1、1Scientific RepoRts|6:38285|DOI:10.1038/ targets-arrestin 2 to reverse morphine tolerance in ratsJian Wang1,Wei Xu1,Tao Zhong1,Zongbin Song1,Yu Zou1,Zhuofeng Ding1,Qulian Guo1,Xinzhong Dong2,3&Wangyuan Zou1Morphine tolerance is a challenging clinical problem that limits its clinical application in p
2、ain treatment.Non-coding microRNAs(miRNAs)modulate gene expression in a post transcriptional manner,and their dysregulation causes various diseases.However,the significance of miRNAs in morphine tolerance is still poorly understood.In the present study,we hypothesized that microRNA-365(miR-365)is a
3、key functional small RNA that reverses morphine tolerance through regulation of-arrestin 2 in rats.Here,microarray analysis and quantitative real-time PCR showed that miR-365 was robustly decreased in the spinal cord after chronic morphine administration.In situ hybridization and immunochemistry dou
4、ble staining showed that miR-365 was expressed in neurons of the spinal cord.We identified-arrestin 2 as the target gene of miR-365 by bioinformatics analysis and luciferase reporter assay.The data showed that overexpression of miR-365 prevented and reversed established morphine tolerance,and increa
5、sed expression of miR-365 caused a decrease in expression of-arrestin 2 protein.miR-365 downregulation is involved in the development and maintenance of morphine tolerance through regulation of-arrestin 2,and miR-365 upregulation provides a promising and novel approach for treatment of morphine tole
6、rance.Morphine is commonly used in clinical management to alleviate moderate to severe pain.However,prolonged and repeated use of morphine leads to tolerance1.In some patients,even the maximum tolerated dose of mor-phine cannot achieve a sufficient analgesic effect2,3.Morphine tolerance is defined a
7、s a gradual loss of drug potency or efficacy and reduced duration of action.When morphine tolerance occurs,dose escalation becomes necessary to maintain the same analgesic effect,which results in an increased likelihood of side effects.Thus far,the mechanisms underlying morphine tolerance are poorly
8、 understood.Multiple factors may be involved in these complex processes,such as opioid receptor desensitization4,changes in glutamate receptor function5,6,protein kinase C(PKC)activation6,and G-protein uncoupling7.In addition,some studies reported that miRNAs play a vital role in regulating morphine
9、 tolerance and chronic pain810.miRNAs bind to 3-UTRs(untranslated region)of target gene mRNA,inhibiting or destabilizing trans-lation of the transcripts1113.miRNAs have been shown to modulate expression of various genes in different systems1417.Recent studies have found miRNAs are also involved in p
10、erioperative medicine,including inflam-mation,ischemia,sepsis and mediating anesthetic toxicity1822.However,the specific role of miRNAs in morphine tolerance is still poorly understood.Studies have reported that miRNAs are highly expressed in the CNS where opioid activity occurs,2326 and some are in
11、volved in morphine tolerance,such as fentanyl increased NeuroD protein level was mediated by miR-1908.He et al.10 have shown that the opioid receptor(MOR)mRNA 3-UTR has a binding site for let-7 and that let-7 could repress MOR expression in mice.Inhibition of let-7 can alleviate morphine tolerance.W
12、u et al.27 found that miRNA-23b could repress MOR translation by interacting with the K box motif in the 3-UTR of MOR 1,and the interaction could suppress receptor translation by blocking polysome-mRNA association.Morphine also decreased miR-133b expression and affected dopaminergic neuron different
13、iation in zebrafish embryos28.Therefore,the roles of miRNAs in regulating morphine tolerance are of growing interest.Here we present evidence that miR-365 is involved in the modulation of morphine tolerance.In the study,we focused on miR-365 for several reasons:Firstly,miR-365 is expressed in the CN
14、S and involved in neurological disorders29,suggesting a potential role of miR-365 in CNS dysfunction.Secondly,based on miRNA microarray profiling,miR-365 was robustly down-regulated in the spinal cord after chronic administration of morphine.1Department of Anesthesiology,Xiangya Hospital,Central Sou
15、th University,Changsha,Hunan 410008,China.2The Solomon H.Snyder Department of Neuroscience,Johns Hopkins University,School of Medicine,Baltimore,Maryland 21205,USA.3Howard Hughes Medical Institute,Johns Hopkins University School of Medicine,Baltimore,Maryland 21205,USA.Correspondence and requests fo
16、r materials should be addressed to W.Z.(email:)received:20 July 2016accepted:07 November 2016Published:06 December 2016OPEN RepoRts|6:38285|DOI:10.1038/srep38285Thirdly,-arrestin 2,which is an important modulator of morphine tolerance30,is the predicted target of miR-365,and there is little informat
17、ion concerning how miR-365 regulates development of morphine tolerance.Therefore,we designed the study to investigate the role of miR-365 in regulating morphine tolerance.We hypothesized that miR-365 in the spinal cord regulates development and maintenance of morphine antinociceptive tolerance by ta
18、rgeting the G-protein-coupled receptor(GPCR)binding protein -arrestin 2.ResultsmiR-365 is down-regulated in the spinal cord of morphine-tolerant rats.To identify miRNAs responsible for morphine tolerance,we profiled miRNA expression patterns through miRNA microarray analysis using L4 5 of spinal cor
19、ds isolated from rats subjected to injection of morphine for 7 consecutive days,which is a classic model to induce morphine tolerance.Morphine tolerance was already established 7 days after morphine injection(Fig.1A).Compared with the normal saline(NS)group(Fig.1B),we found a broad range of miRNA ex
20、pression changes in the morphine treated(MT)spinal cord.miR-365 was one of the most robustly reduced of these miRNAs,and the expression levels of miR-365 were confirmed by qPCR(Fig.1C).miR-365 expression was not altered on day 3 after morphine injection,however,on day 5 it began to decline(Fig.1D),a
21、nd these changes were consistent with development of morphine tolerance.In addition,among the target genes of miR-365,some target genes participate in morphine tolerance(described in the discussion).For this reason,miR-365 was studied further to determine its role in morphine tolerance.To explore fu
22、rther the localization and expression of miR-365 in the spinal cord,miRNA-specific in situ hybridization and staining were employed.The data showed that miR-365 was expressed in the dorsal horn of the spinal cord.Compared with the normal saline group,miR-365 expression was decreased on day 7 after m
23、orphine administration(Fig.1E).The result was consistent with previous quantitative RT-PCR analysis.Co-immunostaining revealed high expression of miR-365 in the neuron specific NeuN-positive neurons of the spinal dorsal horn(Fig.2).Lentivirus-mediated overexpression of miR-365 prevents and reverses
24、morphine tolerance in rats.To confirm delivery of lentivirus(LV)-miR-365 or LV-control into neuronal cells of the spinal cord,transfected cells in the spinal cord were visualized by GFP fluorescence.After intrathecal administration of Figure 1.Downregulation of miR-365 in the spinal cord after induc
25、tion of morphine tolerance.(A)Development of tolerance to morphine-induced antinociception assessed by the tail flick test.Tail flick latency was converted to%MPE.Values are expressed as mean s.e.m.,n=6,*P 0.001 compared with the normal saline group by two-way repeated-measures ANOVA followed by Bon
26、ferroni correction.(B)Heat maps of microRNAs shown by microarray analysis to be significantly upregulated or downregulated in the L4 5 spinal cord of rats following intrathecal injection of morphine compared with normal saline.The heat map diagram shows the result of log2 value of each miRNA microar
27、ray signal in different conditions(morphine or saline injection).Each row represents a miRNA and each column represents a sample.Color scale is shown on the top.(C)Representative examples of microRNAs showing upregulation or downregulation following independent verification with qRT-PCR analyses,n=3
28、.*P 0.01,*P 0.001 relative to the normal saline group by unpaired t-test.(D)Expression level of miR-365 in the L4 5 spinal cord after chronic injection of morphine.Data are expressed as mean s.e.m.,n=6,*P 0.001,versus the normal saline group by two-way repeated-measures ANOVA followed by Bonferroni
29、correction.(E)In situ hybridization images of miR-365 in the spinal cord from normal saline and morphine tolerance groups.n=4,*P 0.001,compared with the normal saline group by Students t-test.Scale bar=200 m.Each experiment was performed in duplicate with the indicated number of samples.MT=morphine
30、tolerance,NS=normal saline,miR=microRNA,MPE=maximum possible RepoRts|6:38285|DOI:10.1038/srep38285LV-miR-365 or LV-control,we found that many neuronal cell bodies were highly fluorescent,suggesting that the lentivirus was successfully transfected into neuronal cells(Fig.3A).miR-365 expression was gr
31、adually increased above basal levels 5 days after intrathecal LV-miR-365 injection and remained increased 10 days after treatment with LV-miR-365(Fig.3B).To explore the role of miR-365 in modulation of morphine tolerance at the behavioral level,we first pretreated rats with intrathecal injection of
32、LV-miR-365,LV-control vector,or vehicle(normal saline)3 days before chronic morphine or normal saline infu-sion.The basal latency of tail flick test was not changed in all groups(data were not shown).Rats treated with saline infusion(Vehicle+NS,LV-control+NS and LV-miR-365+NS)showed no significant d
33、ifference in%MPE dur-ing the 7 days.In rats treated with morphine infusion,rats pretreated with normal saline(Vehicle+Mor)showed 95.6 5.4%(assessed by%MPE)antinociception on day 1;however,from day 3 to day 7,%MPE was significantly reduced compared with day 1.In contrast,rats pretreated with LV-miR-3
34、65 maintained antinociception through day 7 after chronic morphine injection and showed a significant increase in%MPE compared with rats treated with normal saline.Rats pretreated with LV-control vector(LV-control+Mor)had a%MPE similar to the Vehicle+Mor group(Fig.3C).Then we post-treated rats with
35、LV-control or LV-miR-365 vector on day 7 after establishment of morphine tolerance.We found that overexpression of miR-365 significantly reversed morphine tolerance(Fig.3D).These results suggest that miR-365 in the spinal cord contributes to modulation of morphine tolerance.miR-365 directly targets-
36、arrestin 2.To identify target genes of miR-365 relevant to morphine tolerance,we searched for potential candidate targets using bioinformatics tools:TargetScan,miRanda,and miRDB.Among the putative genes identified,we focused on -arrestin 2 because it plays a vital role in morphine tolerance3033.Mice
37、 with a -arrestin 2 null mutation are less likely to develop morphine tolerance30.Therefore,we examined whether -arrestin 2 is a direct target of miR-365 using a luciferase assay.The 3-UTR sequence(including the predicted mir-365 target sequence)of -arrestin 2 was cloned into a reporter vector downs
38、tream of the Renilla luciferase gene.After cotransfection of HEK293 cells with the reporter vector containing miR-365,luciferase activ-ity with the -arrestin 2 3-UTR was attenuated(Fig.4A).To identify further the miR-365 binding sequence within the 3-UTR,we mutated the predicted seed sequence with a
39、 partial deletion(Fig.4B).We found that miR-365 had no effect on luciferase activity(Fig.4A).These findings suggest that miR-365 directly targets the -arrestin 2 3-UTR in a sequence-specific manner.Furthermore,Fig.4B shows that the miR-365 binding site is well conserved among mammals,which indicates
40、 that -arrestin 2 regulation by miR-365 is functionally important.Figure 2.Cell-specific localization of miR-365 in the spinal cord.Cell-specific localization of miR-365 in the spinal cord as assessed by in situ hybridization with subsequent coimmunofluorescence staining of the spinal cord for miR-3
41、65(red),neurons using NeuN antibody(green),astrocytes using GFAP antibody(green),and microglia using Iba1 antibody(green).Scale bar=50 RepoRts|6:38285|DOI:10.1038/srep38285Figure 3.Lentiviral-mediated overexpression of miR-365 prevents and reverses morphine tolerance in rats.(A)Representative image
42、of enhanced green fluorescent protein immunofluorescence in the spinal cord on day 7 after injection of LV-miRNA-365 or LV-control vector.Scale bar=100 m.(B)miR-365 expression levels in the L4 5 spinal cord after lentivirus injection.Rats were injected with control or miR-365 lentivirus vector 3 day
43、s before chronic normal saline infusion.Values are expressed as percentage of values of the LV-control+NS group,n=6,*P 0.001 by two-way repeated-measures ANOVA followed by Bonferroni test.(C and D)Effect of injection of lentivirus miR-365 on the development of morphine tolerance.(C)Groups of rats we
44、re pretreated with vehicle(normal saline),LV-control vector,or LV-miR-365 vector 3 days before chronic injection of morphine or saline.Data are presented as mean s.e.m,n=6,*P 0.001 compared with the Vehicle+NS group.#P 0.001 compared with the LV-control+Mor group by two-way repeated-measures ANOVA f
45、ollowed by Bonferroni test.(D)Groups of rats were injected with morphine twice a day for 17 consecutive days.LV-control vector or LV-miR-365 vector was administered on day 7 after establishment of morphine tolerance.Data are presented as mean s.e.m.,n=6,*P 0.01,*P 0.001 compared with the LV-control
46、group by two-way repeated-measures ANOVA followed by Bonferroni test.Each experiment was performed in triplicate with the indicated number of samples.miR=microRNA,MPE=maximum possible effect,NS=normal saline,Mor=morphine.Figure 4.miR-365 directly targets the-arrestin 2 3-UTR.(A)Activity of luciferas
47、e with the -arrestin 2 3-UTR or a deletion mutation in the 3-UTR in HEK293T cells cotransfected with control microRNA or a miR-365 mimic(n=3).Values are expressed as percentage of values of the control group,*P 0.001 by Students t test.Each experiment was performed in triplicate with the indicated n
48、umber of samples.(B)Schematic representation of the miR-365 sequence and its target sequence within the -arrestin 2 3-UTR.The target sequence is well conserved among mammals.The seed sequence is indicated by bold letters,while the pairing sequence of miR-365 is in red bold letters.miR=microRNA,UTR=u
49、ntranslated region,Del=deletion RepoRts|6:38285|DOI:10.1038/srep38285-arrestin 2 is responsible for miR-365-mediated morphine tolerance.Our luciferase assay results suggest that miR-365 regulates -arrestin 2 expression levels.Therefore,we determined if expression of -arrestin 2 was upregulated durin
50、g development of morphine tolerance.At day 5 and day 7 after morphine injection,-arrestin 2 was gradually increased(Fig.5A)and inversely correlated with miR-365 downregulation(Fig.1E),suggesting that miR-365 and -arrestin 2 are inversely correlated.To explore the functional relevance between miR-365
