Male-specific coordinated changes in expression of miRNA genes, but not other genes within the DLK1-DIO3 locus in multiple sclerosis

Abstract

The role of miRNAs, small non-coding regulatory RNAs, in the molecular mechanisms of multiple sclerosis (MS) development has been intensively studied. MiRNAs tend to be clustered within imprinted regions, and the largest number of miRNA genes is observed in the DLK1-DIO3 locus. Earlier using RNA-seq we identified sex-specific upregulation of the set of miRNA genes from this locus in peripheral blood mononuclear cells (PBMC) of treatment-naive relapsing-remitting MS (RRMS) patients. In the present study we set up to independently investigate the expression of a vast array of genes present in the DLK1-DIO3 imprinted locus. First, we analyzed the expression of miRNA genes, which levels in RRMS were mostly inconsistent based on RNA-seq data and not previously explored using qPCR. We identified that all selected miRNAs - miR-337-3p and -665 from 14q32.2 cluster and miR-370c, -380, -494, -654-3p, -300, -539, -668, and -323b-5p - were upregulated in MS men, but not women when compared to controls, regardless of conflicting RNA-seq data. The expression of miRNAs from the DLK1-DIO3 locus was highly correlated, indicating the existence of a common regulatory mechanism(s) that controls miRNA expression, regardless of the position of their genes within this region. Second, we performed the expression analysis of non-miRNA genes within the locus. The genes encoding proteins (DLK1, DIO3, RTL1), long non-coding RNAs (MEG3, MEG8, and MEG9) and small nucleolar RNAs (SNORD112, SNORD113-5, SNORD113-7, SNORD114-3, SNORD114-8, SNORD114-19) were not dysregulated in RRMS both in men and women. DNA methylation analysis of selected CpG sites within the differentially methylated regions IG-DMR, MEG3-DMR, and MEG8-DMR of the DLK1-DIO3 imprinted locus pointed out that they were not involved in the regulation of miRNA gene expression in RRMS, at least in PBMC population. The question of whether the observed changes in expression of miRNA genes (given that there is a constant expression of other non-miRNA genes of the DLK1-DIO3 locus) are involved in the development of RRMS or are they a consequence of the disease progress, remains open and needs further investigation.

Keywords: DLK1-DIO3 imprinted locus; DNA methylation; Gene expression; Relapsing-remitting multiple sclerosis; Sex-specificity; miRNAs.