Roles of miR-3085-3p in skeletal development and osteoarthritis

Osteoarthritis (OA) is a progressive arthritis catheterized by cartilage degradation, subchondral bone sclerosis and synovial inflammation. MicroRNAs are endogenous non-coding short RNAs, which repress gene expression. Numerous microRNAs have been reported to be involved in pathogenesis of OA. MiR-3085-3p is a novel microRNA that is selectively expressed in human cartilage and targets integrin α5 (ITGA5). This project ravelled roles of miR-3085-3p in the skeletal development and progression of OA, following the optimization of time and dosage across experiments. Overexpression of miR-3085-3p decreased expression of ITGA5 in DF1, SW1353, HeLa and C28/I2 in various assays including qRT-PCR, western blotting and luciferase assay. In luciferase assay, 50nM miR-3085-3p mimic and 72h transfection time are optimal. In qRT-PCR, 50nM and a 48h is recommended for either SW1353 or HeLa, whilst 100nM for C28/I2. In western blotting, 100nM and 72h are reliable, where C28/I2 is not applicable. MiR-3085-3p regulated cartilage homeostasis and chondrocyte function through decreasing the expression of ACAN and COL2A1, and various signalling pathways. MiR-3085-3p increased IL-1β induced MMP13, but transiently downregulated NFκB signalling by targeting MYD88. It repressed TGFβ/SMAD signalling by targeting SMAD2, SAMD3, and SMAD4. MiR-3085-3p potentiates WNT3A-induced TOPFLASH reporter as well as WNT3A-induced AXIN2. MiR-3085-3p has roles in hMSC differentiation. It could depress chondrogenesis and promote IL-1 induced MMP-13 via JNK/AP-1 pathway in primary HACs. MiR-3085-3p also negatively regulates adipogenesis by inhibiting adipogenic marker genes - CEBPα and PPARγ. In contrast, miR-3085-3p enhances osteogenesis by directly targeting CMTM3 and LBH, especially CTDSP2 We did not successfully generate the miR-3085-3p null mice, as a consequence of embryonic lethality. Lethality was also observed in the ITGA5 cartilage-specific knockout mice. Significant developmental defects in skeletal development and possible accelerated progression of osteoarthritis were revelled in ITGA5 cartilage-specific knockout mice, suggesting that ITGA5 is crucial in these processes. MiR-3085-3p potentially has a negative influence on skeletal development and osteoarthritis by targeting ITGA5. defects in skeletal development and possible accelerated progression of osteoarthritis were revelled in ITGA5 cartilage-specific knockout mice, suggesting that ITGA5 is crucial in these processes. MiR-3085-3p potentially has negative effects on skeletal development and osteoarthritis by targeting ITGA5