Abstract：
BACKGROUND: MicroRNA (miRNA) is accepted as a critical regulator of cell differentiation. However, whether microRNA-223 (miR-223) could affect the osteogenic differentiation of periodontal ligament (PDL)-derived cells is still unknown. The aim of this study was to explore the mechanisms underlying the roles of miR-223 in the osteogenesis of PDL-derived cells in periodontitis.
METHODS: Microarray analysis and real-time polymerase chain reaction (RT-PCR) were used to identify difference in miR-223 expression pattern between healthy and inflamed gingival tissue. The target genes of miR-223 were predicted based on Targetscan and selected for enrichment analyses based on Metascape database. The gain-and loss-of-function experiments were performed to discuss roles of miR-223 and growth factor receptor genes in osteogenic differentiation of PDL-derived cells. The target relationship between miR-223 and growth factor receptor genes was confirmed by a dual luciferase assay. Osteogenic differentiation of PDL-derived cells was assessed by Alizarin red staining, RT-PCR and western blot detection of osteogenic markers, including osteocalcin (OCN), osteopontin (OPN) and runt-related transcription factor 2 (Runx2).
RESULTS: MiR-223 was significantly increased in inflamed gingival tissues and down-regulated in PDL-derived cells during osteogenesis. The expression of miR-223 in gingival tissues was positively correlated with the clinical parameters in periodontitis patients. Overexpression of miR-223 markedly inhibited PDL-derived cells osteogenesis, which was evidenced by reduced Alizarin red staining and osteogenic markers expressions. Furthermore, two growth factor receptor genes, including fibroblast growth factor receptor 2 (FGFR2) and transforming growth factor beta receptor 2 (TGFβR2), were revealed to be direct targets of miR-223 and shown to undergo up-regulation in PDL-derived cells during osteogenesis. Moreover, suppression of FGFR2 or TGFβR2 dramatically blocked PDL-derived cells osteogenic differentiation.
CONCLUSIONS: Our study provides novel evidence that miR-223 can be induced by periodontitis and acts as a negative regulator of PDL-derived cells osteogenesis by targeting two growth factor receptors (TGFβR2 and FGFR2).
METHODS: Microarray analysis and real-time polymerase chain reaction (RT-PCR) were used to identify difference in miR-223 expression pattern between healthy and inflamed gingival tissue. The target genes of miR-223 were predicted based on Targetscan and selected for enrichment analyses based on Metascape database. The gain-and loss-of-function experiments were performed to discuss roles of miR-223 and growth factor receptor genes in osteogenic differentiation of PDL-derived cells. The target relationship between miR-223 and growth factor receptor genes was confirmed by a dual luciferase assay. Osteogenic differentiation of PDL-derived cells was assessed by Alizarin red staining, RT-PCR and western blot detection of osteogenic markers, including osteocalcin (OCN), osteopontin (OPN) and runt-related transcription factor 2 (Runx2).
RESULTS: MiR-223 was significantly increased in inflamed gingival tissues and down-regulated in PDL-derived cells during osteogenesis. The expression of miR-223 in gingival tissues was positively correlated with the clinical parameters in periodontitis patients. Overexpression of miR-223 markedly inhibited PDL-derived cells osteogenesis, which was evidenced by reduced Alizarin red staining and osteogenic markers expressions. Furthermore, two growth factor receptor genes, including fibroblast growth factor receptor 2 (FGFR2) and transforming growth factor beta receptor 2 (TGFβR2), were revealed to be direct targets of miR-223 and shown to undergo up-regulation in PDL-derived cells during osteogenesis. Moreover, suppression of FGFR2 or TGFβR2 dramatically blocked PDL-derived cells osteogenic differentiation.
CONCLUSIONS: Our study provides novel evidence that miR-223 can be induced by periodontitis and acts as a negative regulator of PDL-derived cells osteogenesis by targeting two growth factor receptors (TGFβR2 and FGFR2).
摘要：
背景：MicroRNA（miRNA）被认为是细胞分化的关键调节因子。然而,microRNA-223(miR-223)是否会影响牙周膜(PDL)来源细胞的成骨分化尚不清楚.本研究旨在探讨miR-223在牙周炎PDL细胞成骨过程中的作用机制。
方法：使用微阵列分析和实时聚合酶链反应（RT-PCR）来鉴定健康和发炎牙龈组织之间miR-223表达模式的差异。基于Targetscan预测miR-223的靶基因,并基于Metascape数据库选择用于富集分析。进行了功能获得和功能丧失实验，以讨论miR-223和生长因子受体基因在PDL衍生细胞成骨分化中的作用。miR-223与生长因子受体基因之间的靶关系通过双荧光素酶测定得到证实。通过茜素红染色评估PDL衍生细胞的成骨分化，成骨标志物的RT-PCR和Westernblot检测,包括骨钙蛋白（OCN），骨桥蛋白（OPN）和runt相关转录因子2（Runx2）。
结果：在成骨过程中，MiR-223在发炎的牙龈组织中显著增加，而在PDL来源的细胞中下调。miR-223在牙周炎患者牙龈组织中的表达与临床参数呈正相关。过表达miR-223显著抑制PDL衍生细胞成骨,茜素红染色和成骨标志物表达降低证明了这一点。此外，两个生长因子受体基因，包括成纤维细胞生长因子受体2（FGFR2）和转化生长因子β受体2（TGFβR2），被揭示为miR-223的直接靶标，并显示在成骨过程中在PDL衍生的细胞中经历上调。此外，抑制FGFR2或TGFβR2可显著阻断PDL来源的细胞成骨分化.
结论：我们的研究提供了新的证据，表明miR-223可以由牙周炎诱导，并通过靶向两种生长因子受体（TGFβR2和FGFR2）作为PDL衍生细胞成骨的负调控因子。
方法：使用微阵列分析和实时聚合酶链反应（RT-PCR）来鉴定健康和发炎牙龈组织之间miR-223表达模式的差异。基于Targetscan预测miR-223的靶基因,并基于Metascape数据库选择用于富集分析。进行了功能获得和功能丧失实验，以讨论miR-223和生长因子受体基因在PDL衍生细胞成骨分化中的作用。miR-223与生长因子受体基因之间的靶关系通过双荧光素酶测定得到证实。通过茜素红染色评估PDL衍生细胞的成骨分化，成骨标志物的RT-PCR和Westernblot检测,包括骨钙蛋白（OCN），骨桥蛋白（OPN）和runt相关转录因子2（Runx2）。
结果：在成骨过程中，MiR-223在发炎的牙龈组织中显著增加，而在PDL来源的细胞中下调。miR-223在牙周炎患者牙龈组织中的表达与临床参数呈正相关。过表达miR-223显著抑制PDL衍生细胞成骨,茜素红染色和成骨标志物表达降低证明了这一点。此外，两个生长因子受体基因，包括成纤维细胞生长因子受体2（FGFR2）和转化生长因子β受体2（TGFβR2），被揭示为miR-223的直接靶标，并显示在成骨过程中在PDL衍生的细胞中经历上调。此外，抑制FGFR2或TGFβR2可显著阻断PDL来源的细胞成骨分化.
结论：我们的研究提供了新的证据，表明miR-223可以由牙周炎诱导，并通过靶向两种生长因子受体（TGFβR2和FGFR2）作为PDL衍生细胞成骨的负调控因子。
