The function of microRNAs in cartilage and osteoarthritis

MicroRNAs are small double-stranded RNAs, which negatively regulate gene expression and have been shown to have key roles in both chondrocyte development and cartilage homeostasis with age. Deletion of all microRNAs in chondrocytes leads to skeletal growth defects in mice, whilst deletion of specific mi croRNAs, e.g. miR-140, leads to premature articular cartilage degradation and increased susceptibility to posttraumatic osteoarthritis. Studies comparing microRNA expression in normal human articular cartilage compared to osteoarthritic cartilage show differential expression, but varying sample groups make interpretation difficult. MicroRNAs have been proposed as circulating biomarkers of osteoarthritis, but again, this differs amongst patient cohorts. Many micro- RNAs have been shown to have roles in chondrocyte phenotype via signaling pathways, apoptosis, autophagy and senescence. Modulating microRNAs in the joint has been shown to reduce osteoarthritis in animal models and translating this to man as a novel therapeutic strategy will be key

The microRNA-455 null mouse has memory deficit and increased anxiety, targeting key genes involved in Alzheimer’s disease

The complete molecular mechanisms underlying the pathophysiology of Alzheimer’s disease (AD) remain to be elucidated. Recently, microRNA-455-3p has been identified as a cir-culating biomarker of early AD, with increased expression in post-mortem brain tissue of AD patients. MicroRNA-455-3p also directly targets and down-regulates APP, with the overexpres-sion of miR-455-3p suppressing its toxic effects. Here, we show that miR-455-3p expression de-creases with age in the brains of wild-type mice. We generated a miR-455 null mouse utilising CRISPR-Cas9 to explore its function further. Loss of miR-455 resulted in increased weight gain, potentially indicative of metabolic disturbances. Furthermore, performance on the novel object recognition task diminished significantly in miR-455 null mice (p = 0.004), indicating deficits in recognition memory. A slight increase in anxiety was also captured on the open field test. BACE1 and TAU were identified as new direct targets for miR-455-3p, with overexpression of miR-455-3p leading to a reduction in the expression of APP, BACE1 and TAU in neuroblastoma cells. In the hippocampus of miR-455 null mice at 14 months of age, the levels of protein for APP, BACE1 and TAU were all increased. Such findings reinforce the involvement of miR-455 in AD progression and demonstrate its action on cognitive performance

The role of microRNA-3085 in chondrocyte function

MicroRNAs have been shown to play a role in cartilage development, homeostasis and breakdown during osteoarthritis. We previously identified miR-3085 in humans as a chondrocyte-selective microRNA, however it could not be detected by Northern blot. The aim of the current study was to prove that miR-3085 is a microRNA and to investigate the function of miR-3085 in signaling pathways relevant to cartilage homeostasis and osteoarthritis. Here, we confirm that miR-3085 is a microRNA and not another class of small RNA using (1) a pre-miR hairpin maturation assay, (2) expression levels in a Dicer null cell line, and (3) Ago2 pulldown. MicroRNA-3085-3p is expressed more highly in micromass than monolayer cultured chondrocytes. Transfection of miR-3085-3p into chondrocytes decreases expression of COL2A1 and ACAN, both of which are validated as direct targets of miR-3085-3p. Interleukin-1 induces the expression of miR-3085-3p, at least in part via NFκB. In a feed-forward mechanism, miR-3085-3p then potentiates NFκB signaling. However, at early time points after transfection, its action appears to be inhibitory. MyD88 has been shown to be a direct target of miR-3085-3p and may be responsible for the early inhibition of NFκB signaling. However, at later time points, MyD88 knockdown remains inhibitory and so other functions of miR-3085-3p are clearly dominant. TGFβ1 also induces the expression of miR-3085-3p, but in this instance, it exerts a feedback inhibition on signaling with SMAD3 and SMAD4 shown to be direct targets. This in vitro analysis shows that miR-3085-3p functions in chondrocytes to induce IL-1-signaling, reduce TGFβ1 signaling, and inhibit expression of matrix genes. These data suggest that miR-3085-3p has a role in chondrocyte function and could contribute to the process of osteoarthritis

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