The Expression Of MicroRNAs In The Plasma Of METH Addicts And Its Forensic Medicine Significance

METH addiction is a kind of chronic,high relapse encephalopathy caused by repeated contact with drugs.Its main characteristics are compulsive drug use,continuous craving state and high relapse rate after withdrawal,which seriously endangers public health and social security.METH abuse can cause anxiety,depression,mania,cognitive impairment,and psychotic symptoms such as hallucinations and delusions.At present,METH addiction mechanism has not been fully clarified,there is no objective diagnostic index and effective treatment drugs,which brings great difficulties for the country’s drug rehabilitation work and the evaluation and treatment of addicts.Recent studies have shown that non-coding micro RNAs(miRNAs)are not only related to the occurrence and development of neuropsychiatric diseases,but also free from cells and stable in plasma or serum.They have some characteristics of molecular biomarkers of diseases,showing unique value in the diagnosis and prognosis of a variety of diseases.Therefore,to explore the expression changes of miRNAs in the plasma of METH addicts and explore valuable biological markers is of important forensic research significance.Objective: This study was aim to clarify the expression changes of miRNAs in the plasma of METH addicts and the correlation with mental symptoms,to explore the influence of hydrogen intervention on the expression of miRNAs in plasma of METH addicts and to analyze the diagnostic value of different miRNAs in plasma.Which explores the possibility of using them as biological markers for METH addiction.Methods:METH addiction conditioned place preference(CPP)model was established in C57BL/6N mice,and miRNAs sequencing was performed on nucleus accumbens(NAc)of mice.miRNAs whose expression levels were significantly up-regulated in the library were screened,and tissue specificity was verified by q RT-PCR.The downstream target was predicted by bioinformatics and its expression level was detected by Western Blot.The population study included METH addicts(81 cases),normal controls(76 cases)and hydrogen intervention addicts(51 cases).The basic information of METH addicts was collected by questionnaire,and the degree of craving for METH(VAS)was evaluated.The anxiety scores(SAS)and depression scores(SDS)of METH addicts were analyzed.q RT-PCR was used to verify the homologous upregulated expression of miRNAs in human and mouse plasma of METH addicts.ROC curve was used to predict their diagnostic ability.The expression level of miRNAs downstream target proteins in plasma was determined by ELISA kit.Meanwhile,the correlation between the expression level of miRNAs and their target proteins and the mental symptoms of METH addicts was analyzed.Results:1.Animal experiments1)METH addiction CPP of mice showed that the CPP score of METH addiction phase was significantly higher than that of pretest period(P < 0.01),indicating that METH addiction CPP model was successfully established.2)RNA-seq sequencing results showed that METH addicted mice had 36up-regulated miRNAs and 27 down-regulated miRNAs(P < 0.05).3)mmu-miR-206-3p,mmu-miR-592-5p,mmu-miR-9-3p,mmu-let-7b-3p and mmu-miR-155-3p whose expression levels increased significantly in sequencing results were selected as target miRNAs,and q RT-PCR verification results showed that: The expression levels of mmu-miR-206-3p,mmu-miR-592-5p,mmu-miR-9-3p and mmu-let-7b-3p in NAc of METH addicted mice were increased.Tissue specificity verification results of q RT-PCR showed that mmu-miR-592 and mmu-miR-9-3p were specifically expressed in the brain of METH addicted mice,and their expression levels were significantly higher than those of other tissues(P < 0.01).2.Population studiesCombined with the results of animal experiments,human and mouse homologous hsa-miR-206,hsa-miR-592,hsa-miR-9-3p,hsa-let-7b-3p and hsa-miR-155-3p were selected as target miRNAs to verify METH addict plasma.1)q RT-PCR results showed that the expression levels of METH addicts in plasma were significantly higher than those of healthy controls(P < 0.05);2)Bioinformatics analysis showed that 2904 downstream targets were predicted,mainly enriched in the PI3K-Akt signaling pathway,in which BDNF and IGF-1 were enriched for many times.3)ELISA results showed that METH content of BDNF and IGF-1 in plasma of METH addicts decreased to 8.225ng/ml and 544.4mg/ml,respectively(P < 0.01).4)ROC curve analysis showed that,among target miRNAs,the diagnostic efficiency of hsa-miR-9-3p was high,the area under the curve(AUC)was0.756,the 95% confidence interval(CI)was 0.674～0.838,the specificity of the maximum cut-off point was 0.722,and the sensitivity was 0.729.Among the downstream targets,BDNF had the best diagnostic performance,with AUC=0.831(95% CI,0.728-0.933),and the specificity and sensitivity of the maximum cut-off point were(0.968,0.719).logistic regression combined with ROC curve analysis showed that the combined diagnostic efficiency of miR-9-3p and miR-592 was improved,with AUC=0.871(95% CI=0.806-0.935),and the specificity and sensitivity of the maximum cut-off point were(0.785,0.841).5)Spearmen correlation analysis showed that the expression levels of target miRNAs and target proteins were not significantly correlated with the psychiatric symptoms of METH addicts,but the degree of depression of METH addicts was positively correlated with the frequency of drug use and the age of first drug use(r=0.24,P < 0.05).Conclusions:1.The expression levels of hsa-miR-206,hsa-miR-592,hsa-miR-9-3p,hsa-let-7b-3p and their target molecules BDNF and IGF-1 in plasma of METH addicts were changed,and their interactions may affect the process of METH addiction.2.The combination of hsa-miR-9-3p,hsa-miR-592 and BDNF has high diagnostic efficacy for METH addiction,which may be used as potential biological markers for METH addiction.