let-7c-5p drives collagen gene repression linking phenobarbital teratogenicity to cleft palate

The treatment of seizures during pregnancy remains challenging due to the teratogenic effects of many anticonvulsants. Phenobarbital (PB), despite its known risks, is widely used and associated with congenital malformations such as cleft palate (CP). CP is a common craniofacial defect resulting from disrupted palatogenesis, influenced by genetic and environmental factors, including maternal medication. MicroRNAs (miRNAs) are key post-transcriptional regulators implicated in palate development and possibly modulated by teratogenic agents. This study investigated whether miRNA dysregulation observed in CP patients reflects epigenetic mechanisms induced by PB in human embryonic palatal mesenchymal (HEPM) cells. miRNAs dysregulated in palatal tissues from CP patients were previously identified by our group. PB-treated HEPM cells exhibited significant alterations in miRNA expression, notably downregulation of miR-29b-3p and upregulation of let-7c-5p, consistent with the miRNA expression patterns previously observed in CP patient tissues through multiplexed profiling. Bioinformatic analyses predicted collagen fibril organization as a common targeted pathway. Quantitative RT-PCR validation confirmed downregulation of key collagen genes in CP patient tissues and PB-treated HEPM cells. Functional inhibition of let-7c-5p restored COL1A2 and COL3A1 expression, suggesting let-7c-5p mediates collagen gene repression in response to PB and potentially contributes to CP pathogenesis. The findings reveal a shared epigenetic regulatory axis involving let-7c-5p and collagen genes linking PB teratogenicity and CP , providing new insights into extracellular matrix remodeling during palatogenesis.