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.