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Abstract
Ossification of the ligamentum flavum (OLF) is a disorder of heterotopic ossification of spinal ligaments and is the main cause of thoracic spinal canal stenosis. Previous studies suggested that miR-132-3p negatively regulates osteoblast differentiation. However, whether miR-132-3p is involved in the process of OLF has not been investigated. In this study, we investigated the effect of miR-132-3p and its target genes forkhead box O1 (FOXO1), growth differentiation factor 5 (GDF5) and SRY-box 6 (SOX6) on the osteogenic differentiation of ligamentum flavum (LF) cells. We demonstrated that miR-132-3p was down-regulated during the osteogenic differentiation of LF cells and negatively regulated the osteoblast differentiation. Further, miR-132-3p targeted FOXO1, GDF5 and SOX6 and down-regulated the protein expression of these genes. Meanwhile, FOXO1, GDF5 and SOX6 were up-regulated after osteogenic differentiation and the down-regulation of endogenous FOXO1, GDF5 or SOX6 suppressed the osteogenic differentiation of LF cells. In addition, we also found FOXO1, GDF5 and SOX6 expression in the ossification front of OLF samples. Overall, these results suggest that miR-132-3p inhibits the osteogenic differentiation of LF cells by targeting FOXO1, GDF5 and SOX6.
Highlights
Ossification of the ligamentum flavum (OLF) is a rare disorder of heterotopic ossification of spinal ligaments that is almost exclusively reported in Eastern Asian countries
We demonstrated that down-regulation of endogenous forkhead box O1 (FOXO1), growth differentiation factor 5 (GDF5) or sex determining region y-box (SRY-box) 6 (SOX6) suppressed osteogenesis to varying degrees, respectively
Our study provides a comprehensive profiling of miR-132-3p as a novel regulator during the osteogenic differentiation of LF cells
Summary
Ossification of the ligamentum flavum (OLF) is a rare disorder of heterotopic ossification of spinal ligaments that is almost exclusively reported in Eastern Asian countries. TThhee mmRRNNAA aanndd pprrootteeiinn eexxpprreessssiioonn lleevveellss ooff FFOOXXOO11,, GGDDFF55 aanndd SSOOXX66 wweerree ddeetteerrmmiinneedd bbyy qquuaannttiittaattiivveerreeaall--ttiimmeeppoollyymmeerraassee cchhaaiinn rreeaaccttiioonn ((qqRRTT--PPCCRR))((FFiigguurree33AA))aannddWWeesstteerrnnbblolottaannaalylysseess ((FFigiguurree33BB))..AAllltthhrreeeewweerreeuupp--rreegguullaatteeddiinneexxpprreessssiioonnaafftteerroosstteeooggeenniiccddiifffeerreennttiiaattiioonnccoommppaarreeddwwiitthh ddaayy0.0S. FFOOXXOO11, ,GGDDFF55aannddSSOOXX66 kknnoocckkddoowwnn iinnhhiibbiittss oosstteeooggeenniicc ddiiffffeerreennttiiaattiioonn ooff lliiggaammeennttuumm ffllaavvuummcceelllsls. MiR-132-3p regulated osteoblast differentiation in osteoporosis by Besides, miR-132-3p regulated osteoblast differentiation in osteoporosis by targeting EP300 [27] and SIRT1 [28] Whether these two genes were involved in osteogenic differentiation of LF cells requires further study. FOXO1, GDF5 and SOX6 were all found to encourage the osteogenic differentiation of LF cells. During the osteogenic differentiation of LF cells, the three osteogenesis-related genes were up-regulated, and the highest value of each gene expression was found at different times. More research is required to understand the detailed mechanism of these osteogenesis-related genes in LF cells
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