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.
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。