MicroRNAs and predicted targets in the switch from monolayered to spheroids of cholangiocarcinoma cells

Background: Extrahepatic cholangiocarcinoma (eCCA) is characterized by marked molecular heterogeneity and limited therapeutic options. MicroRNAs (miRNAs) are key post-transcriptional regulators of cancer-related pathways, but their contribution to tumor adaptation in physiologically relevant models remains poorly understood. Three-dimensional (3D) tumor spheroids better mimic in vivo conditions than conventional two-dimensional (2D) cultures. Methods: We compared miRNA expression profiles in two eCCA cell lines (Sk-ChA-1 and Mz-ChA-1) grown as monolayers (2D) or multicellular tumor spheroids (3D). MiRNA profiling was performed using NanoString technology. Predicted targets were analyzed by over-representation analysis, and selected miRNAs and genes were validated by RT-qPCR and ELISA-based assays. Results: 3D growth induced extensive miRNA remodeling, with distinct (54 deregulated in Sk-ChA-1 and 29 in Mz-ChA-1 cells) and partially overlapping signatures (miR-1283, miR-577, and miR-2113). Among the shared miRNAs, predicted targets included DUSP10 and RBFOX1, while in spheroids, cell-specific multiple miRNAs converged on shared targets (TNRC6B, SMARCAD1, ATG14, HMGA2, and CLOCK) displaying inverse expression patterns. The transcriptional program impacted MAPK signaling, enhanced EMT, and activated stress-adaptive networks but attenuated proliferation in 3D Sk-ChA-1 cells, while Mz-ChA-1 cells retained a more epithelial and proliferative profile. In this context, we point out the involvement of miR-19b-3p using anti-miR transfection experiments. Conclusion: Our findings reveal a miRNA-driven regulatory landscape associated with 3D growth in eCCA, linking tumor architecture to signaling rewiring and cellular plasticity, and highlight potentially druggable candidate targets and pathways to investigate as candidates using inhibitors or gene therapy-based interventions.