Oka Shunichi, Li Xiaoyan, Zhang Fengzhu, Tewari Nitesh, Ma Ri, Zhong Liangjun, Makishima Makoto, Liu Yi, Bhawal Ujjal K
Department of Anesthesiology, Nihon University School of Dentistry, Tokyo, Japan.
Division of Immunology and Pathology, Dental Research Center, Nihon University School of Dentistry, Tokyo, Japan.
Biochem Biophys Rep. 2020 Dec 31;25:100894. doi: 10.1016/j.bbrep.2020.100894. eCollection 2021 Mar.
MicroRNAs are emerging as critical post-transcriptional modulators in bone remodeling, regulating the functions of osteoblasts and osteoclasts. Intercellular crosstalk between osteoblasts and osteoclasts is mediated by miR-21 that controls the bone homeostasis response, providing potential targets for the maintenance of osteoblast function. The aim of this study was to investigate the effects of miR-21 on osteoblast function, and to explore the underlying mechanism. Increased alkaline phosphatase (ALP) activity and accelerated matrix mineralization was observed in mouse pre-osteoblast MC3T3-E1 cells compared with the non-induction (control) group. MiR-21 positively regulates osteogenic differentiation and mineralization by facilitating the expression of key osteogenic factors (ALP, Runx2, Osteopontin (OPN), Osterix (OSX) and Mef2c) in MC3T3-E1 cells. Furthermore, a deficiency of miR-21 suppresses the expression of those factors at both the mRNA and protein levels, indicating that miR-21 is a positive regulator of osteoblastic differentiation. H-E staining, Azan staining, Masson's Trichrome staining and Toluidine blue staining were performed in jaw and femur tissues of miR-21 knockout (miR-21KO) and wild-type (WT) mice. Immunohistochemical staining revealed substantially lower levels of ALP, Runx2 and OSX expression in jaw and femur tissues of miR-21KO mice. A similar trend was observed in femur tissues using quantitative real-time (RT) PCR. A total of 17 osteogenesis-related mRNAs were found to be differentially expressed in miR-21KO femur tissues using Mouse Gene Expression Microarray analysis. GeneSpring and Ingenuity Pathway Analysis revealed several potential target genes that are involved in bone remodeling, such as IL-1β and HIF-1α. Several important pathways were determined to be facilitators of miR-21, which provides a reliable reference for future studies to elucidate the biological mechanisms of osteoblast function. Taken together, these results lead us to hypothesize a potential role for miR-21 in regulating osteoblast function, thus representing a potential biomarker of osteogenesis.
微小RNA正逐渐成为骨重塑过程中关键的转录后调节因子，调控成骨细胞和破骨细胞的功能。成骨细胞和破骨细胞之间的细胞间串扰由控制骨稳态反应的miR-21介导，为维持成骨细胞功能提供了潜在靶点。本研究的目的是探讨miR-21对成骨细胞功能的影响，并探究其潜在机制。与未诱导（对照）组相比，在小鼠前成骨细胞MC3T3-E1细胞中观察到碱性磷酸酶（ALP）活性增加和基质矿化加速。MiR-21通过促进MC3T3-E1细胞中关键成骨因子（ALP、Runx2、骨桥蛋白（OPN）、osterix（OSX）和Mef2c）的表达来正向调节成骨分化和矿化。此外，miR-21的缺乏在mRNA和蛋白质水平上均抑制了这些因子的表达，表明miR-21是成骨细胞分化的正向调节因子。对miR-21基因敲除（miR-21KO）和野生型（WT）小鼠的颌骨和股骨组织进行苏木精-伊红染色、偶氮染色、马松三色染色和甲苯胺蓝染色。免疫组织化学染色显示，miR-21KO小鼠颌骨和股骨组织中ALP、Runx2和OSX的表达水平显著降低。使用定量实时（RT）PCR在股骨组织中观察到类似趋势。使用小鼠基因表达微阵列分析发现，共有17种与成骨相关的mRNA在miR-21KO股骨组织中差异表达。基因弹簧和 Ingenuity 通路分析揭示了几个参与骨重塑的潜在靶基因，如IL-1β和HIF-1α。确定了几个重要通路是miR-21的促进因子，这为未来阐明成骨细胞功能生物学机制的研究提供了可靠参考。综上所述，这些结果使我们推测miR-21在调节成骨细胞功能方面具有潜在作用，因此代表了一种潜在的成骨生物标志物。
