Circular RNAs (circRNAs) are a new member of noncoding RNAs (ncRNAs) that have recently been described as key regulators of gene expression. Our previous study had identified the negative correlation between circHIPK3 and bladder cancer grade, invasion, as well as lymph node metastasis. However, the roles of circRNAs in cellular proliferation in bladder cancer remain largely unknown. We had analyzed circRNA high-throughout sequencing from human tissues and determined bladder cancer related circRNA-3 (BCRC-3, GenBank: KU921434.1) as a new candidate circRNA derived from PSMD1 gene. The expression levels of circRNAs, mRNAs and miRNAs in human tissues and cells were detected by quantitative real-time PCR (qRT-PCR). The effects of BCRC-3 on cancer cells were explored by transfecting with plasmids in vitro and in vivo. RNA pull down assay, luciferase reporter assay and fluorescence in situ hybridization were applied to verify the interaction between BCRC-3 and microRNAs. Anticancer effects of methyl jasmonate (MJ) were measured by flow cytometry assay, western blot and qRT-PCR. BCRC-3 was lowly expressed in bladder cancer tissues and cell lines. Proliferation of BC cells was suppressed by ectopic expression of BCRC-3 in vitro and in vivo. Mechanistically, overexpression of BCRC-3 induced the expression of cyclin-dependent kinase inhibitor 1B (p27). Importantly, BCRC-3 could directly interact with miR-182-5p, and subsequently act as a miRNA sponge to promote the miR-182-5p-targeted 3’UTR activity of p27. Furthermore, MJ significantly increased the expression of BCRC-3, resulting in an obvious up-regulation of p27. BCRC-3 functions as a tumor inhibitor to suppress BC cell proliferation through miR-182-5p/p27 axis, which would be a novel target for BC therapy.

Cisplatin (CDDP)-based chemotherapy is the first-line treatment for muscle-invasive and metastatic bladder cancer, yet most patients rapidly develop resistance. N6-methyladenosine (m6A) methylation is a pervasive RNA modification, and its specific role and potential mechanism in the regulation of CDDP chemosensitivity in bladder cancer remain unclear. Furthermore, studies have not yet fully elucidated whether circular RNA (circRNA) can directly regulate m6A modification of mRNA. Here we report upregulation of a novel circRNA, hsa_circ_0008399 (circ0008399), by eukaryotic translation initiation factor 4A3 (EIF4A3) in bladder cancer tissues and cell lines. Functionally, circ0008399 inhibited apoptosis of bladder cancer cells. Mechanistically, circ0008399 bound Wilms' tumor 1–associating protein (WTAP) to promote formation of the WTAP/METTL3/METTL14 m6A methyltransferase complex. Circ0008399 increased expression of TNF alpha-induced protein 3 (TNFAIP3) by increasing its mRNA stability in an m6A-dependent manner. In patients with bladder cancer, high expression of circ0008399 and WTAP was associated with poor outcomes. Importantly, activation of the circ0008399/WTAP/TNFAIP3 pathway decreased bladder cancer chemosensitivity to CDDP, and targeting the circ0008399/WTAP/TNFAIP3 axis enhanced the CDDP efficacy. Collectively, these findings give novel insights into circRNA-mediated regulation of m6A modifications and provide potential therapeutic targets for bladder cancer. Significance: A newly characterized circRNA circ0008399 binds WTAP to modulate expression of target RNA through m6A modification and reduce cisplatin sensitivity in bladder cancer, implicating the potential therapeutic value of targeting this axis.

Background: Circular RNAs (circRNAs) have been found to have significant impacts on bladder cancer (BC) progression through various mechanisms. In this study, we aimed to identify novel circRNAs that regulate the function of IGF2BP1, a key m⁶A reader, and explore the regulatory mechanisms and clinical significances in BC. Methods: Firstly, the clinical role of IGF2BP1 in BC was studied. Then, RNA immunoprecipitation sequencing (RIP-seq) analysis was performed to identify the circRNAs interacted with IGF2BP1 in BC cells. The overall biological roles of IGF2BP1 and the candidate circPTPRA were investigated in both BC cell lines and animal xenograft studies. Subsequently, we evaluated the regulation effects of circPTPRA on IGF2BP1 and screened out its target genes through RNA sequencing. Finally, we explored the underlying molecular mechanisms that circPTPRA might act as a blocker in recognition of m⁶A. Results: We demonstrated that IGF2BP1 was predominantly binded with circPTPRA in the cytoplasm in BC cells. Ectopic expression of circPTPRA abolished the promotion of cell proliferation, migration and invasion of BC cells induced by IGF2BP1. Importantly, circPTPRA downregulated IGF2BP1-regulation of MYC and FSCN1 expression via interacting with IGF2BP1. Moreover, the recognition of m⁶A-modified RNAs mediated by IGF2BP1 was partly disturbed by circPTPRA through its interaction with KH domains of IGF2BP1. Conclusions: This study identifies exonic circular circPTPRA as a new tumor suppressor that inhibits cancer progression through endogenous blocking the recognition of IGF2BP1 to m⁶A-modified RNAs, indicating that circPTPRA may serve as an exploitable therapeutic target for patients with BC.