The analysis of miRNA–mRNA regulatory networks using differentially expressed (absolute FC > 1.3 & P < 0.05) and negatively correlated miRNA–mRNA pairs identified in ethanol-exposed hESC-derived cortical interneurons indicated that four AUD-relevant pathways (Sirtuin Signaling, Opioid Signaling, NRF2-mediated Oxidative Stress Response, and IL-1 Signaling) could be regulated by nine miRNAs (3 upregulated and 6 downregulated) and six paired mRNAs (1 upregulated and 5 downregulated) (Fig. S15). In total, 17 canonical pathways potentially regulated by differentially expressed and negatively correlated miRNA–mRNA pairs were identified in eight brain region of AUD subjects and ethanol-exposed hESC-derived cortical interneurons (Table S8). The top three pathways potentially regulated by AUD-associated and negatively correlated miRNA–mRNA pairs in multiple brain regions included CREB Signaling in Neurons, IL-8 Signaling, and Axonal Guidance Signaling. In addition, three AUD-associated miRNA–mRNA regulatory pathways (Opioid Signaling Pathway, NRF-mediated Oxidative Stress Response, and Sirtuin Signaling) were confirmed by the in vitro cellular model study (Table S8).
