Identification of a potentially functional circRNA-miRNA-mRNA regulatory network for investigating pathogenesis and providing possible biomarkers of bladder cancer.

BACKGROUND: Circular RNAs (circRNAs) have received considerable attention in human cancer research. However, many circRNAs remain to be detected. In our study, we determined novel circRNAs and investigated their effects on bladder cancer (BCa). METHODS: Microarray dataset GSE92675 was downloaded from Gene Expression Omnibus (GEO). Then, we combined computational biology with quantitative real-time polymerase chain reaction (qRT-PCR) to select related circRNAs in BCa. The selected circRNA-microRNA (miRNA)-messenger RNA (mRNA) regulatory subnetwork was determined by Gene Oncology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. RESULTS: The regulatory network constructed from the microarray dataset (GSE92675) contained 49 differentially expressed circRNAs (DECs). GO and KEGG analyses showed that the MAPK and PI3K-AKT signaling pathways were statistically significant. On the basis of qRT-PCR and the degree value calculated by the cytoHubba plugin of Cytoscape, hsa_circ_0011385 was finally confirmed. The subnetwork around hsa_circ_0011385 was constructed. In addition, we created a protein-protein interaction (PPI) network composed of 67 nodes and 274 edges after removing independent nodes. GO and KEGG analyses showed that hubgenes were involved in cell cycle activities. Moreover, they could be regulated by miRNAs and play an eventful role in BCa pathogenesis. CONCLUSIONS: We proposed a novel circRNA-miRNA-mRNA network related to BCa pathogenesis. This network might be a new molecular biomarker and could be used to develop potential treatment strategies for BCa.

METTL3 promote tumor proliferation of bladder cancer by accelerating pri-miR221/222 maturation in m6A-dependent manner.

BACKGROUND: METTL3 is known to be involved in all stages in the life cycle of RNA. It affects the tumor formation by the regulation the m6A modification in the mRNAs of critical oncogenes or tumor suppressors. In bladder cancer, METTL3 could promote the bladder cancer progression via AFF4/NF-κB/MYC signaling network by an m6A dependent manner. Recently, METTL3 was also found to affect the m6A modification in non-coding RNAs including miRNAs, lincRNAs and circRNAs. However, whether this mechanism is related to the proliferation of tumors induced by METTL3 is not reported yet. METHODS: Quantitative real-time PCR, western blot and immunohistochemistry were used to detect the expression of METTL3 in bladder cancer. The survival analysis was adopted to explore the association between METTL3 expression and the prognosis of bladder cancer. Bladder cancer cells were stably transfected with lentivirus and cell proliferation and cell cycle, as well as tumorigenesis in nude mice were performed to assess the effect of METTL3 in bladder cancer. RNA immunoprecipitation (RIP), co-immunoprecipitations and RNA m6A dot blot assays were conducted to confirm that METTL3 interacted with the microprocessor protein DGCR8 and modulated the pri-miR221/222 process in an m6A-dependent manner. Luciferase reporter assay was employed to identify the direct binding sites of miR221/222 with PTEN. Colony formation assay and CCK8 assays were conducted to confirm the function of miR-221/222 in METTL3-induced cell growth in bladder cancer. RESULTS: We confirmed the oncogenic role of METTL3 in bladder cancer by accelerating the maturation of pri-miR221/222, resulting in the reduction of PTEN, which ultimately leads to the proliferation of bladder cancer. Moreover, we found that METTL3 was significantly increased in bladder cancer and correlated with poor prognosis of bladder cancer patients. CONCLUSIONS: Our findings suggested that METTL3 may have an oncogenic role in bladder cancer through interacting with the microprocessor protein DGCR8 and positively modulating the pri-miR221/222 process in an m6A-dependent manner. To our knowledge, this is the first comprehensive study that METTL3 affected the tumor formation by the regulation the m6A modification in non-coding RNAs, which might provide fresh insights into bladder cancer therapy.

Combined hepatocellular-cholangiocarcinoma: a population level analysis of incidence and mortality trends.

BACKGROUND: The purpose of this study was to explore trends in incidence, incidence-based (IB) mortality, and survival for combined hepatocellular-cholangiocarcinoma (cHCC-CC) utilizing a population-based database to attract people's attention to this disease. METHODS: The Surveillance, Epidemiology, and End Results (SEER) database was utilized to investigate the incidence and IB mortality for cHCC-CC from 2000 to 2014. Trends in age-adjusted incidence and IB mortality were characterized by the Joinpoint Regression program. The Kaplan-Meier method and log-rank test were utilized to implement survival analyses. Cox regression was utilized to estimate independent predictors of mortality. RESULTS: The incidence of cHCC-CC was 0.26 per 1,000,000 individuals in 2000 and 0.59 per 1,000,000 individuals in 2014, with an annual percent change (APC) (i.e., the extent of increase in incidence) of 3.84% (95% confidence interval [CI] 1.7-6.1; P < 0.05). The IB mortality also displayed a sustained increase (APC was 4.59%, 95% CI 1.9-7.4; P < 0.05). Compared to patients not undergoing surgery, patients undergoing surgical treatment experienced a significant increase in median survival (3 vs. 28 months; P < 0.001). However, the median survival decreased in patients with tumor size > 5 cm (20 vs. 9 months; P < 0.001). Based on univariate Cox regression analysis, African-American race, distant stage, regionalized stage, tumor size ≥ 5 cm, and no surgery were risk factors for death. CONCLUSIONS: We identified an overall steady increase in the incidence of cHCC-CC, which indicates that primary prevention strategies for cHCC-CC have not improved much in recent years and that cHCC-CC needs to be taken seriously.

Simple cholecystectomy is an adequate treatment for grade I T1bN0M0 gallbladder carcinoma: Evidence from 528 patients.

BACKGROUND: T1b gallbladder carcinoma (GBC) is defined as a tumor that invades the perimuscular connective tissue without extension beyond the serosa or into the liver. However, controversy still exists over whether patients with T1b GBC should undergo cholecystectomy alone or radical GBC resection. AIM: To explore the optimal surgical approach in patients with T1b gallbladder cancer of different pathological grades. METHODS: Patients with T1bN0M0 GBC who underwent surgical treatment between 2000 and 2017 were included in the Surveillance, Epidemiology, and End Results database. The Kaplan-Meier method and log-rank test were used to analyze the overall survival (OS) and disease-specific survival (DSS) of patients with T1b GBC of different pathological grades. Cox regression analysis was used to identify independent predictors of mortality and explore the selection of surgical methods in patients with T1b GBC of different pathological grades and their relationship with prognosis. RESULTS: Of the 528 patients diagnosed with T1bN0M0 GBC, 346 underwent simple cholecystectomy (SC) (65.5%), 131 underwent SC with lymph node resection (SC + LN) (24.8%), and 51 underwent radical cholecystectomy (RC) (9.7%). Without considering the pathological grade, both the OS (P < 0.001) and DSS (P = 0.003) of T1b GBC patients who underwent SC (10-year OS: 27.8%, 10-year DSS: 55.1%) alone were significantly lower than those of patients who underwent SC + LN (10-year OS: 35.5%, 10-year DSS: 66.3%) or RC (10-year OS: 50.3%, 10-year DSS: 75.9%). Analysis of T1b GBC according to pathological classification revealed no significant difference in OS and DSS between different types of procedures in patients with grade I T1b GBC. In patients with grade II T1b GBC, obvious survival improvement was observed in the OS (P = 0.002) and DSS (P = 0.039) of those who underwent SC + LN (10-year OS: 34.6%, 10-year DSS: 61.3%) or RC (10-year OS: 50.5%, 10-year DSS: 78.8%) compared with those who received SC (10-year OS: 28.1%, 10-year DSS: 58.3%). Among patients with grade III or IV T1b GBC, SC + LN (10-year OS: 48.5%, 10-year DSS: 72.2%), and RC (10-year OS: 80%, 10-year DSS: 80%) benefited OS (P = 0.005) and DSS (P = 0.009) far more than SC (10-year OS: 20.1%, 10-year DSS: 38.1%) alone. CONCLUSION: Simple cholecystectomy may be an adequate treatment for grade I T1b GBC, whereas more extensive surgery is optimal for grades II-IV T1b GBC.

The role of the HIF-1α/ALYREF/PKM2 axis in glycolysis and tumorigenesis of bladder cancer.

BACKGROUND: As a rate-limiting enzyme of glycolysis, pyruvate kinase muscle isozyme M2 (PKM2) participates in tumor metabolism and growth. The regulatory network of PKM2 in cancer is complex and has not been fully studied in bladder cancer. The 5-methylcytidine (m5C) modification in PKM2 mRNA might participate in the pathogenesis of bladder cancer and need to be further clarified. This study aimed to investigate the biological function and regulatory mechanism of PKM2 in bladder cancer. METHODS: The expression of PKM2 and Aly/REF export factor (ALYREF) was measured by Western blotting, qRT-PCR, and immunohistochemistry. The bioprocesses of bladder cancer cells were demonstrated by a series of experiments in vitro and in vivo. RNA immunoprecipitation, RNA-sequencing, and dual-luciferase reporter assays were conducted to explore the potential regulatory mechanisms of PKM2 in bladder cancer. RESULTS: In bladder cancer, we first demonstrated that ALYREF stabilized PKM2 mRNA and bound to its m5C sites in 3'-untranslated regions. Overexpression of ALYREF promoted bladder cancer cell proliferation by PKM2-mediated glycolysis. Furthermore, high expression of PKM2 and ALYREF predicted poor survival in bladder cancer patients. Finally, we found that hypoxia-inducible factor-1alpha (HIF-1α) indirectly up-regulated the expression of PKM2 by activating ALYREF in addition to activating its transcription directly. CONCLUSIONS: The m5C modification in PKM2 mRNA in the HIF-1α/ALYREF/PKM2 axis may promote the glucose metabolism of bladder cancer, providing a new promising therapeutic target for bladder cancer.