Genetic association and altered gene expression of mir-155 in multiple sclerosis patients

Abstract

Multiple sclerosis (MS) is a complex autoimmune disease of the central nervous system characterized by chronic inflammation, demyelination, and axonal damage. As microRNA (miRNA)-dependent alterations in gene expression in hematopoietic cells are critical for mounting an appropriate immune response, miRNA deregulation may result in defects in immune tolerance. In this frame, we sought to explore the possible involvement of miRNAs in MS pathogenesis by monitoring the differential expression of 22 immunity-related miRNAs in peripheral blood mononuclear cells of MS patients and healthy controls, by using a microbead-based technology. Three miRNAs resulted >2 folds up-regulated in MS vs controls, whereas none resulted down-regulated. Interestingly, the most up-regulated miRNA (mir-155; fold change = 3.30; P = 0.013) was previously reported to be up-regulated also in MS brain lesions. Mir-155 up-regulation was confirmed by qPCR experiments. The role of mir-155 in MS susceptibility was also investigated by genotyping four single nucleotide polymorphisms (SNPs) mapping in the mir-155 genomic region. A haplotype of three SNPs, corresponding to a 12-kb region encompassing the last exon of BIC (the B-cell Integration Cluster non-coding RNA, from which mir-155 is processed), resulted associated with the disease status (P = 0.035; OR = 1.36, 95% CI = 1.05-1.77), suggesting that this locus strongly deserves further investigations.

Keywords: association analysis; expression profile; miRNA; mir-155; multiple sclerosis.

Figure 1

Immune-related miRNA expression pattern in MS patients and healthy subjects. (A) miRNA analysis was performed on total RNA isolated from PBMC samples from 10 MS patients and 6 healthy donors. The heatmap was generated using the dChip software after supervised hierarchical clustering analysis of all unfiltered data. Fold change (FC) is indicated only for those miRNAs showing significant differences in expression levels between cases and controls (P < 0.05; t test for comparing the 2 groups). Differential expression of miRNA patterns is shown by the intensity of red (up-regulation) versus blue (down-regulation); (B) Semi-quantitative real-time RT-PCR analysis of mir-155 and its precursor (BIC) in 10 MS patients and 10 controls. Mir-155 levels were normalized by the endogenous control mir-146a, whereas, for BIC transcripts, the hydroxymethylbilane synthase (HMBS) and beta-actin (ACTB) housekeeping-gene levels were used as calibrators. Results are presented as normalized rescaled values (calculated by the GeNorm software). Significance levels in differences between cases and controls are presented in parenthesis, and were calculated by a one-tailed t test statistics.

Figure 2

Role of mir-155 in the regulation of adaptive/innate immunity. The figure depicts stages in the adaptive (upper part) and innate (lower part) immune responses in which the specific role of mir-155 was demonstrated. HSC, hematopoietic stem cells; CLP, common lymphocyte progenitor; CMP, common myeloid progenitor; MDP, myeloid dendritic progenitor; GMP, granulocyte monocytic progenitor; T-reg, regulatory T cell; Th1, type 1 T helper cell; Th2, type 2 T helper cell; Th17, type 17 T helper cell.

Figure 3

LD haplotype structure of the mir-155 locus. The structure of the genomic region surrounding the mir-155 gene and its precursor MIR155HG (BIC) is shown in the upper part of the figure; exons are represented by boxes, introns by lines, and are drawn to scale (RMPL39 corresponds to the mitochondrial ribosomal protein L39 gene, downstream of the mir-155 locus). Arrows above genes indicate their transcriptional direction. The genomic size is indicated by the ruler at the top of the scheme; the genomic position is depicted on the chromosome 21 ideogram. In the central part of the figure, genotyped SNPs are listed, and their genomic locations are shown by arrows. The identified risk haplotype is indicated with letters (referring to SNP alleles contributing to the haplotype). The haplotype was constructed and phased with PLINK; only phased haplotypes with posterior probability of 1 were included for determining OR and 95% CI (grouping together all the other alleles). In the lower part of the figure, the LD structure of the mir-155 locus is shown. Pair-wise LD values, estimated for the genotyped SNPs, are represented by boxes. The standard color scheme (D’/LOD) of Haploview was used to display the strength of LD: red indicates strong LD, pink intermediate.