Multiple sclerosis (MS) is a chronic immune-mediated inflammatory-degenerative disease of the central nervous system. MicroRNAs (miRNAs) are short sequences of 19-25 non-coding single-stranded RNA nucleotides that regulate transcriptional gene expression by inhibiting the translation of specific messenger RNA targets and protein expression respectively. miRNAs are involved in various physiological and pathological processes, in particular contribute to the activation of the Th1 and Th17 pathways that regulate the immune response. Many studies show that the miRNAs are involved in MS epigenetic mechanisms affecting both the expression of inflammatory cells and myelination factors.
This study aimed to compare the serum miRNAs in relapsing MS patients compared to remitting ones and to healthy controls for better understanding of MS pathogenesis.
We evaluated 3 groups of subjects: 16 relapsing-remitting (RR)MS patients in relapse (Group I); 12 RRMS patients in remission (Group II) and 12 sex- and age-matched healthy controls (Group III). Total extraction of RNA from sera was performed with a column-based method that includes small RNAs and minimizes the carryover of enzyme inhibitors typically contained in biofluids (miRNEeasy serum/plasma kit, QIAGEN). Total RNA was labeled and hybridized with Human miRNA Microarray Release 21 (Agilent) containing probes for 2549 human microRNAs from the Sanger database. Arrays were verified for quality control and extracted by Agilent Feature Extraction 10.7.3.1 software and entirely processed by MATLAB (The MathWorks Inc.) in house-built routines. Deregulated miRNAs were established by permutation test and a false discovery procedure used for multiple comparisons. Unsupervised hierarchical clustering was performed to individuate specific pattern of expression among samples and clusters of miRNAs. The results obtained were validated by Real-Time PCR.
We analyzed 2549 miRNA that were expressed in at least 50% of the samples. After eliminating 10 samples that expressed only a few of these miRNAs, we found 66 expressed miRNAs in the half of remained samples. Microarray analysis identified a signature of 8 deregulated miRNAs in relapsing MS patients compared to controls and remitting MS patients. In particular, among these eight miRNAs, two were up-regulated (miR-2861 and miR-6821-5p) while six were down-regulated (miR-4281, miR-5196-5p, miR-6076, miR-642a -3p, miR-671-5p, miR-6879-5p).
We have found several upregulated or downregulated miRNA to be correlated with disease exacerbation in RRMS patients. Previous studies have reported five of these miRNAs to be involved in other inflammatory disorders. We hypothesize that the miRNA could be useful biomarkers not only to improve diagnosis and disease control, but also to predict the phase of acute exacerbation in MS.