Deduction of Novel Genes Potentially Involved in Osteoblasts of Rheumatoid Arthritis Using Next-Generation Sequencing and Bioinformatic Approaches

The role of osteoblasts in peri-articular bone loss and bone erosion in rheumatoid arthritis (RA) has gained much attention, and microRNAs are hypothesized to play critical roles in the regulation of osteoblast function in RA. The aim of this study is to explore novel microRNAs differentially expressed in RA osteoblasts and to identify genes potentially involved in the dysregulated bone homeostasis in RA. RNAs were extracted from cultured normal and RA osteoblasts for sequencing. Using the next generation sequencing and bioinformatics approaches, we identified 35 differentially expressed microRNAs and 13 differentially expressed genes with potential microRNA–mRNA interactions in RA osteoblasts. The 13 candidate genes were involved mainly in cell–matrix adhesion, as classified by the Gene Ontology. Two genes of interest identified from RA osteoblasts, A-kinase anchoring protein 12 (AKAP12) and leucin rich repeat containing 15 (LRRC15), were found to express more consistently in the related RA synovial tissue arrays in the Gene Expression Omnibus database, with the predicted interactions with miR-183-5p and miR-146a-5p, respectively. The Ingenuity Pathway Analysis identified AKAP12 as one of the genes involved in protein kinase A signaling and the function of chemotaxis, interconnecting with molecules related to neovascularization. The findings indicate new candidate genes as the potential indicators in evaluating therapies targeting chemotaxis and neovascularization to control joint destruction in RA. Overall design: Next-generation sequencing for mRNA and small RNA of normal and RA osteoblasts

成骨细胞在类风湿关节炎（RA）的关节周围骨丢失及骨侵蚀中的作用已受到广泛关注，而微小RNAs（microRNAs）被推测在类风湿关节炎成骨细胞的功能调控中发挥关键作用。本研究旨在筛选在类风湿关节炎成骨细胞中差异表达的新型微小RNAs，并鉴定可能参与类风湿关节炎骨稳态失调的基因。研究人员从培养的正常成骨细胞与类风湿关节炎成骨细胞中提取RNA进行测序。通过下一代测序（next generation sequencing）与生物信息学方法，我们在类风湿关节炎成骨细胞中鉴定出35种差异表达的微小RNAs，以及13种具备潜在微小RNA–mRNA相互作用的差异表达基因。经基因本体（Gene Ontology）分类，该13个候选基因主要参与细胞-基质黏附过程。我们从类风湿关节炎成骨细胞中筛选出两个目标基因：A激酶锚定蛋白12（A-kinase anchoring protein 12，AKAP12）与富含亮氨酸重复序列15（leucin rich repeat containing 15，LRRC15），二者在基因表达综合数据库（Gene Expression Omnibus，GEO）的相关类风湿关节炎滑膜组织芯片中表达更为稳定，且分别被预测可与miR-183-5p、miR-146a-5p产生相互作用。经Ingenuity通路分析（Ingenuity Pathway Analysis，IPA），AKAP12是参与蛋白激酶A信号通路及趋化功能的基因之一，可与血管新生相关分子产生相互作用。本研究结果提示，新的候选基因可作为评估靶向趋化作用与血管新生以控制类风湿关节炎关节破坏的治疗方案的潜在指标。整体实验设计：对正常成骨细胞与类风湿关节炎成骨细胞的mRNA及小RNA进行下一代测序。