β-catenin/TCF4/NANOG axis controls miR-302 transcription in colorectal cancer cells

The miR-302 cluster, a key pluripotency-associated non-coding RNA, has been implicated in stem cell homeostasis and tumourigenesis. However, its regulatory mechanisms in cancers, including colorectal cancer (CRC) remain poorly understood. Here, we demonstrate that the β-catenin/TCF4 complex significantly enhances miR-302 expression through direct promoter activation in CRC cells. We hypothesized that the β-catenin/TCF4 complex directly activates the miR-302 promoter and cooperates with NANOG in a transcriptional feedback loop sustaining stem-like traits in CRC cells. Using a combination of promoter-driven luciferase reporter assays, chromatin immunoprecipitation (ChIP), and molecular dynamics simulations, we identify a regulatory axis involving Wnt signalling and the transcription factor NANOG. Our data show that individual members of the miR-302 cluster activate the NANOG promoter, while both NANOG and β-catenin/TCF4 synergistically enhance miR-302 promoter activity, suggesting the presence of a positive feedback loop. Structural simulations further elucidate the binding interactions between TCF4, NANOG, and the miR-302 promoter, corroborating our experimental observations. Together, these findings position miR-302 as a downstream effector of Wnt/β-catenin signalling and an integral component of NANOG-mediated transcriptional networks in CRC stem-like cells. This work advances our understanding of non-coding RNA regulation in cancer and highlights potential therapeutic opportunities for targeting stemness-associated pathways.