mu-Opioid receptor agonists differentially regulate the expression of miR-190 and NeuroD

Abstract

The agonists of mu-opioid receptor (OPRM1) induce extracellular signal-regulated kinase (ERK) phosphorylation through different pathways: morphine uses the protein kinase C (PKC)-pathway, whereas fentanyl functions in a beta-arrestin2-dependent manner. In addition, the two pathways result in the different cellular location of phosphorylated ERK and the activation of different sets of transcriptional factors. In the current study, the influence of the two pathways on the expression of microRNAs (miRNAs) was investigated. After treating the primary culture of rat hippocampal neurons and the mouse hippocampi with morphine or fentanyl for 3 days, seven miRNAs regulated by one or two of the agonists were identified. One of the identified miRNAs, miR-190, was down-regulated by fentanyl but not by morphine. This down-regulation was attenuated by 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene (U0126), which blocks the phosphorylation of ERK. When fentanyl-induced but not morphine-induced ERK phosphorylation was blocked in the primary cultures from beta-arrestin2(-/-) mouse, fentanyl did not decrease the expression of miR-190. However, a PKC inhibitor that blocked morphine-induced ERK phosphorylation specifically had no effect on the miR-190 down-regulation. Therefore the decrease in miR-190 expression resulted from the agonist-selective ERK phosphorylation. In addition, the expressional changes in one of the miR-190 targets, neurogenic differentiation 1 (NeuroD), correlated with those in miR-190 expression, suggesting the OPRM1 could regulate the NeuroD pathways via the control of miR-190 expression.

Fig. 1.

Morphine and fentanyl are used at equivalent doses. A and B, dose-dependent curves of morphine (A) and fentanyl (B) to induce ERK phosphorylation in rat primary cultures. Cultures were treated with indicated concentrations of agonists for 5 min. The immunoreactivities of phosphorylated ERK were normalized against those of total ERK. Then the normalized results were compared with those obtained from untreated rat primary cultures (control). EC₅₀ was calculated by averaging the EC₅₀ values generated in each repetition of the experiments. Experiments were repeated for four times. The error bar represents S.D. C and D, dose- and time-dependent curves of morphine (C) and fentanyl (D) to induce analgesia effect in CD1 mouse. Mice were subcutaneously injected with the indicated doses of agonists for indicated times. The %MPE and ED₅₀ were determined as described under Materials and Methods. The ED₅₀ values of morphine and fentanyl were calculated depending on the %MPEs at 30 min and at 15 min (the peaks of analgesia effect), respectively. Each point in the graph included 8 to 12 mice. The error bar presented the S.E.

Fig. 2.

Agonist-selective regulation on miR-190 expression. A, time-dependent abilities of morphine and fentanyl to modulate the expression of miR-190 in rat primary cultures. Cultures were treated with 1 μM morphine or 10 nM fentanyl for indicated times. The expression of miR-190 was determined by real-time PCR and normalized against the mRNA level of β-actin as described under Materials and Methods. The normalized results were further normalized against the results in untreated cultures (0 h). B, dose-dependent curves of morphine and fentanyl to modulate the expression of miR-190 in rat primary cultures. Cultures were treated with indicated doses of agonists for 3 days. The expression of miR-190 was determined as in A. Experiments were repeated four times. Data were analyzed by one-way ANOVA with post hoc Dunnett test for comparisons. Error bars, S.D.; *, significant changes.

Fig. 3.

ERK phosphorylation is required for the down-regulation of miR-190l. A, rat primary cultures were treated with PBS, 10 μM naloxone, 10 μM CTOP, or 10 μM TIPPψ for 3 h. Then the cultures were treated with 1 μM morphine or 10 nM fentanyl for an additional 3 days. The expression of miR-190 was determined by real-time PCR. The results of miR-190 were normalized against those of β-actin and further normalized against the result obtained from untreated cultures (control) in the PBS group. B, rat primary cultures were treated with PBS, 0.2% DMSO, 2 μM U0126, 4 μM Ro-31-8425, 10 μM PP2, or 10 μM U73122 for 3 h. Then the cultures were treated with 1 μM morphine or 10 nM fentanyl for an additional 3 days. The expression of miR-190 was determined as in A. Experiments were repeated four times. Data were analyzed by two-way ANOVA with post hoc Bonferroni test for comparisons. Error bars, S.D.; *, significant changes.

Fig. 4.

β-arretsin2-mediated ERK phosphorylation is required for the down-regulation of miR-190. Primary cultures from wildtype C57/BL6 mice were treated with PBS, 0.2% DMSO, 2 μM U0126 or 4 μM Ro-31-8425 for 3 h. Primary cultures from β-arretsin2−/− C57/BL6 mice were treated with PBS for 3 h. Then the cultures were treated with 1 μM morphine or 10 nM fentanyl for additional three days. The expression of miR-190 was determined by real-time PCR. The results of miR-190 were normalized against those of β-actin, and further normalized against the result obtained from untreated cultures (control) in “PBS+wild type” group. Experiments were repeated for four times. Data were analyzed by two-way ANOVA with post hoc Bonferroni test for comparisons. Error bars, S.D.; *, significant changes.

Fig. 5.

NeuroD expression was consistent with miR-190 expression. Rat primary cultures were treated with PBS, 10 μM naloxone, 10 μM CTOP, 10 μM TIPPψ, or 2 μM U0126 for 3 h. Then the cultures were treated with 1 μM morphine or 10 nM fentanyl for an additional 3 days. The mRNA levels of NeuroD were determined by real-time PCR (A). The PCR results of NeuroD were normalized against those of β-actin and further normalized against the result obtained from untreated cultures (control) in the PBS group. The protein levels of NeuroD (B) were determined by immunoblotting after nuclear extraction as described under Materials and Methods. The immunoreactivities of NeuroD were normalized against those of total ERK and further normalized against the result obtained from untreated cultures (control) in the PBS group. Experiments were repeated for four times. Data were analyzed by two-way ANOVA with post hoc Bonferroni test for comparisons. Error bars, S.D.; *, significant changes.

Fig. 6.

NeuroD is one target of miR-190. A and B, rat primary cultures were transfected with control RNA, miR-190 mimic, or miR-190 inhibitor by using Lipofectamine 2000. Two days after transfection, the mRNA (A) and protein (B) levels of NeuroD were determined by real-time PCR and immunoblotting, respectively. The results were normalized against internal control (β-actin for mRNA and total ERK for protein) and further normalized against the results obtained from cultures transfected with control RNA. Data were analyzed by one-way ANOVA with Dunnett test as post hoc test to do comparisons. C, schematics of the 3UTR and 3UTRmu reporters. The first nucleotide after the stop codon of rat NeuroD mRNA is designated as number 1. D, HEK293 cells were transfected with one of the RNAs, one of the reporters, and the luciferase reporter system by using Lipofectamine 2000. RNAs included control RNA, miR-190 mimic, and miR-190 inhibitor. Reporters included vector, 3UTR, and 3UTRmu. The luciferase expression was determined as described under Materials and Methods. The results were normalized against internal control (R. reniformis luciferase) and further normalized against the results obtained from cultures transfected with control RNA in each group. Data were analyzed by two-way ANOVA with post hoc Bonferroni test for comparisons. Experiments (A–D) were repeated four times. Error bars, S.D.; *, significant changes.