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Forkhead box O1 (FOXO1) is a key regulator of osteogenesis. The aim of this study was to identify the mechanisms of microRNAs (miRNAs) targeting FOXO1 in osteogenic differentiation of human bone marrow mesenchymal stem cells (hMSCs). Three miRNA target prediction programs were used to search for potential miRNAs that target FOXO1. Quantitative real‐time polymerase chain reaction was conducted to detect the expression of miR‐1271‐5p and FOXO1 during osteogenic differentiation. Target gene prediction and screening, luciferase reporter assay was used to verify the downstream target gene of miR‐1271‐5p. The expression levels of FOXO1 and Runx2 were detected by RT‐qPCR and Western blot analysis. Alkaline phosphatase (ALP) activity and matrix mineralization were detected by biochemical methods. The expression levels of Runx2, ALP, and osteocalcin were detected by RT‐qPCR. Our results showed that miR‐1271‐5p was downregulated during osteogenic induction. And the expression levels of miR‐1271‐5p were higher in osteoporotic tissues than that in adjacent nonosteoporotic tissues. The expression levels of FOXO1 were lower in osteoporotic tissues than that in adjacent nonosteoporotic tissues. And a negative correlation was found between miR‐1271‐5p and FOXO1 in osteoporotic tissues. Overexpression of miR‐1271‐5p downregulated FOXO1 and inhibited osteogenic differentiation in hMSCs. Overexpression of miR‐1271‐5p downregulated the expression of osteogenic markers and reduced ALP activity. In addition, ectopic expression of FOXO1 reversed the effect of miR‐1271‐5p on osteogenic differentiation. In conclusion, miR‐1271‐5p functioned as a therapeutic target of osteogenic differentiation in hMSCs by inhibiting FOXO1, which provides valuable insights into the use of miR‐1271‐5p as a target in the treatment of osteoporosis and other bone metabolic diseases. [ABSTRACT FROM AUTHOR]

Up-regulated HIF-2α (hypoxia induced factor 2) had been demonstrated to contribute to Osteoarthritis (OA) development via inducing the expression of matrix-degrading enzymes. However, the HIF-2α also could promote primary cilia loss through HIF-2α/AURKA (Aurora kinase A)/NEDD9 pathway. And the primary cilia dysfunction is another characteristic of the OA. Thus, we investigated here whether the HIF-2α also contributes the OA development through mediating the primary cilia loss. The primary chondrocytes were isolated from the experimental OA mice induced by destabilization of the medial meniscus (DMM). Chondrocytes were cultured under normoxia (21% O 2) or hypoxia (2% O 2) conditions. The HIF-1α and HIF-2α expressions were assessed by western blot. The cilia formation was counted by immuno-staining the acetylated tubulin. The contribution of HIF-1α or HIF-2α to the primary cilia loss was assessed by knocking-down the HIF-1α or HIF-2α individually. The HIF-2α/AURKA/NEDD9 pathway was validated through over-expressing or knocking-down specific components of the pathway and then counting the primary cilia number. Finally, the pathway was further confirmed in the OA mice. Hypoxia could induce the expression of both HIF-1α and HIF-2α, and also reduce the number of primary cilia on the chondrocytes isolated from the experimental OA mice. Knocking-down or over-expressing HIF-1α or HIF-2α individually showed that the HIF-2α could induce the primary cilia reduction rather than the HIF-1α. Manipulating the HIF-2α expression could positively affect the AURKA and NEDD9 expression. Manipulating the AURKA and NEDD9 expressions could reverse the function of HIF-2α on primary cilia. In the mice, knocking-down both AURKA and NEDD9 could alleviate the OA development significantly. Up-regulated HIF-2α contributes to the Osteoarthritis development through mediating the primary cilia loss, which might be developed as therapeutic targets for OA treatment. • Up-regulated HIF-2α contributes to the Osteoarthritis development • HIF-2α induces primary cilia loss in OA through HIF2α/AURKA/ NEDD9 pathway • HIF-1α does not involve into the primary cilia regulation under hypoxia in OA [ABSTRACT FROM AUTHOR]