Circular RNA SMAD4 coordinates with m⁶A-dependent IGF2BP2 to promote gallbladder cancer development

Circular RNAs (circRNAs) have emerged as important regulators in tumorigenesis through microRNA sponging, protein translation and interaction with RNA-binding proteins. Here, we identified a novel circRNA termed circSMAD4 that was generated by alternative splicing from exon 2 to exon 10 and back to the beginning of exon 2 of SMAD4 pre-mRNA, constituting a 1435-nucleotide circular RNA. circSMAD4 was upregulated in gallbladder cancer (GBC) tissues and positively correlated with cancer progression. Elevated expression and tumorigenic activity of circSMAD4 mainly relied on its interaction with IGF2BP2, where m⁶A-binding motifs located at KH3 and KH4 of IGF2BP2 recognized and bound to the flanking intron sequences of circSMAD4. In the cytoplasm, IGF2BP2 stabilized circSMAD4 levels, and circSMAD4 in turn inhibited ubiquitination and degradation of IGF2BP2, thereby creating a positive feedback loop for both elevated expressions. Collaboration of circSMAD4 and IGF2BP2 activated JAK2/STAT3 and epithelial-mesenchymal transition (EMT) signalling. Notably, treatment of xenografted mouse models with CWI1-2, a small molecule for interruption of IGF2BP2 binding to circRNA, resulted in decreases in circSMAD4-induced tumour development. Collectively, our findings suggest that circSMAD4 cooperates with IGF2BP2 to promote tumour progression, highlighting the circSMAD4-IGF2BP2 complex as a potential therapeutic target in GBC.