Opioid-Induced Regulation of Cortical Circular- Grin2b _011731 Is Associated with Regulation of circGrin2b Sponge Target miR-26b-3p

Abstract

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_circGrin2b_011731 (circGrin2b), is upregulated in the OFC of rats following chronic self-administration (SA) of the opioid heroin. circGrin2b is derived from Grin2b, which encodes the regulatory subunit of the glutamate ionotropic NMDA receptor, GluN2B. However, the upstream regulatory mechanisms of circGrin2b biogenesis and the downstream consequences of circGrin2b dysregulation remain unknown. We hypothesized that opioid-induced elevation of circGrin2b is accompanied by regulation of circRNA biogenesis enzymes, and that circGrin2b 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 circGrin2b expression following exposure to the opioid morphine. We measured mRNA expression of known circRNA splicing factors and observed significant downregulation of Fused in Sarcoma (Fus), a negative regulator of circRNA biogenesis, following 90 min or 24 h of morphine exposure. Downregulation of Fus at 24 h post-morphine was accompanied by upregulation of circGrin2b and downregulation of miR-26b-3p, a predicted miRNA target of circGrin2b. Luciferase reporter assays confirmed interaction of miR-26b-3p with circGrin2b. Finally, we report a significant negative relationship between circGrin2b and miR-26b-3p expression in the OFC of rats following heroin SA. We conclude that regulation of circGrin2b is an opioid-induced neuroadaptation that may impact downstream signaling of miRNA pathways in the frontal cortex.

Keywords: circular RNA; microRNA; morphine; opioids; splicing.

Figure 1

Morphine exposure regulates expression of enzymes involved in circRNA biogenesis. (A) Overview of experimental design and treatment of primary cortical cells with morphine. (B–G) Expression of putative circRNA biogenesis enzymes following 24 h morphine exposure (B,D,F) and 90 min (C,E,G) morphine exposure for Fus (B,C), Adar1 (D,E), and Qki (F,G), Error bars indicate SEM. ** and * above the solid line indicate one-way ANOVA, p < 0.01 and p < 0.05, respectively. ** and * directly above histogram indicate p < 0.01 and p < 0.05 versus vehicle, respectively, for post hoc tests. ns, not significant.

Figure 2

Morphine exposure regulates expression of circGrin2b in primary cortical cultures. (A–D) Expression of circGrin2b (A,C) and linear Grin2b (B,D) at 24 h (A,B), or 90 min (C,D) after morphine exposure. Error bars indicate the standard error of the mean (SEM). * above the solid line indicates one-way ANOVA, p < 0.05. # above the solid line denotes one-way ANOVA p < 0.1 but p > 0.05; * directly above histogram indicate p < 0.05 versus vehicle post hoc test.

Figure 3

Morphine exposure regulates expression of miRNAs with putative circGrin2b binding sites. miRNA expression in primary cortical cultures following morphine treatment for miR-26b-3p (A), miR-100-3p (B), miR-350 (C), and miR-382-5p (D). Error bars indicate the standard error of the mean (SEM). # above the solid line denotes one-way ANOVA p < 0.1 but p > 0.05; * directly above histogram indicate p < 0.05 versus vehicle post hoc test. ns, not significant.

Figure 4

circGrin2b sponges miR-26b-3p. (A) Relative luminescence values in HEK293T cells following co-transfection of circGrin2b pMirTarget vector, a miR-26b-3p mimic, or a non-targeting control mimic. Mimics alone were used as a negative control and did not produce chemiluminescence. (B) Proposed mechanisms for circGrin2b-mediated opioid-induced neuroadaptations. Following 24 h morphine exposure, an increase in circGrin2b expression results in an elevation of circGrin2b available to interact with other targets, such as miR-26b-3p. * p < 0.05, *** p < 0.001, ns, not significant.

Figure 5

circGrin2b is negatively correlated with miR-26b-3p levels in the rat frontal cortex following opioid self-administration. (A) The average lever responses on the active lever paired with saline or heroin infusions, or an inactive lever during SA. Each dot represents the average lever responses for one day of SA. (B,C) Correlation between OFC circGrin2b expression with the putative miRNA target miR-26b-3p in rats that self-administered saline (B) or heroin (C).