Opioids are widely used in the clinic to treat moderate to severe pain. However, the development of tolerance after chronic treatment posed major medical concern. Since the cloning of opioid receptors (μ, δ, and κ or mu, delta, and kappa) in the early 90s, their spatial and temporal regulation has been intensively studied. μ-opioid receptor (MOR) is the major molecular target of opioids and understanding its regulation is critical to unravel the mechanism underlying the pharmacological effects of opioid drugs. Gene regulation can be exerted at both transcriptional and post-transcriptional levels. Early studies have demonstrated a lack of correlation between morphine treatment and MOR expression at the transcriptional level. The post-transcriptional regulation of MOR remains unclear.
In this study, we identified the major transcript of MOR (MOR1) and its contiguous 3'-UTR, which extends more than 10 kb from the stop codon. The MOR 3'-UTR uses a single poly (A) signal 10153 bp downstream of the stop codon and is cleaved at the CA cleavage site 26 bp downstream of the poly (A) signal. The MOR1 poly (A) signal demonstrates a distinct preference to its own promoter in the luciferase assay, indicating a functional coupling between the promoter and poly (A) signal. When cloned into a reporter construct, the MOR1 3'-UTR showed a robust inhibitory effect independent of the promoter type and cellular background, which suggests a translational suppression. This is confirmed by the fact that MOR1 3'-UTR did not change the total mRNA level of the reporter gene but sharply decreased its polysome mRNA level. To identify the cis - and trans - interactions in the 3'-UTR, we predicted three putative cis -acting motifs (two K Boxes and one Brd Box) using the UTRscan program (http://bighost.ba.itb.cnr.it/BIG/UTRScan). The mutagenesis analysis confirmed the function of K Box1 (3802-3807 bp downstream of the stop codon). miRNA23b was predicted to interact with the K Box motif. Over-expressing miRNA23b in NS20Y cells inhibits the luciferase activity in the plasmid with MOR1 3'-UTR, but not in the one without MOR1 3'-UTR. K Box1 mutation diminished the effect of miRNA23b, suggesting a specific interaction between miRNA23b and K Box1 in the MOR1 3'-UTR. miRNA23b represses the translation efficiency of the reporter mRNA by inhibiting its association with polysomes. Knocking down the miRNA23b in NS20Y cells induced a significant increase of MOR protein level, thereby confirming the role of miRNA23b in regulating endogenous MOR expression. Interestingly, chronic morphine treatment increases the expression of miRNA23b and subsequently decreases the polysome association of MOR1 mRNA in which the MOR receptor and MOR1 3'-UTR are required.
In summary, this study identified the 3'-UTR of MOR1 and revealed a novel type of trans -acting factor for MOR gene, miRNA23b that binds to the K Box in the 3'-UTR and represses translation by inhibiting the polysome mRNA association. Chronic morphine treatment increases the miRNA23b expression and inhibits the polysome association of MOR1 mRNA. This suggests a pathway that transduces the membrane signal to regulate the intracellular MOR expression as part of the post-transcriptional regulation of μ-opioid receptor.