| Addiction is a chronic,relapsing brain disorder characterized by intense drug craving and repeated drug use despite negative consequences.The high rate of relapse is a major challenge in treating drug addiction.Exposure to drug-associated cues and contexts is considered the important trigger for drug craving to relapse after prolonged abstinence.Researcher reported that cue-induced craving progressively increases during the early days of abstinence and stay elevated after prolonged time periods.This phenomenon,termed ‘incubation of drug craving’,is also observed in humans that cue-induced craving increases during abstinence for nicotine,alcohol,methamphetamine and cocaine.Over the decades,researchers have discovered several critical mechanisms of incubation of drug craving.In contrast,the mechanism underlying incubation of craving for drugs is in infancy and not well known.The nucleus accumbens(NAc)is thought to be an important nucleus involved in drug addiction acquiring,reward reinforcement,drug craving and relapse.The dopaminergic neural projection in the nucleus accumbens is closely related to drug reward and reinforcement,while the glutaminergic neural projection is involved in drug craving.The relevant molecular changes within the nucleus accumbens neuron maybe as biomarkers to evaluate drug craving and the relapse rate.Circ RNAs are produced in a non-canonical splicing form in gene in organisms.Circ RNAs could serve as miRNA sponges to modulate cellular function and disease progression.In neurons,Circ RNAs are mainly located in synapses and involved in synaptic formation and neuronal activity.Recent studies have shown that circRNAs play an important role in the development of Parkinson’s disease,Alzheimer’s disease,Ischemic brain injury and other diseases.However,the role of circRNAs in drug addiction,especially in incubation drug craving,have not been reported.Based on the background,this study intends to elucidate the role of circRNA—miRNA—m RNA axis regulation mechanism in the incubation of morphine.It provides a potentially effective therapeutic strategy for cue-induced opioids craving after prolonged withdrawal.Part Ⅰ Establishment of morphine induced incubation of craving model and screening of target circRNAObjective: In this study,to establish morphine induced incubation of craving model by CPP apparatus.And to observe the changes of circRNA expression profile in incubation of morphine craving in NAc by high-throughput sequencing technology.To identify and screen the target circRNA by qRT-PCR,FISH and bioinformatics analysis.Methods: Firstly,CPP was used to establish induced incubation of morphine craving.The mice(C57BL/6J)were conditioned immediately after the injection of morphine(1mg/kg,5mg/kg or 10mg/kg s.c.)for 45 minutes6 consecutive days.CPP scores were measured on the first day after morphine withdrawal(T1/WD1)and the 14 th day(T2/WD14)to evalute the incubation of craving model.Then,the expression of circRNAs in NAc of morphine craving mice were detected by high-throughput sequencing.The increased expression of circRNAs in the sequencing results were verified by qRT-PCR.Further more,FISH verified the expression changes of target circRNA and identified its cell location.qRT-PCR was performed after RNase R digested to verify the target circRNA was circular.Results:1.The CPP scores of mice conditioned with high-dose morphine(5,10mg/kg)in WD14 were significantly higher than WD1,but not the low-dose morphine(1mg/kg).2.Circ RNA sequencing data showed that 16 circRNAs significant changes in the incubation of morphine craving.There were 7 up-regulated circRNAs and 9 down-regulated circRNAs.3.qRT-PCR results showed that the expressions of circtmeff-1,circ Dmxl2 and circ Pik3 c in the WD14 group were significantly higher than WD1 group.4.The incubation of craving progressively increased during abstinence.CPP scores in WD14 and WD28 were significantly higher than WD1.Circtmeff-1 expression in the nucleus NAc was consistent with the trend of CPP scores.5.FISH images showed that the expression of circtmeff-1 in group WD14 was significantly higher than group WD1.And circtmeff-1 was mainly expressed in the cytoplasm.6.After digested by RNase R,circtmeff-1 and tmeff-1 m RNA were amplified with divergent primer and convergent primer,respectively.RT-PCR results showed that circtmeff-1 tolerated RNase R digestion,and the m RNA was digested by RNase R.Summary: By CPP model,high dose morphine(5,10mg/kg)injection can induce the incubation of drug craving.Circ RNAs expression profile were changed in incubation of morphine craving in NAc,and circtmeff-1 was confirmed as a circRNA by divergent primers,RNase R digestion and other methods.The expression of circTmeff-1 was positively correlated with CPP scores.Part Ⅱ The functional of circTmeff-1 in cue-induced incubation of morphine cravingObjective: To investigate the expression pattern of circTmeff-1 in cue-induced incubation of morphine craving.And to observe the effects of knockdown or overexpression circTmeff-1 in the NAc core or shell of mice on drug craving,spontaneous activity,spatial memory and anxiety.Methods: First,qRT-PCR was performed to verify the expression of circTmeff-1 in the NAc core or shell in cue-induced morphine craving.we detected the circTmeff-1 expression pattern in NAc core or shell on WD14 with or without CPP test.Next,In WD14 CPP test,the walls and/or floors of CPP apparatus were changed from conditioning phase.CPP scores were observed with the change of cues,and the expression of circTmeff-1 was detected by qRT-PCR.AAV was used to regulate the down-regulate or overexpression of circTmeff-1.The mice were injected AAV into the NAc core or shell(0.2ul)with stereotaxic locators.qRT-PCR and western blot were used to verify the transfection effect of AAV at different time in NAc core and shell of mice.Then,CPP model was used to observe the effect of down-regulate or overexpression of circTmeff-1 on cue-induced incubation of morphine craving.The Barnes maze,open field test and light and dark box experiment were used to observe the effects of down-regulation or overexpression of circTmeff-1 in NAc core or shell on spatial memory,spontaneous activity and anxiety.Results:1.qRT-PCR results showed that the expression of circTmeff-1 in the NAc core was significantly higher in WD14 test than in the WD1,but not in the NAc shell.Meanwhile,the expression of circTmeff-1 in NAc core was significantly higher in the test group than no test group.2.CPP scores results showed that changing the cues of drug conditioning(wall and/or floor)could reduce the drug craving of mice.The CPP scores of floor changed group significantly lower than the control group.In addition,the expression of circTmeff-1 was consistent with the CPP score.3.qRT-PCR results showed that AAV could significantly overexpression or down-regulation of circTmeff-1 expression at the injection site.And western blot showed that the expression of GFP or mcherry was detected 1week after AAV injection,and could be sustained 4 weeks at least.4.CPP scores showed that down-regulation circTmeff-1 in NAc core could inhibit the incubation of drug craving induced by 5mg/kg morphine,and overexpression of circTmeff-1 in NAc core could increase the incubation of drug craving induced by 1mg/kg morphine.5.The the latent time of Barnes maze showed that it is no effect down-regulation or ovexpression circTmeff-1 in NAc core or shell on spatial memory.6.The results of open field test showed that it is no effect downregulation or ovexpression circTmeff-1 in NAc core or shell on movement distance of mice in 30 mins.7.The light and dark test results showed that retention time in light box of down-regulation or ovexpression circTmeff-1 in NAc core or shell was no difference compared with the control group.Summary: The expression of circTmeff-1 in NAc core of mice was increased in the cue-induced incubation of morphine craving,but there was no significant change in the NAc shell.The expression of morphine craving can be induced by drug paired cues and the circTmeff-1was consistent with the CPP scores.Dow-regulation circTmeff-1 in NAc core decreased 5mg/kg morphine-induced incubation of drug craving,while overexpression of circTmeff-1 in NAc core increased 1mg/kg morphine-induced incubation of craving.The overexpression or down-regulation of circTmeff-1 in NAc core or shell of mice had no effect on spatial memory,spontaneous activity and anxiety.Part Ⅲ The mechanism of circTmeff-1 in regulating cue-induced incubation of morphine cravingObjective: To observe witch miRNA could binding to circTmeff-1regulating the incubation of morphine craving,and explore the role of miRNA on m RNA.To elucidate the circTmeff-1-miRNA-m RNA regulatory network in incubation of morphine craving.Methods: Fristly,bioinformatics analysis was used to predict miRNA binding sites to circTmeff-1 by miRanda,Target Scan and RNAhybrid databases and verified that by qRT-PCR.Then,double luciferase reporting assay,RNA pulldown,FISH were preformed to locate circTmeff-1 and determined binding effect with targeted miRNA in NAc core.Further more,bioinformatics analysis was used to predict the co-binding m RNAs with miR-541-5p and miR-6934-3p by miRanda and miRDB databases,and the m RNAs were verified in the NAc core by qRT-PCR.Western blot was performed to determine the expression changes of target proteins in cue-induced incubation of drug craving and the effects of circTmeff-1 and miR-541-5p/miR-6934-3p on target proteins expression.Results:1.Bioinformatics prediction results showed that there were a total of 33 miRNAs combined with circTmeff-1 by miRanda,Target Scan and RNAhybrid databases,and 10 miRNAs with highs cores were verified by qRT-PCR in NAc core There were 4 miRNAs were consistent with the predicted results.2.Dual-luciferase reporting results showed that miR-541-5p and miR-6934-3p could significantly inhibit fluorescence expression,and the mutant circTmeff-1binding sites of miR-541-5p/miR-6934-3p could reverse the inhibitory effect of miR-541-5p and miR-6934-3p on fluorescence expression.3.RNA pulldown results showed that the expression levels of miR-541-5p and miR-6934-3p in the pulldown group were significantly higher than that in the control group.4.FISH fluorescence image showed that circTmeff-1 was mainly expressed in cytoplasm and co-located with miR-541-5p and miR-6934-3p.5.CPP score showed that miR-541-5p could significantly reduce CPP score in WD14 mice,while miR-6934-3p showed no statistical significance.At the same time,both miR-541-5p and miR-6934-3p inhibitor could reverse the effect of down-regulation of circTmeff-1on CPP scores.6.Bioinformatics prediction results showed that 5 m RNAs were bound to miR-541-5p and miR-6934-3p by miRanda and miRDB databases.Through the qRT-PCR verification,NAc core Nfasc and Vamp1 expression is in WD14 significantly higher than and WD1.7.Western blot results showed the expression of VAMP1 and NFASC increased significantly in WD14;Overexpression circTmeff-1can significantly increased VAMP1 expression,and miR-541-5 p and miR-6934-3 p can reverse circTmeff-1 on VAMP1 expression;Meanwhile,miR-541-5p could decrease the expression of NFASC,while miR-6934-3p was not.In addition,the expression of BDNF in WD14 was significantly higher than that in WD1.The expression of AMPA2 in WD14 was lower than WD1.The expression of AMPA1 in WD14 was not significantly different from WD1.Overexpression of circTmeff-1 can decrease the expression of AMPA2 and increase the expression of BDNF,and these effects can be reversed by miR-541-5p and miR-6934-3p.Summary: It was confirmed that circTmeff-1 could bind to mir-541-5p and mir-6934-3p and inhibit their effect.And miR-541-5p and miR-6934-3p can inhibit VAMP1 and NFASC expression.circTmeff-1-miR-541-5 p/miR-6934-3p-VAMP1 / NFASC regulatory network role was observed in cueinduced incubation of drug craving.Conclusions: The expression profile of circRNA in NAc of mice was changed in incubation of morphine craving,and the expression of circTmeff-1was significantly increased.Inhibition of circtmeff-1 expression in NAc core was significantly inhibited the cue-induced incubation of morphine craving.CircTmeff-1 regulated incubation of morphine craving by circTmeff-1-miR-541-5p/miR-6934-3p-VAMP1/NFASC axis.This regulatory axis enriches the understanding of incubation of drug craving and provides a new target for the prevention and treatment of relapse. |
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