Toward Understanding the Role of miRNAs in Cleft Palate Only: Observations from Patient Tissues and In Vitro Assays

Cleft palate only (CPO) is a multifactorial craniofacial malformation with significant genetic and epigenetic contributions. Among these, microRNAs (miRNAs) have emerged as key regulators of palate development, although their precise role in CPO pathogenesis remains incompletely elucidated. In this study, we performed a comprehensive miRNA expression analysis on palatal tissues from an Italian cohort of non-syndromic CPO patients, compared with a human embryonic palate mesenchymal (HEPM) cell line. Using the NanoString® nCounter platform for miRNA profiling, we identified significant deregulation of several miRNAs, notably the upregulation of miR-205-5p and miR-200c-3p, and the downregulation of miR-125a-5p. Based on these expression changes, a functional analysis was carried out to identify potential target genes. Validation in primary cell cultures derived from patient tissues confirmed these expression patterns. Functional analyses and target predictions implicated PAX9, a key transcription factor essential for palatogenesis, as a probable target of miR-205-5p, while miR-125a-5p was associated with the regulation of PRTG and PRSS35, genes involved respectively in neural crest cell biology and extracellular matrix remodeling. Although modulation of certain predicted targets of miR-200c-3p was observed, in vitro inhibition experiments did not show significant changes in gene expression, highlighting the complexity of miRNA regulatory networks and the need for further studies to unravel these interactions. Altogether, these findings advance the understanding of miRNA-mediated molecular mechanisms in CPO and identify novel candidate pathways for further mechanistic and therapeutic investigation.