ACCELERATED COMMUNICATION Long-Term Morphine Treatment Decreases the Association of -Opioid Receptor (MOR1) mRNA with Polysomes through miRNA23b

-Opioid receptor (MOR) mediates most of the pharmacological effects of opioid drugs. The expression of MOR is temporarily and spatially regulated at both the transcriptional and post-transcriptional levels. Long-term morphine treatment that induces tolerance does not alter MOR mRNA expression, suggesting no direct link between agonist treatment and MOR gene transcription. We previously identified the 3 -untranslated region (3 -UTR) of the major transcript of -opioid receptor (MOR1) and revealed a novel trans-acting factor, miRNA23b, that binds to the K box motif in the 3 -UTR. The interaction between miRNA23b with the MOR1 3 -UTR suppressed receptor translation by inhibiting polysome–mRNA association. In this report, we demonstrate that long-term morphine treatment increases miRNA23b expression in a doseand time-dependent manner and represses the association of MOR1 mRNA with polysomes through the MOR1 3 -UTR. The translational luciferase reporter assay shows a suppression effect of morphine on reporter activity that requires the MOR1 3 -UTR. This suggests a potential link between MOR expression and morphine treatment at the post-transcriptional level in which a specific miRNA, miRNA23b, is involved. Opioid drugs are widely used clinically to treat moderate to severe pain. Three major opioid receptors, , , and , belong to the G-protein-coupled receptor superfamily (Kieffer, 1995). -Opioid receptor (MOR) mediates most of the pharmacological effects of opiates; its regulation is of great importance to unravel the molecular mechanisms underlying the physical responses to opioid treatment, such as tolerance and dependence. In addition to the multiple cis-acting elements that regulate the transcription of MOR (Hwang et al., 2004; Kim et al., 2006; Choi et al., 2007; Song et al., 2007), a recent study on the 3 -UTR of the major -opioid receptor mRNA (MOR1) has started to address the regulation of MOR at the post-transcriptional level (Wu et al., 2008). The phenomenon of MOR down-regulation has been observed in various cell lines and neurons caused by long-term agonist treatment (Yabaluri and Medzihradsky, 1997; Tao et al., 1998; Yamamoto et al., 2008). Down-regulation results primarily from the sequestration of membrane receptors to the cytosol via clathrin-coated pits and dynamin after receptor phosphorylation; the internalized endosomes merge with intracellular lysosomes, and the receptors are degraded proteolytically, resulting in a decrease of the total number of receptors in the cell (Binyaminy et al., 2008). In addition to this classic model, accumulating evidence shows the involvement of many other factors in the processes [e.g., protein kinase C (Kramer and Simon, 1999), mitogen-activated protein kinase (Schmidt et al., 2000) and Ca /calmodulin-dependent protein kinase (Koch et al., 1997)]. However, whether a decreased receptor biosynthesis is involved is still under debate. One study measured the receptor turnover This work was supported by the National Institutes of Health National Institute on Drug Abuse [Grants DA00564, DA01583, DA11806, DA11190, K05-DA00153, K05-DA70554, K02-DA13926] and the F&A Stark Fund of the Minnesota Medical Foundation. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.108.053462. □S The online version of this article (available at http://molpharm. aspetjournals.org) contains supplemental material. ABBREVIATIONS: MOR, -opioid receptor; 3 -UTR, 3 -untranslated region; MOR1, major -opioid receptor transcript; RT-PCR, reverse transcription-polymerase chain reaction; qPCR, quantitative polymerase chain reaction; qRT-PCR, quantitative real-time polymerase chain reaction; HA, hemagglutinin; SBA, sodium butyric acid; miRNA, microRNA. 0026-895X/09/7504-744–750$20.00 MOLECULAR PHARMACOLOGY Vol. 75, No. 4 Copyright © 2009 The American Society for Pharmacology and Experimental Therapeutics 53462/3451742 Mol Pharmacol 75:744–750, 2009 Printed in U.S.A. 744 http://molpharm.aspetjournals.org/content/suppl/2009/01/14/mol.108.053462.DC1 Supplemental material to this article can be found at: at A PE T Jornals on Jne 0, 2017 m oharm .aspeurnals.org D ow nladed from rate in mouse neuronal N2A cells expressing a cloned -opioid receptor (N2A-MOR) and found that the down-regulation of MOR caused by agonist stimulation is the sum of both accelerated receptor degradation and decreased receptor biosynthesis (Afify, 2002). The expression of MOR can be regulated at both the transcriptional and post-transcriptional levels. It is widely accepted that long-term morphine treatment does not alter MOR mRNA levels (Brodsky et al., 1995), indicating that morphine has no significant effect on the transcription of MOR gene. Nonetheless, it is not known whether morphine can regulate MOR mRNA at the post-transcriptional level, possibly through interactions between trans-acting factors and its 3 -UTR. Although morphine can induce discrete and fluctuating expression of important factors related to MOR function (Ammon-Treiber and Hollt, 2005), whether these factors are involved in the post-transcriptional regulation of MOR remains unknown. Our recent study identified miRNA23b as a trans-acting factor that represses MOR translation efficiency through interaction with the 3 -UTR of MOR1 (Wu et al., 2008). Little is known about the mechanisms responsible for regulating miRNA expression. In some cells, the production of miRNAs seems to be actively regulated (Woods et al., 2007; Boyd, 2008). It would be interesting to investigate whether activating a membrane receptor could control the expression of a miRNA, thereby regulating expression of the receptor gene. In this report, we investigated whether morphine treatment could change miRNA23b expression and consequently regulate the translation of MOR mRNA. miRNA23b interacts with the MOR1 3 -UTR through binding to a K Box motif (Wu et al., 2008). The MOR1 3 -UTR is absent from the plasmid DNA sequences in all cell lines that express cloned MOR, such as N2A-MOR and HEK-MOR (Chakrabarti et al., 1995; Wu and Wong, 2005). N2A cells are not known to express any opioid receptors (Im et al., 2001); N2A-MOR cells were established by stably transfecting MOR1 into N2A cells. MOR1, a 1.4-kb insert, was subcloned into the expression vector pRC/CMV; the cDNA sequence contains the entire coding region of the MOR, together with 200 base pairs of 5 noncoding region and only 30 base pairs of 3 noncoding region (i.e., the 3 -UTR). The cloned MOR (without the major part of MOR1 3 -UTR sequence) behaves like native receptor in terms of its desensitization and downregulation effects (Chakrabarti et al., 1995). However, it has not yet been determined whether the absence of MOR1 3 UTR influences the regulation of MOR gene. In this report, we investigated the effect of morphine on miRNA23b expression in N2A-MOR cells and the resulting changes in MOR1 mRNA translation efficiency. In addition, we revealed the critical roles of MOR receptor and MOR1 3 -UTR in this pathway. Materials and Methods Cell Culture, Transfection, and Luciferase Reporter Assay. Mouse neuronal cells N2A and N2A-MOR (Chakrabarti et al., 1995) and human neuronal cells NMB and SHSY-5Y were maintained in advanced Dulbecco’s modified Eagle’s medium or RPMI 1640 medium (for NMB) (Invitrogen, Carlsbad, CA) with 5% heat-inactivated fetal bovine serum in an atmosphere of 10% (for N2A and N2A-MOR) or 5% (for NMB and SHSY-5Y) CO2 at 37°C. The medium for N2AMOR was supplemented with 0.2% G418 (Geneticin). Transfections of anti-23b or anti-miR negative control primer (Ambion, Austin, TX) were performed using Lipofectamine 2000 (Invitrogen) as described previously (Wu et al., 2008). For the luciferase reporter assay, cells were plated at a density of 0.5 10 cells per well in 24-well plates 24 h before transfection; 2 ng of Renilla reniformis luciferase plasmid pCMV-Rluc (a gift from Dr. Yan Zeng, University of Minnesota, Minneapolis, MN) was included for normalization. Morphine was added 3 h before transfecting pSVUTR or pSVPA plasmids (Wu et al., 2008). Twenty-four hours after transfection, the firefly and R. reniformis luciferase activities were determined by a luminometer (Berthold, Oak Ridge, TN) using Dual-Luciferase Reporter Assay systems (Promega, Madison, WI) according to the manufacturer’s protocol. RT-PCR, Real-Time qPCR, and qRT-PCR. RNA was isolated from cells using TRI reagent (Molecular Research Center, Cincinnati, OH) and treated with Turbo DNase I (2 U/ g of RNA) (Ambion) before being reverse-transcribed. One-step RT-PCR was performed using the OneStep RT-PCR Kit (QIAGEN, Valencia, CA) and the following primers: mouse MOR1, 5 -CTGCTCGAATCCGTCAAAACA-3 (sense) and 5 -AGCAACCTGATTCCAAGTAGA-3 (antisense); HA-MOR1, 5 -CTGCTCGAATCCGTCAAAACA-3 (sense) and 5 GGCAACTAGAAGGCACAGTC-3 (antisense); and mouse -actin, 5 -TGGCCTTAGGGTGCAGGGGG-3 (sense) and 5 -GTGGGCCGCTCTAGGCACCA-3 (antisense). For MOR1 RNA, the product from the one-step RT-PCR was re-amplified for a second round using Taq polymerase (New England Biolabs, Ipswich, MA) and primers: 5 -CTGCTCGAATCCGTCAAAACA-3 (sense) and 5 -GTAGATGGCAGCCTCTAA-3 (antisense). PCR was performed on a GeneAmp PCR System 9600 (PerkinElmer Life and Analytical Sciences, Waltham, MA) using 30 cycles (for MOR1 and HA-MOR1) or 20 cycles (for -actin) of 94°C for 1 min, 55°C for 1 min, and 72°C for 1 min followed by 72°C for 10 min. The linear range of PCR cycles for each gene had been predetermined using relative PCR, and cycle numbers for PCR and RT-PCR were optimized according to the results. PCR products were electrophoresed in 1 or 2% agarose gels, quantified by ImageQuant 5.2 (GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK) and verified by DNA sequence analysis. miRNA-enriched RNA was extracted and reverse transcribed followed by qPCR as described before (Wu et al., 2008). One-tenth of the reverse transcription mix was used for real-time qPCR. The miRNA primer sets hsa-miR23b and snoRNA234 (as an internal control) (Applied Biosystems) were used for reverse transcription, and qPCR was performed according to the manufacturer’s protocol. Real-time qPCR and qRT-PCR were performed on an iCycler (BioRad Laboratories, Oakland, CA) using either an iQ Supermix Kit (Bio-Rad) for miRNA23b and snoRNA234 or a Quantitect SYBR Green RT-PCR kit (QIAGEN) for MOR1, HA-MOR1 and -actin. The relative expression levels of miRNA23b were calculated using the Gene Expression Macro (Bio-Rad Laboratories, Hercules, CA) normalized to those of snoRNA234; and the levels of MOR1 and HAMOR1 were calculated against those of -actin. Polysome mRNA Extraction. Polysome mRNA extraction was conducted as described previously (Wu et al., 2008). Polysomal mRNA was isolated from pellets using TRI reagent (Molecular Research Center) following the manufacturer’s protocol. Statistics. Data are presented as mean values S.D. Comparisons between groups were performed using the Student’s t test. P 0.05 was taken as significant.