A miRNA screen reveals a transcriptional network controlling the initiation of mammary organoids

Organoid formation is driven by poorly understood intrinsic cellular properties and transcriptional programs that govern plasticity and differentiation. Deciphering these regulatory networks is essential for understanding normal tissue homeostasis and tumor initiation. Using a 3D organotypic model, which better recapitulates cell-matrix interactions and biochemical cues, we performed a miRNA-based screening strategy to identify key regulators of organoid initiation from human primary mammary epithelial cells. Our findings reveal that miR-106a-3p acts as a central modulator of mammary epithelial plasticity, enriching for stem/progenitor-like cells (CD44^high/CD24^low phenotype), driving organoid expansion, fostering K14⁺/K19⁺ lineage intermixing, and promoting branching morphogenesis characteristic of early ductal development. Further analysis revealed a core transcriptional network involving CBFB, NF-YA, GATA3, and REST, which supports organoid-forming potential. This regulatory program also induces a hybrid epithelial-mesenchymal transition (EMT) state, enhancing cellular plasticity while preserving organoid structural integrity. Extending these findings to cancer, we demonstrate that enforced expression of miR-106a-3p significantly increases tumoroid formation, suggesting that the tumor microenvironment, as modeled by 3D culture, promotes miR-106a-3p expression and functional relevance in tumorigenic processes. Collectively, these data indicate that miR-106a-3p drives a transient expansion of progenitor-like states and orchestrates transcriptional reprogramming during organoid initiation, with broader implications for breast tissue homeostasis and pathophysiological remodeling in cancer.