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. We aimed to investigate the effect of several microRNAs on the differentiation of oligodendrocyte progenitor cells (OPCs). Therefore CG-4 cells (an OPC cell line) were transfected with each microRNA mimic separately and were then cultured in differentiation medium for 48h We performed RNA-sequencing on the transfected cells (5 different microRNAs) and controls (proliferation vehicle control, differentiation vehicle control, differentiation microRNA negative control) We compared each microRNA condition to each control regarding differentially expressed genes and Gene Ontology enrichment analysis

多发性硬化（multiple sclerosis, MS）的髓鞘再生从疾病初始阶段便存在缺陷，并随疾病进展显著下降，这主要源于少突胶质前体细胞（oligodendrocyte progenitor cells, OPCs）受到损伤，进而引发神经退行性变并导致不可逆的神经功能缺损。当前MS治疗方案中，促进髓鞘再生的治疗策略仍处于初步阶段且相对匮乏。在既往研究中，我们已鉴定出21种主要在复发型和/或缓解型MS患者脑脊液（cerebrospinal fluid, CSF）中异常表达的微小RNA（microRNAs, miRNAs）。本研究中，我们观察到在上述21种微小RNA中，13种经转染的微小RNA模拟物（microRNA mimics）可降低CG-4少突胶质前体细胞系的分化能力。通过RNA测序（RNA sequencing, RNA-seq）及独立的实时定量逆转录聚合酶链反应（RT-qPCR）分析，我们证实miR-33-3p、miR-34c-5p、miR-124-5p及miR-145-5p可阻碍OPC分化，具体表现为髓鞘前期少突胶质细胞（premyelinating oligodendrocyte, OL）标志物（Tcf7l2、Cnp[miR-145-5p除外]）及成熟少突胶质细胞标志物（Plp1、Mbp、Mobp）的表达下调；而仅miR-214-3p可促进OPC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介导的细胞命运变化进行了全面探索。最终，我们通过RT-qPCR分析验证了每种微小RNA的多个预测信使RNA（messenger RNA, mRNA）靶基因的下调情况，这些靶基因可能通过极具特异性的机制参与调控OPC分化，其中部分机制在OPC/OL生理学中尚未被阐明。本研究为OPC分化、脱髓鞘/髓鞘再生（demyelination/remyelination）的病理生理学研究开辟了新视角，甚至有望为MS新型促髓鞘再生治疗策略的研发提供思路。本研究旨在探究多种微小RNA对少突胶质前体细胞（OPCs）分化的影响。为此，我们将CG-4细胞（一种OPC细胞系）分别转染每种微小RNA模拟物，随后在分化培养基中培养48小时。我们对转染后的细胞（涉及5种不同微小RNA）及对照组（增殖载体对照组、分化载体对照组、分化微小RNA阴性对照组）进行了RNA测序。我们针对差异表达基因及基因本体富集分析，将每种微小RNA处理组与各对照组进行了比较分析。