| Objective: Drug addiction was a chronic brain disease,which characterized by out of control for forced medication and persisting in drug craving.A plenty of evidence indicated that noncoding RNA of epigenetics played a key role in regulating drug addiction.Heroin addiction acted as one of the most common opioid addition,which had become one of the social public health problems in China.Therefore,it would have an important basic and clinical significance to explore the molecular mechanism of heroin addiction and relapse.miRNA was a kind of non-coding RNA,which had been proved that it was enriched in the nervous system and involved in the regulation of neuromodulation in the brain,including the release of synaptic mediators,regulating the genes expression.Hence,the aim of this study was to explore the epigenetic mechanism of miR-181 a in heroin addiction and relapse,which would provide the biomarkers for diagnosing and treating of heroin addiction and offer new clues and methods for preventing relapse.Experiment 1: Based on the results of the miRNA microarray of human plasma to verify the expression of hsa-miR-181aMethods: The plasma samples collected from healthy control people(Control,n=49)and heroin addiction patients(Heroin addiction,n=53)based on the strict inclusion and exclusion,which were used to miRNA microarray analysis.Total RNA was extracted from plasma to verify the results of miRNA microarray by RT-PCR analysis.Results: The expression of hsa-miR-181 a were detected by RT-PCR,and hsa-miR-181 a expression in Heroin addiction group was significantly higher than that in Control group [t(100)=54.956,P<0.001].The results of RT-PCR were consistent with the that of miRNA microarray.Experiment 2: Based on the results of miRNA microarray of NAc brain regions from heroin self-administration rats to verify the expression level of miR-181 a in ratsMethods: Male Sprague Dawley(SD)rats underwent the operation of jugular vein intubation,after one week of recovery,and then conducted the heroin self-administration training(0.2mg/ml)for 4 hours per day lasting 14 days.After the heroin self-administration rats model established successfully and the SD rats were abstinent for 1 day and 14 days.The test of heroin relapse induced by cues for 2 hours,and these rats were divided into two groups,cue induced relapse on first day group(CS1,n=8)and cue induced relapse on 14 days group(CS14,n=8).Meanwhile,the saline instead of heroin was underwent self-administration training,and these rats belong to control group(Control,n=7).The rats were decapitated and isolating their NAc immediate after cue induced relapse test,extracting RNA and verifying the results of miRNA microarray using RTPCR analysis.Results: RT-PCR analysis found that the expression level of miR-181 a was statistically significant among three groups[F(2,11)=4.074,P=0.047].LSD analysis showed that compared with Control group,the expression of miR-181 a was increased in CS1 group,however,that was no statistically significant(P>0.05).Compared with CS1 group,the expression of miR-181 a was significantly decreased in CS14 group(P<0.05).The results were consistent with the results of miRNA microarray.Experiment 3: The effect of rno-miR-181 a on the expression of target MeCP2 gene by Luciferase assayMethods: According to the TargetScan database to predict the binding site of miR-181 a and target MeCP2 gene,structured the MeCP2-WT plasmid and MeCP2-MU plasmid and then transfected into 293 T cells along with miR-181 a plasmid or negative control,respectively.After 48 hours,the Dual-Luciferase Reporter System Assay was used detect the luciferase expression of each group,and observed whether miRNA had an inhibitory effect on the expression of MeCP2.Results: Dual-Luciferase Reporter Assay System analysis found that the expression of MeCP2 in MeCP2-WT+miR-181 a group was significantly lower than that in MeCP2-WTNC+miR-181 a group [t(4)=11.480,P=3.28×10-4],meanwhile,the expression of MeCP2 in MeCP2-WT+miR-181 a group was also significantly decreased than that in MeCP2-WT+miR-181a-NC group [t(4)=3.645,P=0.022].Those results demonstrated that rno-miR-181 a could bind with 3’UTR of MeCP2 directly and inhibit the expression of MeCP2.Experiment 4: To detect the expression of MeCP2 protein of heroin self-administration rats after cue-induced relapse.Methods: After the heroin self-administration rats model established successfully,the rats were randomly divided into two groups,cue induced relapse for 1 day group(CS1,n=8)and cue induced relapse for 14 days group(CS14,n=8),and the saline instead of heroin was acted as control group(Control,n=7).The rats from three groups were decapitated and isolating their NAc immediate after cue induced relapse test,western blot analysis was used to detect the expression of MeCP2 protein.Results: Western blot analysis showed that the expression of MeCP2 protein was statistically significant among three groups[F(2,6)=34.467,P=5.13×10-4].The results of LSD analysis found that the expression of MeCP2 in CS14 group was significantly increased than that in Control group and CS1 group(P<0.05)Experiment 5: To detect the expression of mRNA and protein of MeCP2 in NAc brain region of heroin self-administration rats after over-expressing rno-miR-181aMethods: After the heroin self-administration rats model established successfully,the rats were randomly divided into three groups,over-expression miR-181 a withdrawal for 14 days by cue induced relapse group(LV-miR-181 a,n=8),negative expression miR-181 a withdrawal for 14 days by cue induced relapse group(LV-GFP,n=8),and withdrawal for 14 days by cue induced relapse control group(CS14,n=8).Brain microinjection method was used to inject lentiviral in NAc brain region of rats from LV-miR-181 a group and LV-GFP group,and then conducted the 2 hours heroin addiction cue induced relapse test after two weeks.The rats NAc regions were isolated after decapitating.RT-PCR analysis was used to detect the expression of MeCP2 mRNA,and the expression of MeCP2 protein was detected by western blot.Results: 1.RT-PCR analysis found that the expression of MeCP2 mRNA was reduced in NAc brain region after over-expressing rno-miR-181 a,however,there was no statistically significant among these groups[F(2,12)=0.500,P=0.618].2.The expression of MeCP2 was reduced significantly after LV-miR-181 a [F(2,6)=41.008,P=3.17×10-4].LSD analysis showed that the expression of MeCP2 in LV-miR-181 a group was declined significantly compared with CS14 group and LV-GFP group(P<0.05).Experiment 6: To observe the change of cue induced heroin seeking behavior after overexpressing rno-miR-181 a in the NAc of heroin self-administration ratsMethods: After the heroin self-administration rats model established successfully,the rats were randomly divided into three groups,over-expression miR-181 a withdrawal for 14 days by cue induced relapse group(LV-miR-181 a,n=8),negative expression miR-181 a withdrawal for 14 days by cue induced relapse group(LV-GFP,n=8),and withdrawal for 14 days by cue induced relapse control group(CS14,n=8).Brain microinjection method was used to inject lentiviral in NAc brain region of rats from LV-miR-181 a group and LV-GFP group,and then conducted the 2 hours heroin addiction cue induced relapse test after two weeks.Results: There was a significant difference in the cue induced heroin seeking behavior of rats among three groups[F(2,21)=7.559,P=0.03].LSD analysis showed that the cue induced heroin seeking behavior in LV-miR-181 a group was declined significantly compared with CS14 group and LV-GFP group(P<0.05).These results suggested that over-express miR-181 a in NAc brain region could significantly inhibit the cue induced heroin seeking behavior.Experiment 7: Effect of LV-siRNA-MeCP2 injected in the NAc region of rat on the cue induced heroin seeking behavior and the MeCP2 mRNA and protein expressionMethods: After the heroin self-administration rats model established successfully,the rats were randomly divided into three groups,MeCP2 inhibit expression group(LV-siRNA-MeCP2,n=8),negative virus group(LV-GFP,n=8)and withdrawal for 14 days by cue-induced relapse group(CS14,n = 8).Brain microinjection method was used to inject lentiviral in NAc brain region of rats from LV-miR-181 a group and LV-GFP group,and then conducted the 2 hours heroin addiction cue induced relapse test after two weeks.The rats NAc regions were isolated after decapitating.RT-PCR analysis was used to detect the expression of MeCP2 mRNA,and the expression of MeCP2 protein was detected by western blot.Results: 1.The cue induced heroin seeking behavior of rats in LV-siRNA-MeCP2 group was decreased significantly compared with LV-GFP group and CS14 group [F(2,21)=3.851,P=0.038].2.There was a significant difference of the expression of MeCP2 mRNA among three groups [F(2,12)=4.302,P=0.039].LSD analysis showed that the expression of MeCP2 in LV-siRNAMeCP2 group was declined significantly compared with CS14 group and LV-GFP group(P<0.05).3.Western blot analysis showed that the expression of MeCP2 protein was statistically significant among CS14 group(n = 3),LV-GFP group(n=2)and LV-siRNA-MeCP2 group(n=2)[F(2,6)=43.717,P=2.65×10-4].LSD analysis showed that the expression of MeCP2 protein in LVsiRNA-MeCP2 group was declined significantly compared with CS14 group and LV-GFP group(P<0.01).Conclusions: The results of this study shows that peripheral plasma hsa-miR-181 a levels of heroin addictions are significantly higher than controls.Meanwhile the expression miR-181 a of the heroin self-administration rats in the NAc region increased after 1 day of withdrawal,but there was no significant difference in the heroin self-administration rats compared with the control group,.But,The expression miR-181 a levels in the NAc region of the heroin self-administration rats was significantly decreased after 14 day of withdrawal(P<0.05).Furthermore,it was found that overexpression of rno-miR-181 a levels in the NAc region of heroin self-administration rats could significantly inhibit in cue-induced heroin seeking behavior after withdrawal,and through the database and Dual-Luciferase was confirmed that rno-miR-181 a could directly target 3’UTR of MeCP2 and inhibit the MeCP2 expression.At the same time,it was further demonstrated that the reduction of NAc region MeCP2 protein by LV-siRNA-MeCP2 over-expression could significantly inhibit cue-induced heroin seeking behavior.Based on the experimental results,it demonstrated that the epigenetic mechanism of non-coding miR-181 a target MeCP2 gene in heroin addiction and relapse,and also provided a new biomarkers and a potential therapeutic target for the prevention and treatment of clinical heroin addiction and relapse. |
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