背景:m6A调节因子在多种自身免疫性疾病中具有重要作用,但是它们在硬皮病中的潜在功能,一种难治性结缔组织病,尚不清楚。已知生腱蛋白C(TNC)是硬皮病发展中促进胶原沉积的因子,但TNC和m6A监管机构之间的监管关系尚不清楚。
方法:我们提取了包括41名健康对照和61名硬皮病患者的GSE33463数据,我们分析了21种m6A调节因子的表达水平以及它们之间的关联。此外,我们获得了随机森林(RF)和列线图模型来预测硬皮病的相似性。接下来,我们通过共识聚类对m6Aclusters和基因簇进行了分类,我们对每个簇进行了免疫细胞浸润分析。最后,我们将腺病毒注射到博来霉素(BLM)诱导的硬皮病小鼠模型中,用于过度表达FTO和TNC。我们使用病理染色并测量其羟脯氨酸含量和胶原蛋白mRNA来评估小鼠样品中皮肤纤维化的程度。
结果:我们最初确定了十四个差异表达的m6A调节剂(WTAP,RBM15,CBLL1,FTO,ALKBH5,YTHDC1,YTHDC2,YTHDF1,YTHDF2,YTHDF3,RBMX,HNRNPC,IGFBP1和IGFBP2)。我们发现ALKBH5与CBLL1和RBM15呈正相关,而FTO与WTAP呈负相关。此外,我们使用RF模型确定了四个M6A稳压器(CBLL1、IGFBP1、YTHDF2和IGFBP2),我们设计了一个列线图模型,根据校准曲线和临床影响曲线,这些变量被证明是可靠的。我们发现m6AclusterA与1型T辅助细胞浸润相关,而GeneclusterA与调节性T细胞浸润相关。最后,我们表明FTO过表达下调了TNC的m6A和mRNA水平,减轻硬皮病小鼠模型的皮肤纤维化。因此,我们的过表达实验提供了初步证据,表明TNC是硬皮病的一个不利因素.
结论:我们的方法可能是一种新的、准确的硬皮病诊断方法。此外,我们的结果提示FTO/TNC可能是硬皮病治疗的新靶点.
BACKGROUND: m6A regulators have important roles in a variety of autoimmune diseases, but their potential function in
scleroderma, a refractory connective tissue disease, remains unclear. Tenascin C (TNC) is known to be a factor promoting collagen deposition in the development of
scleroderma, but the regulatory relationship between TNC and m6A regulators is unknown.
METHODS: We extracted GSE33463 data consisting of forty-one healthy controls and sixty-one patients with scleroderma, and we analyzed the expression levels of twenty-one m6A regulators as well as the associations between them. In addition, we obtained random forest (RF) and nomogram models to predict the likehood of
scleroderma. Next, we categorized the m6Aclusters and geneclusters by consensus clustering, and we performed an immune cell infiltration analysis for each cluster. Finally, we injected adenoviruses into a bleomycin (BLM)-induced mouse model of
scleroderma, which was used to overexpress FTO and TNC. We assess the extent of skin fibrosis in the mice samples using pathology stains and measuring their hydroxyproline content and collagen mRNA.
RESULTS: We initially identified fourteen differentially expressed m6A regulators (WTAP, RBM15, CBLL1, FTO, ALKBH5, YTHDC1, YTHDC2, YTHDF1, YTHDF2, YTHDF3, RBMX, HNRNPC, IGFBP1 and IGFBP2). We found ALKBH5 to be positively associated with CBLL1 and RBM15, and FTO to be negatively associated with WTAP. In addition, we identified four m6A regulators (CBLL1, IGFBP1, YTHDF2 and IGFBP2) using a RF model, and we designed a nomogram model with those variables that proved reliable according to the calibration curve and clinical impact curve. We found that the m6Acluster A was correlated with Type 1 T helper cell infiltration and the genecluster A was correlated with regulatory T cell infiltration. Finally, we showed that FTO overexpression downregulated the m6A and mRNA levels of TNC, and alleviated skin fibrosis in the mouse model of scleroderma. Thus, our overexpression experiments provide preliminary evidence suggesting that TNC is an adverse factor in scleroderma.
CONCLUSIONS: Our approach might be useful as a new and accurate scleroderma diagnosis method. Moreover, our results suggested that FTO/TNC might be a novel
scleroderma therapeutic target.