Opioid-Induced Regulation of Cortical Circular-Grin2b_011731 Is Associated with Regulation of circGrin2b Sponge Target miR-26b-3p

Opioid use induces neurobiological adaptations throughout mesolimbic brain regions, such as the orbitofrontal cortex (OFC), which mediates decision-making and emotional-cognitive regulation. Previously, we showed that a circular RNA (circRNA) species, rno_<i>circGrin2b</i>_011731 (<i>circGrin2b</i>), is upregulated in the OFC of rats following chronic self-administration (SA) of the opioid heroin. <i>circGrin2b</i> is derived from <i>Grin2b</i>, which encodes the regulatory subunit of the glutamate ionotropic NMDA receptor, GluN2B. However, the upstream regulatory mechanisms of <i>circGrin2b</i> biogenesis and the downstream consequences of <i>circGrin2b</i> dysregulation remain unknown. We hypothesized that opioid-induced elevation of <i>circGrin2b</i> is accompanied by regulation of circRNA biogenesis enzymes, and that <i>circGrin2b</i> may sponge microRNAs (miRNAs), as miRNA sponging is a well-described characteristic of circRNAs. To test these hypotheses, we established an in vitro primary cortical cell culture model to examine alterations in <i>circGrin2b</i> expression following exposure to the opioid morphine. We measured mRNA expression of known circRNA splicing factors and observed significant downregulation of Fused in Sarcoma (<i>Fus</i>), a negative regulator of circRNA biogenesis, following 90 min or 24 h of morphine exposure. Downregulation of <i>Fus</i> at 24 h post-morphine was accompanied by upregulation of <i>circGrin2b</i> and downregulation of <i>miR-26b-3p</i>, a predicted miRNA target of <i>circGrin2b</i>. Luciferase reporter assays confirmed interaction of <i>miR-26b-3p</i> with <i>circGrin2b</i>. Finally, we report a significant negative relationship between <i>circGrin2b</i> and <i>miR-26b-3p</i> expression in the OFC of rats following heroin SA. We conclude that regulation of <i>circGrin2b</i> is an opioid-induced neuroadaptation that may impact downstream signaling of miRNA pathways in the frontal cortex.