microRNAs dysregulated in multiple sclerosis affect the differentiation of CG-4 cells, an oligodendrocyte progenitor cell line

Remyelination in multiple sclerosis (MS) is from the start of the disease imperfect, and strongly decreases with its progression, mainly due to the harm on oligodendrocyte progenitor cells (OPCs), causing neurodegeneration and resulting in irreversible neurological deficits. Therapeutic strategies promoting remyelination are still very preliminary and lack within the current treatment panel of MS. In a previous study we have identified 21 microRNAs dysregulated mostly in the CSF of relapsing and/or remitting MS patients. Here, we observed that among these, the majority of 13 transfected microRNA mimics decreased the differentiation of an OPC cell line called CG-4. Herein we support by RNA sequencing and independent RT-qPCR analyses that miR-33-3p, miR-34c-5p, miR-124-5p, and miR-145-5p impede OPC differentiation as evidenced by the downregulation of premyelinating oligodendrocyte (OL) (Tcf7l2, Cnp [except for miR-145-5p]) and mature OL (Plp1, Mbp, Mobp) markers, whereas only miR-214-3p promotes OPC differentiation. We further propose a comprehensive exploration of their change in cell fate through Gene Ontology enrichment analysis. We finally confirm by RT-qPCR analysis the downregulation of several predicted mRNA targets for each microRNA that possibly support their effect on OPC differentiation by very distinctive mechanisms, of which some are still unexplored in OPC/OL physiology. We hereby open new perspectives in the research on OPC differentiation and the pathophysiology of demyelination/remyelination, and possibly even in the research on new remyelinating therapeutic strategies in the scope of MS.多发性硬化（multiple sclerosis, MS）患者的髓鞘再生自疾病起始便存在缺陷，且随疾病进展显著恶化，其主要原因是少突胶质细胞前体细胞（oligodendrocyte progenitor cells, OPCs）受到损伤，进而引发神经变性并造成不可逆的神经功能缺损。当前多发性硬化的临床治疗方案中，可促进髓鞘再生的治疗策略仍处于初步探索阶段，且选择十分有限。在既往的一项研究中，我们已鉴定出21种主要在复发缓解型多发性硬化患者脑脊液（cerebrospinal fluid, CSF）中异常表达的微小RNA（microRNA, miRNA）。本研究观察到，在上述异常表达的微小RNA中，13种经转染的微小RNA模拟物的大部分均可抑制名为CG-4的少突胶质细胞前体细胞系的分化。本研究通过RNA测序与独立的实时定量聚合酶链反应（reverse transcription quantitative polymerase chain reaction, RT-qPCR）分析证实，miR-33-3p、miR-34c-5p、miR-124-5p及miR-145-5p可阻碍少突胶质细胞前体细胞分化，该效应可通过髓鞘前期少突胶质细胞（oligodendrocyte, OL）标志物（Tcf7l2、Cnp，miR-145-5p除外）与成熟少突胶质细胞标志物（Plp1、Mbp、Mobp）的表达下调得以验证；而仅有miR-214-3p可促进少突胶质细胞前体细胞分化。我们进一步通过基因本体富集分析（Gene Ontology enrichment analysis）对这些微小RNA介导的细胞命运改变展开了全面探究。最终我们通过RT-qPCR分析验证了每种微小RNA的多个预测mRNA靶标的表达下调，这些靶标可能通过极具特异性的机制参与调控少突胶质细胞前体细胞分化，其中部分机制在少突胶质细胞前体细胞/少突胶质细胞的生理学研究中尚未被阐明。本研究为少突胶质细胞前体细胞分化研究、脱髓鞘/髓鞘再生的病理生理学研究，乃至多发性硬化领域新型促髓鞘再生治疗策略的开发提供了全新的研究视角。