Background: The response rate to immunotherapy in patients with bladder cancer (BCa) remains relatively low. Considering the stable existence and important functions in tumour metabolism, the role of circRNAs in regulating immune escape and immunotherapy sensitivity is receiving increasing attention. Methods: Circular RNA (circRNA) sequencing was performed on five pairs of BCa samples, and circFAM13B (hsa_circ_0001535) was screened out because of its remarkably low expression in BCa. Further mRNA sequencing was conducted, and the association of circFAM13B with glycolysis process and CD8⁺ T cell activation was confirmed. The functions of circFAM13B were verified by proliferation assays, glycolysis assays, BCa cells-CD8⁺ T cell co-culture assays and tumorigenesis experiment among human immune reconstitution NOG mice. Bioinformatic analysis, RNA–protein pull down, mass spectrometry, RNA immunoprecipitation, luciferase reporter assay and fluorescence in situ hybridization were performed to validate the HNRNPL/circFAM13B/IGF2BP1/PKM2 cascade. Results: Low expression of circFAM13B was observed in BCa, and it was positively associated with lower tumour stage and better prognosis among patients with BCa. The function of CD8⁺ T cells was promoted by circFAM13B, and it could attenuate the glycolysis of BCa cells and reverse the acidic tumour microenvironment (TME). The production of granzyme B and IFN-γ was improved, and the immunotherapy (PD-1 antibodies) sensitivity was facilitated by the inhibition of acidic TME. Mechanistically, circFAM13B was competitively bound to the KH3-4 domains of IGF2BP1 and subsequently reduced the binding of IGF2BP1 and PKM2 3’UTR. Thus, the stability of the PKM2 mRNA decreased, and glycolysis-induced acidic TME was inhibited. The generation of circFAM13B was explored by confirming whether heterogeneous nuclear ribonucleoprotein L (HNRNPL) could promote circFAM13B formation via pre-mRNA back-splicing. Conclusions: HNRNPL-induced circFAM13B could repress immune evasion and enhance immunotherapy sensitivity by inhibiting glycolysis and acidic TME in BCa through the novel circFAM13B/IGF2BP1/PKM2 cascade. Therefore, circFAM13B can be used as a biomarker for guiding the immunotherapy among patients with BCa.

Background: The modification and regulation of N6-methyladenosine (m⁶A) at mRNA level can affect the development and progression in various tumors. ALKBH5, as an m⁶A demethylase, plays different roles in tumors by regulating the m⁶A modification of mRNA. However, its role in renal cell carcinoma (RCC) remains unclear. Methods: First, levels of ALKBH5 in RCC tissues and cell lines were verified by qRT-PCR and western blot. We analyzed the relationship between ALKBH5 and the clinicopathological characteristics of RCC patients and the influence of ALKBH5 on the prognosis of patients. Then we generated ALBKH5-overexpression, ALBKH5-knockdown stable RCC cell lines and their control cell lines. Through cell proliferation assay, colony formation assay, cell invasion and tumor migration assay, cell cycle assay and xenograft studies, we studied the ALKBH5 roles in RCC cell lines. AURKB was predicted to be its potential target based on TCGA database analysis and verified by western blot. The role of AURKB in RCC was verified by TCGA database and Kaplan-Meier analysis with TMA immunohistochemical analysis. Finally, the specific molecular mechanism of ALKBH5 targeting AURKB was explored by dual-luciferase reporter assay, RNA immunoprecipitation (RIP), m⁶A dot-blot assay, m⁶A RNA Immunoprecipitation (MeRIP) assay, and mRNA stability assay. Results: We found that ALKBH5 was highly expressed in both RCC tumor tissues and cell lines. Clinicopathological analysis showed that high ALKBH5 expression was associated with larger tumor volume (P=0.017) and higher TNM staging (P=0.006), and worse prognosis (log rank: P=0.0199). The cellular functional assays showed that stably overexpression ALKBH5 could promote the cell proliferation, colony formation, cell migration and cell invasion of renal cell carcinoma cells in vitro and promote tumor growth in vivo. In contrast, ALKBH5 knocked down inhibited cell proliferation, colony formation, migration and invasion of renal cell carcinoma cells in vitro. Based on TCGA database analysis, AURKB was predicted highly expressed in RCC and a potential target of ALKBH5. Both database prediction and TMA immunohistochemical analysis supported that AURKB could affect the prognosis of RCC patients (P values of 5.5e-08 and 0.0004, respectively) and was regulated by ALKBH5 expression level. Subsequent mechanism experiments showed that ALKBH5 regulated the expression of AURKB by regulating the stability of AURKB mRNA in the m⁶A-dependent manner, and finally promoted cell proliferation. Furthermore, we found that hypoxia-induced HIF could up-regulate both expressions of AURKB and ALKBH5. Conclusions: Our findings suggest that ALKBH5 may play a carcinogenic role in renal cell carcinoma by stabilizing AURKB mRNA in a m⁶A-dependent manner. These data suggest that ALKBH5 may play a key role in RCC and targeting the ALKBH5 signaling pathway may be a promising strategy for the treatment of RCC.

Background: Tumour-derived exosomes have recently been shown to participate in the formation and progression of different cancer processes, including tumour microenvironment remodelling, angiogenesis, invasion, metastasis, and drug resistance. However, the function and mechanism of exosome-encapsulated nucleic acids and proteins in bladder cancer remain unclear. This study aimed to investigate the effects of tumour-derived exosomes on the tumorigenesis and development of bladder cancer. Methods: In this study, gene expression profiles and clinical information were collected from The Cancer Genome Atlas (TCGA) database and two independent Gene Expression Omnibus (GEO) datasets. The nucleic acids and proteins encapsulated in bladder cancer-derived exosomes were obtained from the ExoCarta database. Based on these databases, the expression patterns of exosomal mRNAs and proteins and the matched clinicopathological characteristics were analysed. Furthermore, we carried out a series of experiments to verify the relevant findings. Results: A total of 7280 differentially expressed mRNAs were found in TCGA data, of which 52 mRNAs were shown to be encapsulated in bladder cancer-derived exosomes. Survival analysis based on the UALCAN database showed that among the top 10 upregulated and the top 10 downregulated exosomal genes, only the expression of KRT6B had a statistically significant effect on the survival of bladder cancer patients. Additionally, clinical correlation analysis showed that the elevated level of KRT6B was highly associated with bladder cancer stage, grade, and metastasis status. GSEA revealed that KRT6B was involved not only in epithelial–mesenchymal transition-related pathways but also in the immune response in bladder cancer. Ultimately, our experimental results were also consistent with the bioinformatic analysis. Conclusion: KRT6B, which can be detected in bladder cancer-derived exosomes, plays an important role in the epithelial–mesenchymal transition and immune responses in bladder cancer. Further research will enable its potentially prognostic marker and therapeutic target for bladder cancer.

N6-methyladenosine (m6A) is important in the physiological processes of many species. Methyltransferase-like 16 (METTL16) is a novel discovered m6A methylase, regulating various tumors in an m6A-dependent manner. However, its function in bladder cancer (BLCA) remains largely unclear. In the present study, we found that low expression of METTL16 predicted poor survival in BLCA patients. METTL16 inhibited the proliferation and cisplatin-resistance function of bladder cancer cells in vitro and in vivo. In addition, METTL16 reduced the mRNA stability of prostate transmembrane protein androgen induced-1 (PMEPA1) via binding to its m6A site in the 3'-UTR, thereby inhibited the proliferation of bladder cancer cells and increased the sensitivity of cisplatin through PMEPA1-mediated autophagy pathway. Finally, we found that hypoxia-inducible factor 2α (HIF-2α) exerted its tumor-promoting effect by binding the METTL16 promoter region to repress its transcription. Taken together, High expression of METTL16 predicted better survival in BLCA. METTL16 significantly inhibited bladder cancer cell proliferation and sensitized bladder cancer cells to cisplatin via HIF-2α-METTL16-PMEPA1-autophagy axis in a m6A manner. These findings might provide fresh insights into BLCA therapy.

Background: The immunotherapy sensitivity of patients with bladder cancer (BCa) remains low. As the role of protein methylation in tumorigenesis and development becomes clearer, the role of lysine N-methyltransferase SET domain containing 7 (SETD7) in the progression and immune escape of BCa is worth studying.