Abstract：
BACKGROUND: Periodontal ligament stem cells (PDLSCs) are the most potential cells in periodontal tissue regeneration and bone tissue regeneration. Our prior work had revealed that WD repeat-containing protein 72 (WDR72) was crucial for osteogenic differentiation of PDLSCs. Here, we further elucidated its underlying mechanism in PDLSC osteogenic differentiation.
METHODS: Human PDLSCs, isolated and identified by flow cytometry, were prepared for osteogenic differentiation induction. Levels of WDR72, long non-coding RNA X-Inactive Specific Transcript (XIST), upstream stimulatory factor 2 (USF2), and osteogenic marker genes (Runx2, Osteocalcin, and Collagen I) in human PDLSCs and clinical specimens were detected by RT-qPCR. Protein expressions of WDR72, Runx2, Osteocalcin, and Colla1 were tested by Western blot. The interactions among the molecules were verified by RIP, RNA pull-down, ChIP, and luciferase reporter assays. Osteogenic differentiation was evaluated by alkaline phosphatase (ALP) and alizarin red staining (ARS).
RESULTS: WDR72 was decreased in periodontal tissues of periodontitis patients, and overexpression reversed TNF-α-mediated suppressive effects on PDLSC osteogenic differentiation. Mechanically, XIST recruited the enrichment of USF2 to the WDR72 promoter region, thereby positively regulating WDR72. WDR72 silencing overturned XIST-mediated biological effects in PDLSCs.
CONCLUSIONS: WDR72, regulated by the XIST/USF2 axis, enhances osteogenic differentiation of PDLSCs, implying a novel strategy for alleviating periodontitis.
METHODS: Human PDLSCs, isolated and identified by flow cytometry, were prepared for osteogenic differentiation induction. Levels of WDR72, long non-coding RNA X-Inactive Specific Transcript (XIST), upstream stimulatory factor 2 (USF2), and osteogenic marker genes (Runx2, Osteocalcin, and Collagen I) in human PDLSCs and clinical specimens were detected by RT-qPCR. Protein expressions of WDR72, Runx2, Osteocalcin, and Colla1 were tested by Western blot. The interactions among the molecules were verified by RIP, RNA pull-down, ChIP, and luciferase reporter assays. Osteogenic differentiation was evaluated by alkaline phosphatase (ALP) and alizarin red staining (ARS).
RESULTS: WDR72 was decreased in periodontal tissues of periodontitis patients, and overexpression reversed TNF-α-mediated suppressive effects on PDLSC osteogenic differentiation. Mechanically, XIST recruited the enrichment of USF2 to the WDR72 promoter region, thereby positively regulating WDR72. WDR72 silencing overturned XIST-mediated biological effects in PDLSCs.
CONCLUSIONS: WDR72, regulated by the XIST/USF2 axis, enhances osteogenic differentiation of PDLSCs, implying a novel strategy for alleviating periodontitis.
摘要：
背景：牙周膜干细胞（PDLSCs）是牙周组织再生和骨组织再生中最有潜力的细胞。我们先前的工作表明，含WD重复序列的蛋白72（WDR72）对于PDLSCs的成骨分化至关重要。这里,我们进一步阐明了其在PDLSC成骨分化中的潜在机制。
方法：人类PDLSCs，通过流式细胞术分离和鉴定，准备用于成骨分化诱导。WDR72，长链非编码RNAX-失活特异性转录本（XIST）的水平，上游刺激因子2（USF2），和成骨标记基因(Runx2，骨钙蛋白，通过RT-qPCR检测人PDLSCs和临床标本中的胶原蛋白I）。WDR72、Runx2、骨钙蛋白的表达,和Colla1通过蛋白质印迹测试。通过RIP验证了分子之间的相互作用，RNA下拉，ChIP,和荧光素酶报告基因测定。通过碱性磷酸酶（ALP）和茜素红染色（ARS）评估成骨分化。
结果：牙周炎患者牙周组织中WDR72减少，过度表达可逆转TNF-α介导的对PDLSC成骨分化的抑制作用。机械上,XIST将USF2富集到WDR72启动子区，从而积极调节WDR72。WDR72沉默推翻了XIST介导的PDLSCs生物学效应。
结论：WDR72，受XIST/USF2轴调节，增强PDLSCs的成骨分化，暗示了一种缓解牙周炎的新策略。
方法：人类PDLSCs，通过流式细胞术分离和鉴定，准备用于成骨分化诱导。WDR72，长链非编码RNAX-失活特异性转录本（XIST）的水平，上游刺激因子2（USF2），和成骨标记基因(Runx2，骨钙蛋白，通过RT-qPCR检测人PDLSCs和临床标本中的胶原蛋白I）。WDR72、Runx2、骨钙蛋白的表达,和Colla1通过蛋白质印迹测试。通过RIP验证了分子之间的相互作用，RNA下拉，ChIP,和荧光素酶报告基因测定。通过碱性磷酸酶（ALP）和茜素红染色（ARS）评估成骨分化。
结果：牙周炎患者牙周组织中WDR72减少，过度表达可逆转TNF-α介导的对PDLSC成骨分化的抑制作用。机械上,XIST将USF2富集到WDR72启动子区，从而积极调节WDR72。WDR72沉默推翻了XIST介导的PDLSCs生物学效应。
结论：WDR72，受XIST/USF2轴调节，增强PDLSCs的成骨分化，暗示了一种缓解牙周炎的新策略。
