglycan syndecan-1, CD138, is known to be associated with cell proliferation, adhesion, and migration in malignancies. We previously reported that syndecan-1 (CD138) may contribute to urothelial carcinoma cell survival and progression. We investigated the role of heparanase, an enzyme activated by syndecan-1 in human urothelial carcinoma. Using human urothelial cancer cell lines, MGH-U3 and T24, heparanase expression was reduced with siRNA and RK-682, a heparanase inhibitor, to examine changes in cell proliferation activity, induction of apoptosis, invasion ability of cells, and its relationship to autophagy. A bladder cancer development mouse model was treated with RK-682 and the bladder tissues were examined using immunohistochemical analysis for Ki-67, E-cadherin, LC3, and CD31 expressions. Heparanase inhibition suppressed cellular growth by approximately 40% and induced apoptosis. The heparanase inhibitor decreased cell activity in a concentration-dependent manner and suppressed invasion ability by 40%. Inhibition of heparanase was found to suppress autophagy. In N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN)-induced bladder cancer mice, treatment with heparanase inhibitor suppressed the progression of cancer by 40%, compared to controls. Immunohistochemistry analysis showed that heparanase inhibitor suppressed cell growth, and autophagy. In conclusion, heparanase suppresses apoptosis and promotes invasion and autophagy in urothelial cancer.

Circular RNA circKIF4A Sponges miR-375/1231 to Promote Bladder Cancer Progression by Upregulating NOTCH2 Expression.

Circular RNAs (circRNAs) have been found to be important mediators of many biological processes in the growth and metastasis of various cancers. However, the potential roles of most circRNAs in the progression of bladder cancer remain unclear. In this research, we investigate the role of circKIF4A (hsa_circ_0007255) in the development and progression of bladder cancer. Detected by qRT-PCR analysis, circKIF4A was significantly upregulated in bladder cancer tissues and cell lines. We conducted CCK-8, colony-formation, transwell and mouse xenograft assays to explore the function of circKIF4A in bladder cancer. Functionally, knockdown of circKIF4A inhibited the proliferation and colony-formation ability of bladder cancer cells. Migration and metastatic ability were dramatically decreased after transfection with small interfering RNA targeting circKIF4A in both in vitro and in vivo assays. Mechanically, luciferase reporter assays and RNA immunoprecipitation assays were carried out to elucidate the underlying molecular mechanism of circKIF4A. The results revealed that circKIF4A sponges miR-375/1231 to promote bladder cancer progression by upregulating NOTCH2. Generally, our research unveils the essential role of circKIF4A-miR-375/1231-NOTCH2 axis in bladder cancer progression possibly via the competing endogenous RNA mechanism.

GRHL2 Acts as an Anti-Oncogene in Bladder Cancer by Regulating ZEB1 in Epithelial-Mesenchymal Transition (EMT) Process.

PurposeGRHL2 played important roles in different cancers. In this study, we aimed to investigate the roles of GRHL2 in bladder cancer.MethodsThe immunohistochemistry assay was performed to detect the expression of GRHL2 in bladder cancer tissues and adjacent noncancerous tissues and the expression levels of GRHL2 and zinc finger E-box binding homeobox (ZEB1) mRNA in tissues were determined by qRT-PCR. In addition, qRT-PCR and Western blotting were applied to detect the expression levels of GRHL2 and ZEB1 in bladder cancer cell lines (RT4, BIU-87, 5637, T24) and immortalized human bladder epithelial cell line (SV-HUC-1). The cell models with up-regulated and down-regulated expression of GRHL2 were constructed using bladder cancer cell lines T24 and 5637 to investigate the underlying roles of GRHL2 on the proliferation, migration, invasion and EMT process of bladder cancer cells. After that, cell proliferation was evaluated by CCK8 assay, cell cycle assay and colony formation assay. Transwell assay and wound healing assay were performed to determine the invasion and migration ability of the bladder cancer cells. The expressions of epithelial-mesenchymal transition (EMT) related proteins (E-cadherin, Vimentin, Slug and Snail) were assessed by Western blot analysis. Moreover, ZEB1 and GRHL2 were co-transfected into T24 and 5637 cells and their effects on EMT process and invasive capacity of cells were examined.ResultsThe expression of GRHL2 was down-regulated in bladder cancer tissues and human bladder cancer cell lines compared with the normal bladder tissues and immortalized human bladder epithelial cell line. Besides, down-regulation of GRHL2 improved the proliferation ability of bladder cancer cells and promoted the EMT process through up-regulation of ZEB1. The overexpression of ZEB1 partially reversed the inhibitory effect of GRHL2 on EMT.ConclusionGRHL2 acts as an anti-oncogene to regulate bladder cancer cell proliferation and inhibit EMT by targeting ZEB1. This study may provide a theoretical basis for further research.

Long Non-Coding RNA BCAR4 Binds to miR-644a and Targets TLX1 to Promote the Progression of Bladder Cancer.

BackgroundBladder cancer is a serious threat to human health. It is meaningful to study the pathogenesis of bladder cancer. Long non-coding RNAs (lncRNAs) are reported to promote or inhibit bladder cancer development. However, the role of lncRNA BCAR4 in the regulation of bladder cancer remains unclear.PurposeThis study was to explore the role of lncRNA BCAR4 in the progression of bladder cancer cell.MethodsRT-PCR was used to examine the expression of BCAR4 and miR-644a. CCK8 assay, colony formation assay, Transwell assay were used to detect the progression of bladder cancer cells after transfecting of indicated plasmids.ResultsThe expression of BCAR4 was higher in bladder cancer cell lines than normal urothelial cell line. Moreover, the expression of BCAR4 was associated with the advanced stage and metastasis of bladder cancer. Through knockdown of BCAR4, we discovered that knockdown of BCAR4 significantly decreased the proliferation, migration and invasive abilities of bladder cancer cells. Mechanically, we showed that BCAR4 can bind to miR-644a directly and targets TLX1. Moreover, we also showed that miR-644a was also highly expressed in bladder cancer cells and inhibition of miR-644a or overexpression of TLX1 can increased the migration abilities of bladder cancer caused by knockdown of BCAR4.ConclusionWe showed that BCAR4 sponged miR-644a to modulate the expression of TLX1 and promote bladder cancer development.