Abstract

Objective

   To perform a comprehensive multicompartment analysis of microRNA
   (miRNA) expression in multiple sclerosis (MS) linked to disease
   activity and compared with other neuroinflammatory diseases through a
   retrospective cross-sectional study.

Methods

   One hundred twenty-seven miRNAs were measured by PCR arrays on pooled
   CSF, serum, and peripheral blood mononuclear cell (PBMC) samples of 10
   patients with relapsing MS and 10 controls. Sixty-four miRNAs were then
   measured by quantitative PCR on individual CSF samples of patients with
   relapsing or remitting MS and controls (n = 68). Fifty-seven miRNAs
   were analyzed in the CSF from a second cohort (n = 75), including
   patients with MS, neuroinfectious, or neuroinflammatory diseases and
   controls. MiRNAs significantly dysregulated in the CSF were analyzed on
   individual serum/PBMC samples (n = 59/48) of patients with relapsing or
   remitting MS and controls. Post hoc analysis consisted of principal
   component analysis (PCA), gene set, and pathway enrichment analysis.

Results

   Twenty-one miRNAs were differentially expressed, mainly upregulated in
   the CSF during MS relapses. Relapsing MS and
   neuroinfectious/inflammatory diseases exhibited a partially overlapping
   CSF miRNA expression profile. Besides confirming the association of
   miR-146a-5p/150-5p/155-5p with MS, 7 miRNAs uncharacterized for MS
   emerged (miR-15a-3p/124-5p/149-3p/29c-3p/33a-3p/34c-5p/297). PCA showed
   that distinct miRNA sets segregated MS from controls and relapse from
   remission. In silico analysis predicted the involvement of these miRNAs
   in cell cycle, immunoregulation, and neurogenesis, but also revealed
   that the signaling pathway pattern of remitting MS is more akin to
   controls rather than patients with relapsing MS.

Conclusions

   This study highlights the CSF-predominant dysregulation of miRNAs in MS
   by identifying a signature of disease activity and intrathecal
   inflammation among neuroinflammatory disorders.
     __________________________________________________________________

   Multiple sclerosis (MS) is characterized by multifocal inflammatory
   lesions inducing myelin sheath damage and axonal degeneration. MS
   results from a complex interplay between genetic susceptibility and
   environmental and epigenetic factors, but its molecular determinants
   remain elusive. A predominance of patients present initially with a
   relapsing-remitting disease course.^[31]1

   MicroRNAs (miRNAs) are single-stranded noncoding RNAs regulating
   posttranscriptionally the expression of a large spectrum of genes
   during various biological processes.^[32]2 Several miRNAs have been
   associated with MS, but only few were linked to disease
   activity.^[33]3,[34]4 Most studies were performed in small cohorts, in
   single biological compartments, rarely compared with other neurologic
   disorders or were not replicated, hence large heterogeneity between
   study populations and analysis methods. Few studies have focused on CSF
   miRNAs,^[35]5 although these might be more relevant to understanding
   disease regulation.

   We sought to characterize miRNA expression in 3 biological
   compartments, i.e., CSF, serum, and peripheral blood mononuclear cells
   (PBMCs) of patients with MS according to disease activity compared with
   that of controls and other neurologic disorders in the CSF. MiRNAs
   known for their involvement in immunity, inflammation,
   neurodegeneration, and lipid metabolism were measured by quantitative
   PCR (qPCR). Finally, a principal component analysis (PCA) and a pathway
   enrichment analysis were performed, altogether leading to a
   comprehensive multicompartment characterization of the miRNA expression
   profile in MS.

Methods

Patients

   CSF and blood samples were collected at the Cliniques Universitaires
   Saint-Luc (Brussels) between March 2005 and March 2018, processed
   according to international guidelines within 1 hour before storage at
   −80°C until miRNA extraction.^[36]6,[37]7 PBMCs paired with serum were
   processed as previously described.^[38]8 The study was conducted
   stepwise ([39]figure 1). A group size of at least 9 patients was
   calculated using the CSF fold changes of relapsing MS vs controls in
   the 2 miRNA arrays, with alpha error at 0.05 and power of 0.80
   ([40]stat.ubc.ca/∼rollin/stats/ssize/n2.html). Patient cohorts were
   classified according to the BioMS-eu consortium definitions (table e-1,
   [41]links.lww.com/NXI/A188).^[42]9 The 2017 McDonald criteria^[43]10
   were used for MS diagnosis. Relapse was defined clinically and/or by
   the presence of gadolinium-enhancing lesions (GELs) on MRI for 93%–100%
   of patients, depending on the cohort.^[44]11 Remitting patients were
   clinically stable for 1 month up to 21 years. To reduce bias, patients
   were age and sex matched to controls, and none of the patients with MS
   were under any disease-modifying treatment (DMT). None of the relapsing
   patients had received high-dose IV methylprednisolone before sample
   collection. Clinical and paraclinical features of the patients are
   summarized in [45]tables 1 and e-1.^[46]12,[47]13 Diagnoses of other
   disease categories are listed in table e-2. Data were collected between
   February 2015 and July 2018.

Figure 1. Workflow summary of different study steps.

   [48]Figure 1
   [49]Open in a new tab

   AUC = area under the curve; HC = healthy control; Infect/Inflam-ND =
   patients with infectious or inflammatory neurologic disorders; KEGG =
   Kyoto Encyclopedia of Genes and Genomes; mRNA = messenger RNA; miRNA =
   microRNA; PBMC = peripheral blood mononuclear cell; PCA = principal
   component analysis; qPCR = quantitative PCR; Rel MS = relapsing MS; Rem
   MS = remitting MS; ROC = receiver operating characteristic; RRMS =
   relapsing-remitting MS; SC = symptomatic control.

Table 1.

   Baseline characteristics of patients and controls included in the CSF
   microRNA study

   [50]graphic file with name NEURIMMINFL2019023325t1.jpg
   [51]Open in a new tab

MiRNA isolation

   MiRNAs were isolated from CSF (400 μL) and serum (200 μL) using the
   miRNeasy Serum/Plasma Kit, and from 11-13.10^6 PBMCs using the miRNeasy
   Mini Kit, following the manufacturer's protocol (Qiagen, Hilden,
   Germany). Caenorhabditis elegans miR-39 mimic (Qiagen) was added to CSF
   and serum samples as spike-in control for relative quantification.
   MiRNA extraction, reverse transcription (RT), and PCR were performed
   sequentially.

MiRNA PCR array

   MiRNA profiling was kindly performed by the manufacturer with
   “Inflammatory Response & Autoimmunity” and “T-cell & B-cell Activation”
   miScript miRNA PCR Arrays (Qiagen) on a pool of 10 patients with
   relapsing MS and 10 symptomatic controls (SCs, for CSF) or 10 healthy
   controls (HCs) (for serum and PBMCs).

RT, preamplification, and qPCR

   RT was performed on 5 μL of miRNA using the miScript II RT Kit. One
   microliter of complementary DNA was preamplified using the miScript
   PreAmp PCR Kit following the manufacturer's recommendations (Qiagen).
   The spike-in control C. elegans miR-39 mimic was not preamplified. qPCR
   reactions were performed in duplicate, except for miR-24-3p in CSF, a
   putative internal reference gene,^[52]14 which was assayed in
   triplicate. Patients' subgroups and controls were equally distributed
   across each run to minimize interrun bias for a single miRNA target.

qPCR analysis

   Sample selection was based on Ct variability within duplicates of a
   same sample and between samples, as well as melt curve morphology. Ct
   was set at 40 in case of inefficient amplification. The miRNA
   transcripts were normalized using references (exogenous miR-39 for