The role of microRNAs in cartilage homeostasis and osteoarthritis arthritis treatment

摘要 软骨细胞在肢体发育、出生后骨骼生长和维持方面发挥着重要作用 成人生活中的软骨组织。软骨细胞基因表达异常是骨质疏松症发生的关键事件 骨关节炎的发病机制。MicroRNAs是一类非编码RNA，在进化过程中 通过转录后机制保守和调节基因表达。许多MIR显示组织特异性 表达模式，表明这些miRs在组织特异的生理或 发育过程。然而，软骨细胞特异性MIR及其上游分子信号和靶点 基因的识别才刚刚开始。我们和其他人报道了miR-140，一种软骨特异的miRNA， 在软骨发育和动态平衡中起关键作用，而在骨性关节炎中表达降低 在发病机制上有贡献。 在我们的初步实验中，miR-455(miR-455-5P和miR-455-3P)以及miR-140的两条链 被鉴定为软骨特异基因的主要转录因子Sox9的靶标。MiR-基因的表达 与正常软骨相比，455-5P/3P在人骨关节炎软骨中表达下调。使用CRISPR/CAS9 系统中，我们产生了miR-455-5p/3p基因敲除小鼠。这些小鼠在6个月时表现出严重的OA表型。 微阵列分析揭示了软骨细胞中一组可能的miR-455靶基因，包括HIF2A，它 是OA发病机制中的关键转录因子，也是miR455两条链的靶点。重要的是，HIF2A 在6个月时，miR-455基因缺失的小鼠关节软骨中几乎所有的软骨细胞都有较强的表达。 这些观察结果支持miR-455-5p/3p是软骨的关键调节因子的假设。 动态平衡及其表达和功能的变化在影响疾病中发挥重要作用 关节软骨。我们提出了以下具体目标： 目的1：利用miR-455 KO小鼠和人分析miR-455-5p/3p在骨性关节炎发病机制中的作用 关节软骨细胞。 目的2：在关节软骨细胞中鉴定直接调控软骨的miR-455-5p/3p下游靶点 动态平衡与骨性关节炎发病机制。 目的：观察关节腔内注射miR-455s对骨性关节炎动物模型的治疗作用。 拟议中的研究有可能揭示重要的新的调控途径 软骨发育和动态平衡为疾病机制和治疗开辟了新的视角 干预措施。

ABSTRACT Chondrocytes play an important role in limb development, postnatal skeletal growth and in maintaining cartilage tissues during adult life. Dysregulation of gene expression in chondrocytes is a key event in the pathogenesis of osteoarthritis (OA). MicroRNAs are a family of noncoding RNAs that are evolutionarily conserved and regulate gene expression by posttranscriptional mechanisms. Many miRs show tissue specific expression patterns, suggesting that these miRs play a crucial role in tissue specific physiological or developmental processes. However, chondrocyte specific miRs, their upstream molecular signals, and target genes are only beginning to be identified. We and others reported that miR-140, a cartilage specific miRNA, plays a critical role both in cartilage development and homeostasis while the reduced expression in OA contributes to pathogenesis. In our preliminary experiments, both strands of miR-455 (miR-455-5p and miR-455-3p) as well as miR-140 were identified as targets of Sox9, a main transcription factor for cartilage specific genes. Expression of miR- 455-5p/3p was down-regulated in human OA cartilage compared with normal cartilage. Using CRISPR/Cas9 system, we generated miR-455-5p/3p knockout mice. These mice showed a severe OA phenotype at 6 months. Microarray analyses revealed a set of possible miR-455 target genes in chondrocytes, including HIF2A, which is a key transcription factor in OA pathogenesis, and a target of both strands of miR455. Importantly, HIF2A was strongly increased in almost all chondrocytes in articular cartilage of miR-455 null mice at 6 months. These observations support the hypothesis that miR-455-5p/3p are critical regulators of cartilage homeostasis and that changes in their expression and function play an important role in diseases affecting articular cartilage. We propose the following specific aims: Aim 1: Analyze the function of miR-455-5p/3p in OA pathogenesis using miR-455 KO mice and human articular chondrocytes. Aim 2: Identify in articular chondrocytes the direct miR-455-5p/3p downstream targets that regulate cartilage homeostasis and OA pathogenesis. Aim 3: Examine the therapeutic effects of intraarticular miR-455s administration in animal models of OA. The proposed studies have the potential to reveal important new regulatory pathways that control cartilage development and homeostasis and open new insight on disease mechanisms and therapeutic interventions.