| Objective: Heroin addiction is a chronic brain disease characterized by recurrence.Studies have shown that the pathogenesis is of heroin addiction is accounted for the neuroadaptive changes of brain structure and function after long-term exposure to heroin,such as impaired learning and memory substrates or neuronal plasticity and altered expression of signaling molecules.Up to now,the main treatment for heroin addicts is to control opioid withdrawal syndrome,but there is no effective measures to prevent heroin relapse.If more specific biomarkers are found,which will guide and effectively intervene the relapse behavior.mi RNA is a class of non coding RNAs with a length of about 22 nucleotides,it can regulate target gene translation,closely related to drug addiction.However,at present,there are few studies on the neurobiological mechanism between mi RNA and heroin relapse behavior.In this study,we chosed mi R-206 as the target to explore its' effect on heroin relapse behavior,meanwhile,we observed the effect of mi R-206 on the expression of the targeted genes in order to clarify its main regulatory mechanism underlying heroin relapse.Experiment 1: Microarray analysis of mi RNA expression profiles in nucleus accumbens of heroin addicted rats and targeted genes analysis.Methods: SD male rats were subjected to 14 consecutive days of heroin self-administration training(FR1,heroin dose was 0.05 mg/injection/kg).The heroin trained rats were divided into three groups: control group(Control,n=3),cue-induced relapse on first day group(CS1,n=3)and cue induced relapse on 14-th day group(CS14,n =3),The rats in the CS1 and CS14 group were respectively decapitated after test of relapse behavior withdrawan in 1 day,14 day from heroin self-administration.The samples of nucleus accumbens(NAc)in three groups were sent to the gene company to carry out the mi RNA expression analysis,to complete the screening of the gene chip,this experiment focused on the selection of increasing expression of mi RNA following the withdrawal.Targeted genes were analyzed through the online database including mi Rbase,Targetscan,mi RWalk,microcosm Targets and other databases,and mi R-206 binding target genes were focused and accessed to literature to be understood the target genes.Results: A series of mi RNA expression profiles were obtained by gene chip technology.The mi R-206 expression in CS1 group was less than that of CS14 group.The mi R-206 was chosed and the BDNF/Me CP2/GABBR1 protein was identified as its' targeted genes according to the databases.Experiment 2: The expression of mi R-206 was verified in nucleus accumbens of heroin addicted rats.Methods: The experimental rats were divided into three groups:control group(Control,n=4),cue induced relapse on first day group(CS1,n=4)and cue induced relapse on 14-th day group(CS14,n=4),the control group was decapitated on the first day of withdrawal,the CS1 and CS14 group after test of cue-induced relapse was respectively decapitated in 1-th day,14-th day of the withdrawal.Assay of q RT-PCR was used to verify whether the expression of mi R-206 in NAc was consistent with the results of gene chip analysis.Results:One-way ANOVA showd that the expression of mi R-206 was statistically different between the groups(F(2,10)=5.44,P<0.05).q RT-PCR assay showed that the expression of mi R-206 in CS1 group was significantly higher than that in control group(P<0.05),and compared with CS1 group,the expression of mi R-206 in NAc of rats in CS14 group was higher.(P<0.05).The results of this experiment are consistent with the mi R-206 expression profile in gene chip.Experiment 3:Effects of down-or up-regulation of miR-206-3p expression in NAc on the cue-induced relapse behavior.Methods: The thirty-four heroin addicted rats,randomly divided into four groups according to the active poke: cue-induced relapse on 14-th day group(CS14,n=8),negative control group(LVGFP,n=8),overexpression of mi R-206 group(LV-mi R-206-3p,n=9),low expression of mi R-206group(LV-mi R-206-3p inhibition,n=9).By rat stereotaxic technique,the rats were respectively micro-injected LV-mi R-206-3p,LV-mi R-206-3p inhibition,or LV-GFP lentivirus into NAc site of the corresponding group within two days after the end of self-administration training,the rats recovered by continuous administration of penicillin for three days(I.m,qd,0.3ml/),then the four groups of rats were carried out the two hours of cue induced relapse behavior test on 14-th withdrawal day.Followed the heroin relapse behavior,the rats were decapitated and taken the samples of NAc tissue.The expression level of mi R-206-3p was detected by q RT-PCR,and the expression of BDNF/Me CP2/GABBR1 protein was tested by Western blot.Results: One-way ANONA found a significant effect of the active pokes between groups(F(3,32)=3.42,P<0.05),but there was no significant effect of the inactive pokes(P>0.05);q RT-PCR data showed the expression of mi R-206 was significantly different between the groups(F(3,13)=30.86,P<0.05).One-way ANONA also found a significant effect of the BDNF expression level(F(2.12)=29.75,P<0.05),Me CP2 expresion level(F(2.12)=3.90,P<0.05)and GABBR1 expresion level(F(3,14)=46.96,P<0.05),respectively.Compared with the LV-GFP group,the expression level of BDNF and Me CP2 of the LV-mi R-206-3p inhibition group was significantly increased(p < 0.05,respectively),while the expression level of GABBR1 was significantly decreased(p < 0.05).Experiment 4: Effect of mi R-206-5p inhibition in NAc on the cue-induced heroin seeking behavior.Methods:The twenty-four heroin addicted rats,randomly divided into three groups according to the active poke: cue induced relapse on 14-th day group(CS14,n=8),negative control group(LV-GFP,n=8),low expression of mi R-206-5p group(LV-mi R-206-5p,n=8),the first day at the end of administration training,injected LV-mi R-206-5p inhibition,LV-GFP lentivirus into NAc of the corresponding group respectively.The recovered rats were were unified two hours of cueinduced relapse behavior test on 14-th withdrawal day.Perfused brain tissues were to observe the transfection efficiency of lentivirus.Results: There was no significant difference in the active pokes between groups(F(2.22)=0.97,P>0.05),which revealed that inhibition of the mi R-206-5p gene expression in the NAc failed to affect the cue-induced heroin seeking behavior.Experiment 5: The effect of microinjection of Gabbr1 si RNA,into the NAc on heroin relapse behavior Methods: The twenty-six heroin addicted rats,randomly divided into three groups in the light of the active poke: cue-induced relapse on 14-th day group(CS14,n=8),negative control group(LV-GFP,n=9),Gabbr1-si RNA group(LV-Gabbr1-si RNA,n=9).Last two groups were respectively injected with LV-mi R-206-5p inhibition or LV-GFP lentivirus into the NAc by stereotaxic technique within two days after completing the heroin training.After recovery,these three groups of rats were tested the cue-induced relapse behavior on 14-th withdrawal day,and rats brain tissues were perfused to observe the transfection efficiency of lentivirus.Results: There was no significant difference in the active pokes between groups(F(2.24)=1.20,P>0.05),which revealed that inhibition of the gabbr1 gene expression in the NAc by its specific si RNA had no remarkable regulation effects on heroin relapse behavior.Experiment 6: Effect of overexpression of Me CP2 in the NAc on heroin seeking behavior in rats.Methods: The twenty-four rats established successfully the heroin self-administration were randomly divided into 3 groups: cue-induced relapse on 14-th day group(CS14,n=8),negative contral group(LV-GFP,n=8),overexpression of Me CP2group(LV-Me CP2,n=8),LV-GFP group and LV-Me CP2 group were injected lentivirus into the NAc by stereotaxic technique on first withdrawal day.After 14 days of withdrawal,the three groups were subjected to test of CS 2h,finally the NAc tissues were removed and the q RT-PCR and Western-blot experiments were performed.Results: The results showed rats active pokes between groups have significant difference(F(2,22)=3.57,P<0.05),but not the inactive pokes(P>0.05).The expression level of BDNF protein(F(2.12)=25.65,P<0.05)and Me CP2 protein(F(2.12)=11.01,P<0.05)between groups had a statistical significant difference too.Compared with the LV-GFP group,the expression level of BDNF and Me CP2 of the LV-Me CP2 group was significantly increased(p < 0.05,respectively).Experiment 7 Effects of mi R-206 on the expression of Me CP2 gene by double Luciferase assay.Method: The experiment was divided into 6 groups in the culture cells: control+mi RNA-206,control+mi RNA-206-NC,Me CP2+mi RNA-206,Me CP2WT+mi RNA-206-NC,Me CP2MU+mi RNA-206 and Me CP2MU+mi RNA-206-NC.The plasmid of Me CP2 gene,plasmid of mi RNA-206 and Renilla plasmid were transfected into 293 T cells by ROCHE kit.After transfection with 48 h,the fluorescence value of each group was detected by Promega to observe the effect of mi R-206 on the expression of fluorescent protein in Me CP2 WT group.Results: Compared with Me CP2WT+mi RNA-206-NC group,the fluorescent expression of Me CP2WT+mi RNA-206 group decreased(P=0.025),the results suggested that mi R-206 could partially inhibit the expression of Me CP2 gene.Conclusion: The present studies demonstrated that the expression of mi R-206 in the NAc increased following the heroin withdrawal from heroin self-administration,and the cue-induced heroin seeking behavior enhanced by inhibiting the expression of mi R-206 in the NAc.These results suggested that the overexpression of mi R-206 after withdrawal may play a protective role in the heroin relapse behavior induced by cues which associated previsiously with heroin reward following the withdrawal.Through the analysis of the predicted target genes of mi R-206,mi R-206 could regulate mainly the expression of BDNF and Me CP2,decreasing the expression of mi R-206 increased the expression of Me CP2 in vivo.Moreover,double luciferase assay showed that mi R-206 slightly and directly down-regulated the expression of Me CP2.Therefore,mi R-206 may regulate the expression of Me CP2 and BDNF to protect from the relapse behavior. |
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