Effetto dell’esposizione cronica volontaria di eroina e WIN55,212-2 sull’espressione di micro RNA nelle aree cerebrali coinvolte nella dipendenza da sostanze d’abuso

Micro RNAs (miRNA) are 21-25 nucleotide endogenous non coding RNAs that regulate gene expression at the post-transcriptional level through binding to a target complementary mRNA. Targeted mRNAs are translationally inhibited or subjected to degradation by miRNAs, depending on the complementarity in the 3’UTR of the mRNAs. Long-term intake of drugs of abuse results in persistent perturbation of synaptic plasticity, progressing towards high risk, drug-seeking behavior and relapse. The molecular mechanisms leading to addiction are still poorly understood but emerging evidence suggests that drug induced neuroplasticity depends on epigenetic changes in gene expression and post-transcriptional regulation. MiRNAs have been implicated in the mechanisms of drug addiction, and further studies may be central for a better understanding of their potential role as novel therapeutic targets of this disorder. Different miRNAs may respond to different drugs and regulate diverse pathways. Thus they can specifically affect the development of synaptic connections and plasticity, direct dendrite formation in neurons, and have an important role in the development of addiction-related behaviors. The aim of this study was to identify the correlation between miRNAs regulation and addiction in four reward related areas (NAc shell, NAc core, PFCX, CPu) following heroin and WIN55,212-2 self-administration (SA). The SA model used in this study is a well-established drug addiction model that provides great face validity to human drug intake. In order to evaluate changes in miRNAs expression we performed miRNome microarray analyses on RNA. We also measured the mRNA levels of 11 different genes, known to be involved in addiction and which were predicted as potential targets of some miRNAs found dysregulated in our esperiment. Lastly, we performed a western blot analysis in order to assay the proteins expression profile following heroin SA. Future studies are required to evaluate protein expression following WIN55,212-2 SA. We identified several miRNAs in which the expression was modified after heroin SA. We found 39 miRNAs dysregulated in NAc shell: 16 upregulated (FC>2) and 23 downregulated (FC<-2); 114 miRNAs dysregulated in NAc core: 97 upregulated and 17 downregulated; 125 miRNAs dysregulated in the caudate putamen: 105 upregulated and 20 downregulated and 51 miRNAs dysregulated in the prefrontal cortex, 39 upregulated and 12 downregulated. Following WIN55,212-2 SA we observed that 95 miRNAs were differentially expressed in NAc shell: 40 upregulated (FC>2) and 55 downregulated (FC<-2); 96 miRNAs were dysregulated in NAc core: 13 upregulated and 83 downregulated; 110 miRNAs dysregulated in the caudate putamen: 11 upregulated and 99 downregulated; and 53 miRNAs in the prefrontal cortex: 26 upregulated and 27 downregulated. Gene expression analysis in the heroin group showed 2 genes downregulated in NAc shell (BDNF, FC=-2.78 and CCKBR, FC=-1.85), 3 downregulated in NAc core (DRD3, FC=-2.38, MeCP2, FC=-2.14 and NOS1 FC=- 1.94), 2 upregulated in the caudate putamen (BDNF, FC=2.74 and CREB, FC=2.09). No changes were found in the prefrontal cortex following heroin SA. As regard WIN55,212-2 SA, only 1 gene was upregulated in NAc core (BDNF, FC=2.16). A strong dysregulation was observed in the caudate putamen: 9 were upregulated (CREB, FC=7.20; CLOCK, FC=3.44; DRD3, FC=4.23; NOS2, FC=6.75; SIRT2, FC=11.53; NOS1, FC=11.05; MECP2, FC=11.03; SIRT1, FC=6.30; OPRM1, FC=3.89) and 1 downregulated (BDNF, FC=-2.23). We identified only 1 gene downregulated in the prefrontal cortex (BDNF, FC=-2.15). No changes were observed in NAc shell. Protein expression analysis revealed that the levels of BDNF, MeCP2 and pCREB were decreased in NAc shell. Only CCKBR protein expression was increased in NAc core, whereas MECP2, NOS1, CLOCK, CREB, pCREB, BDNF, SIRT2 and MOR1 proteins were downregulated. MeCP2 was upregulated and CREB, BDNF, CLOCK, pCREB, NOS1 and SIRT1 were downregulated in the caudate putamen. In the prefrontal cortex 4 proteins showed downregulation: BDNF, p-CREB, SIRT1 and MOR1. This study provides insight into the effect of heroin and WIN55,212-2 on miRNA and gene expression in key brain areas associated with addiction, supporting the hypothesis that long-lasting changes may occur in the expression of genes after opioids and cannabinoids consumption. The influence of miRNAs on the regulation of gene expression suggests that miRNAs may play a role in the vulnerability to addiction. The present findings give a hint for further studies and are important in terms of their translational potential. In fact, understanding how miRNAs affect gene regulation may be helpful for developing therapeutic strategies for the treatment of addiction.