A Study On The Mechanism Of The Targeted Regulation MiR-592-3p On TMEFF1 In Cue-induced Incubation Of Morphine Craving

The high relapse rate after drug withdrawal is a serious public health problem.Incubation of craving is relevant to re-exposure to environmental cues related to drug use,which is an important factor leading to relapse.TMEFF1(tomoregulin-1)is a novel transmembrane protein.Studies have shown that TMEFF1 is a regulatory factor of addiction-related protein(addicsin).And it is involved in morphine addiction.However,little is known regarding the role of TMEFF1 in morphine relapse,especially the incubation of morphine craving.Therefore,this study focuses on the role of TMEFF1 protein in morphine incubation craving.Protein functions are regulated by many factors,in which Micro RNA(miRNA)play an important role.MicroRNA refers to the category of single-stranded small noncoding RNAs that are approximately 22 nucleotides in length,which mainly regulate gene expression at the post-transcriptional level.In this study,high-throughput sequencing,bioinformatics analysis,transcriptomics,and other technologies were used to clarify the targeted regulation of MiR-592-3p on TMEFF1 in the incubation of morphine craving.Objective: In this study,TMEFF1 was taken as the target to observe its effect on the incubation of craving in morphine.Meanwhile,the regulation process and its mechanism of upstream miR-592-3p was a new idea for prevention of morphine relapse.Methods: Mice were received a subcutaneous injection(s.c.)of saline or morphine(1 or 5?mg/kg).And then they were alternating training for 6 days.Then CPP scores were measured on the first day(T1/WD1)after morphine withdrawal and the 14 th day(T2/WD14)aims to establish the incubation of morphine craving model.At the same time,Western Blot were used to detect the expression of TMEFF1 in nucleus accumbens core(NAcC)and nucleus accumbens shell(NAcSh)of model mice.Then,adeno-associated virus(AAV)was constructed to inhibit TMEFF1 expression.And the effect of TMEFF1 changes was observed in the nucleus accumbens(NAc)on incubation of craving in mice by Conditioned place preference(CPP)test.We screened down-regulated miRNAs through miRNA high throughput sequencing.Next,Targetscan databases were used to predict miRNAs that can regulate TMEFF1 gene.And q RT-PCR was used to detect the target miRNA—miR-592-3p.The AAV were constructed to over-expression miR-592-3p to observe the effect on incubation of morphine craving in mice.At the same time,dual luciferase assay was used to determine whether there was a binding site between miR-592-3p and 3′UTR region of TMEFF1 m RNA.Results:1.The CPP test demonstrated that the CPP score of the T2 was significantly higher than that of T1(P <0.01),which have indicated that the model of incubation of craving was successfully established.2.The expression of NAcC TMEFF1 in T2 of model mice was significantly higher than that of T1(P <0.05),but NAcSh was unchanged.After inhibiting the expression of TMEFF1 in NAcC,the CPP score of T2 in model mice were significantly lower than that of T1(P <0.001).It is suggested that the morphine craving of mice was drastically reduced,but the inhibition of NAcSh TMEFF1 was not changed.3.8 miRNAs that were downregulated in mice models of T2 stage,which were screened by miRNA sequencing and Target Scan databases.And miR-592-3p decreased most significantly(P<0.001).By q RT-PCR verification,we identified miR-592-3p as the research object.Over-expression of NAc C miR-592-3p,the results showed that the CPP score of T2 phase of mice model was significantly lower than that of T1(P<0.001).It was suggesting that over-expression of miR-592-3p can inhibit morphine-induced incubation of craving.While over-expression of NAc Sh miR-592-3p,there was no change of morphine craving.4.The results of dual luciferase reporter assay showed that miR-592-3p could significantly reduce the related luciferase activity of reporter construct compared with the control group(P<0.01).It was suggesting that miR-592-3p can specially bind to the 3′UTR region of TMEFF1 m RNA.Conclusion1.TMEFF1 could regulate incubation of morphine craving through the nucleus accumbens core.2.miR-592-3p may negatively regulate the expression of TMEFF1,which plays an important role in morphine incubation of craving.