Paloviita, P.; Hautala, E.; Granskog, R.; Nykänen, S.; Pulkkanen, R.; Kirjanov, I.; Santaniemi, R.; Grym, H.; Hyden-Granskog, C.; Tuuri, T.; Vuoristo, S.

MicroRNA (miRNA) levels increase during human embryonic genome activation (EGA) and have been implicated in the first lineage decisions. Re-analyzing single-cell and bulk small RNA-seq (sRNA-seq) from human and mouse embryos, together with new sRNA-seq of naive human embryonic stem cells (hESCs) differentiated into hypoblast-like cells (HLCs) and extra-embryonic mesoderm (EXM)-like cells (EXMCs), we identify hsa-miR-92a-3p as highly expressed from oocyte to morula stage and dynamically regulated during HLC and EXMC formation. Functional inhibition of hsa-miR-92a-3p delays RACL (HLC/EXMC) differentiation, maintains epiblast-like and promotes trophectoderm (TE)-like transcriptional features, and reduces hypoblast and EXM marker acquisition. Transcriptome analyses revealed derepression of hsa-miR-92a-3p targets, including FGF2, and shifts in developmental and cell-cycle programs. The patterns of FGF protein stainings and flow cytometry-based cell-cycle analysis further implicate these pathways in RACL differentiation. Our findings position hsa-miR-92a-3p as a central regulator coordinating signaling and cell-cycle cues during extra embryonic lineage progression.