MicroRNA-7188-5p and miR-7235 regulates Multiple sclerosis in an experimental mouse model

Highlights

• Eleven miRNA’s were overexpressed in EAE mice and it was correlated in MS patient’s samples.

• In vivo and in vitro experiments were performed to identify miRNAs could be used as an effective therapeutic biomarkers.

• MiRNA targeted genes such as FXBO33, SGMS-1, ZDHHC-9, GABRA-3 and NRXN-2 were negatively expressed in lymphoid cells.

• The silencing of miRNA-7188-5p and miR-7235 changed the CNS pathophysiology in EAE conditions.

Abstract

The short non-coding microRNAs (miRNAs) have emerged as reliable modulators of various pathological conditions including autoimmune diseases in mammals. The current study, aims to identify new potential differential expressed miRNAs and their downstream mRNA targets of the autoimmune disease, Multiple sclerosis (MS). The study identifies a new set of miRNA(s) that are probably implicated in MS using computational tools. The study further carried-out different in vivo and in vitro experiments to check these identified miRNAs could be role in as therapeutic and prognostic applications. Preliminary insilico screening revealed that miR-659-3p, miR-659-5p, miR-684, miR-3607-3p, miR-3607-5p, miR-3682-3p, miR-3682-5p miR-4647, miR-7188-3p, miR-7188-5p and miR-7235 are specifically elevated in the secondary lymphoid cells of EAE mice. In addition, expression of the downstream target mRNA of these miRNAs such as FXBO33, SGMS-1, ZDHHC-9, GABRA-3, NRXN-2 were reciprocal to miRNA expression in lymphoid cells. These confirmed by applying the mimic and silencing miRNA models, suggesting new inflammatory target genes of these promising miRNA markers. The in vivo adoptive transfer model revealed that the suppression of miRNA-7188-5p and miR-7235 changed the pattern of astrocytes and CNS pathophysiology. The current study opens a new miRNA and their mRNA targets in MS disease. The absence of miRNA-7188-5p and miR-7235 enhanced the disease alleviation, confirms the regulatory effect of these targets. These optimized results highlights new set of miRNA’s with therapeutic potential in experimental MS. Further studies are required to confirm these miRNA as therapeutic biomarker.

Introduction

Multiple sclerosis (MS) is a neurological autoimmune disease characterized by infiltration of T lymphocytes and macrophages into the central nervous system (CNS) that leading to multifocal areas of demyelination in the CNS and microglial mediated pathological conditions (Prineas et al., 2001; Macrez et al., 2016). Clinically, MS patients showed a variable pattern of relapsing remittance and intermittent inflammatory exacerbations. Markers for diagnosing MS are usually a proteins or specific antibodies such as chemokines, glycoproteins, IgG and IgM antibodies, and cell surface markers of inflammation (Harris et al., 2017). However, these biomarkers are not well-correlated with the disease course of MS (Martinez and Peplow, 2020)), indicating an urgent need in developing and validating biomarkers that correlate MS using different detection methods (Ziemssen et al., 2019).

Although the pathological events of MS are well-established, the epigenetic involvement in the pathogenesis of MS is still not completely understood (Orefice et al., 2020; Compston and Coles, 2008; Huang et al., 2017). miRNAs, a class of non-coding single-stranded RNA, is 19–24 nucleotides that regulate post-transcriptional modulation in the host genome. MiRNA could interact to 3′ untranslated region (UTR), or rarely 5′ UTRs, of mRNA transcripts, which made perfect and imperfect complementary binding, leads to redundant translational inhibition of mRNA targets (Bartel, 2009; Thomson et al., 2011; Wang and Chen, 2019; Mycko and Baranzini, 2020). Besides, the computational approach in the identification of novel and disease-related miRNA has been considered as a reliable and cost-effective tool (Saravanan et al., 2015; Thirugnanasambantham et al., 2013, 2015), that reflect the progression of the disease in clinical samples (Chen et al., 2018a). Strategies such as qPCR, next-generation sequencing (NGS), and microarray analysis has been utilized to analyze the miRNA expression pattern in various body fluids, such as blood, serum, plasma, cerebrospinal fluid (CSF), and urine in MS patients (Venkatesha et al., 2018). The levels of miRNAs are significantly altered in clinical fluids with MS progression and in responses to treatment (Zhang et al., 2014, 2015; Naghavian et al., 2015; Jagot and Davoust, 2016). These researches implicate miRNAs are promising biomarkers in the MS autoimmune diseases, and studying the role of miRNAs in MS has attracted attention in recent years (Mycko and Baranzini, 2020)

Similar to clinical samples, dysregulated miRNA expression is noticed in the experimental autoimmune encephalomyelitis (EAE) animal model, a well-characterized experimental model for the human MS disease, that exhibited CNS inflammation and pathological features of MS including ascending paralysis and motor neuron damage and death (Bannerman et al., 2005; Vogt et al., 2009). Typically, the EAE model is induced through active immunization with myelin-derived proteins or peptides (e.g., myelin oligodendrocyte glycoprotein (MOG) in an adjuvant to sensitize the T cells in the peripheral lymphoid tissue (Farooqi et al., 2010). Moreover, in vivo injection of the lipopolysaccharides (LPS) induces EAE in T cell receptor (TCR) transgenic mice and relapse of encephalomyelitis in normal mice (Chen et al., 2018b). Recent reports also identified miR-155 as a key regulator of these inflammatory responses, in mice which resulted in a decrease in Th1 and Th17 cellular differentiation in the CNS as well as peripheral lymphoid organs. However, animal models for MS can produce chronic inflammatory events, the in vivo approach for studying miRNAs role in neuronal degeneration and regeneration is still lacking. In this study, we aim to fill this knowledge gap. Here, we applied a bioinformatics approach to recognize a new set of miRNAs that correlate with MS progression and quantified the expression levels of identified miRNAs and their targets in the EAE/MS disease mice model.