Casomorphine-10 (CM-10) Peptide Orchestrates Circadian and Neurodevelopmental Gene Clusters via δ-Opioid Receptor Signaling: Insights from Transcriptome Analysis with δ-Opioid Receptor-Expressing HEK293 Cells

<b>Background:</b> β-casomorphin-10 (CM-10), a peptide fragment derived from milk casein with the sequence YPFPGPIPNS, has demonstrated notable anxiolytic activity in BALB/c mice. Yet, its cellular responses and mechanistic pathways remain largely uncharacterized. <b>Methods:</b> We performed RNA-seq analysis to profile gene expression changes in δ-opioid receptor-expressing HEK293 cells (DOR-HEK), comparing CM-10-treated and untreated conditions. <b>Results:</b> CM-10 exposure led to differential expression of 1714 genes in DOR-HEK cells, with 34 upregulated (>1.4-fold) (1.9%) and 1680 downregulated (<0.71-fold) (98.1%), based on a predicted <i>p</i>-value threshold of <0.05. Notably, we identified 10 clusters that were associated with reduced cyclic AMP (cAMP) in DOR-HEK cells following CM-10 treatment. These clusters particularly involved genes related to regulatory subunits of cAMP-dependent protein kinases, such as <i>PRKAR2A</i>, cAMP-responsive element-binding pathway, circadian rhythms, such as <i>CLOCK</i>, <i>ARNT1</i>, <i>CRY2</i>, <i>PER1</i>, and <i>PER2</i>, and anxiety and depression, such as <i>NOTCH1</i>, <i>NOTCH2</i> and <i>ANK2</i>. A network with these selected genes was confirmed by STRING analysis. <b>Conclusions:</b> These findings indicate that CM-10 may activate DOR-mediated signaling by suppressing cAMP levels, implicating a distinct molecular cascade in HEK293 cells.