Epigenetic Dysregulation of microRNA-142 and Upregulation of Multiple microRNAs That Target Oxytocin Receptor in the Frontal Cortex of Individuals with Autism

Background:  MicroRNAs are small RNA molecules that can bind messenger RNA (mRNA) and inhibit the translation of proteins.  While mounting evidence has shown that levels of specific genes and mRNAs are dysregulated in the brains of individuals with autism, there is little information about the dysregulation of microRNAs in the brain.  Since any one microRNA can possibly target many mRNAs, the dysregulation of a single microRNA can have a very high impact on cellular and neuronal function.  

Objectives:  Our first objective was to identify microRNAs that are dysregulated in the brain of individuals with autism.  The second objective is to determine the probably targets of these microRNAs, and to determine the biological implications of the dysregulation of these specific microRNAs.

Methods:  In order to identify differentially expressed microRNAs in the autistic brain, we performed high throughput small RNA sequencing on RNA samples from 12 autistic and 12 control prefrontal cortex samples.  Results were validated with real time PCR and interesting microRNAs were further studied for changes in epigenetic regulation.  Bioinformatic analysis (DIANALAB and microrna.org) were used to reveal potential target genes for the dysregulated microRNAs, and to identify their biological patways (gene ontology).  Luciferase assays in cells were performed to validate possible mRNA targets, and real time PCR was performed on the target mRNAs to understand the relationship between microRNA expression and mRNA expression.

Results:  High throughput sequencing revealed a list of 23 microRNAs that were differentially expressed between control and autistic brains.  Using real time PCR, we validated three of the top seven of these microRNAs, mir-142, mir-21, and mir-451.  We further discovered that the promoter of mir-142 is hypomethylated in these same brain samples, therefore suggesting an interaction between epigenetic dysregulation and microRNA expression.    Gene ontology analysis revealed that the main mRNA targets of the three microRNAs are involved in axon guidance.  Additional bioinformatic analysis revealed that both mir-21and mir-451 can target the oxytocin receptor gene.   Using luciferase assays, we provide evidence that mir-21 and mir-451 can inhibit translation of the OXTR transcript in cells. Of interest, we found that oxytocin receptor gene expression levels are increased in our autism brain samples, and that there is a direct positive correlation between oxytocin gene expression levels and the levels of microRNA-21.

Conclusions:  We have identified three microRNAs that are overexpressed in our autistic brain cohort.  We provide significant evidence that these microRNAs are involved in molecular mechanisms that are highly related to brain function and social behavior.  Specifically, the finding that mir-21 and mir-451 target OXTR provides a direct link between microRNA dysregulation and molecular mechanisms that regulate social behavior.  Therefore, we provide evidence that microRNAs may play an integral role in the etiology of autism spectrum disorders.