BACKGROUND Periodontal ligament stromal cells (PDLSCs) are ideal cell sources for periodontal tissue repair and regeneration, but little is known about what determines their osteogenic capacity. Long non-coding RNAs (lncRNAs) are important regulatory molecules at both transcriptional and post-transcriptional levels. However, their roles in the osteogenic differentiation of PDLSCs are still largely unknown. METHODS The expression of lncRNA Fer-1-like family member 4 (FER1L4) during the osteogenic differentiation of PDLSCs was detected by quantitative reverse transcription polymerase chain reaction. Overexpression or knockdown of FER1L4 was used to confirm its regulation of osteogenesis in PDLSCs. Alkaline phosphatase and Alizarin red S staining were used to detect mineral deposition. Dual luciferase reporter assays were used to analyze the binding of miR-874-3p to FER1L4 and vascular endothelial growth factor A (VEGFA). Bone regeneration in critical-sized calvarial defects was assessed in nude mice. New bone formation was analyzed by micro-CT, hematoxylin and eosin staining, Masson's trichrome staining, and immunohistochemical analyses. RESULTS FER1L4 levels increased gradually during consecutive osteogenic induction of PDLSCs. Overexpression of FER1L4 promoted the osteogenic differentiation of PDLSCs, as revealed by alkaline phosphatase activity, Alizarin red S staining, and the expression of osteogenic markers, whereas FER1L4 knockdown inhibited these processes. Subsequently, we identified a predicted binding site for miR-874-3p on FER1L4 and confirmed a direct interaction between them. Wild-type FER1L4 reporter activity was significantly inhibited by miR-874-3p, whereas mutant FER1L4 reporter was not affected. MiR-874-3p inhibited osteogenic differentiation and reversed the promotion of osteogenesis in PDLSCs by FER1L4. Moreover, miR-874-3p targeted VEGFA, a crucial gene in osteogenic differentiation, whereas FER1L4 upregulated the expression of VEGFA. In vivo, overexpression of FER1L4 led to more bone formation compared to the control group, as demonstrated by micro-CT and the histologic analyses. CONCLUSION FER1L4 positively regulates the osteogenic differentiation of PDLSCs via miR-874-3p and VEGFA. Our study provides a promising target for enhancing the osteogenic potential of PDLSCs and periodontal regeneration.

中文翻译：

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长非编码RNA FER1L4通过miR-874-3p和血管内皮生长因子A促进人牙周膜间质细胞的成骨分化。

背景技术牙周膜间质细胞（PDLSC）是用于牙周组织修复和再生的理想细胞来源，但是对于决定其成骨能力的知之甚少。长非编码RNA（lncRNA）在转录和转录后水平上都是重要的调节分子。但是，它们在PDLSC的成骨分化中的作用仍然是未知的。方法采用定量逆转录聚合酶链反应检测PDLSCs成骨分化过程中lncRNA Fer-1样家族成员4（FER1L4）的表达。FER1L4的过表达或敲低被用来确认其在PDLSCs中对成骨的调控。碱性磷酸酶和茜素红S染色用于检测矿物沉积。使用双重荧光素酶报告基因分析来分析miR-874-3p与FER1L4和血管内皮生长因子A（VEGFA）的结合。在裸鼠中评估临界大小的颅骨缺损中的骨再生。通过微型CT，苏木精和曙红染色，Masson三色染色和免疫组化分析来分析新的骨形成。结果在连续的成骨诱导PDLSCs过程中，FER1L4水平逐渐升高。如碱性磷酸酶活性，茜素红S染色和成骨标记物的表达所揭示，FER1L4的过表达促进了PDLSC的成骨分化，而FER1L4的抑制则抑制了这些过程。随后，我们确定了FER1L4上miR-874-3p的预测结合位点，并确认了它们之间的直接相互作用。野生型FER1L4报告基因活性受到miR-874-3p的显着抑制，而突变FER1L4报告基因则不受此影响。MiR-874-3p通过FER1L4抑制PDLSCs的成骨分化并逆转成骨作用。此外，miR-874-3p靶向VEGFA，这是成骨分化中的关键基因，而FER1L4上调了VEGFA的表达。在体内，与对照组相比，FER1L4的过表达导致更多的骨形成，如微型CT和组织学分析所证实。结论FER1L4通过miR-874-3p和VEGFA积极调节PDLSC的成骨分化。我们的研究为增强PDLSCs的成骨潜能和牙周再生提供了有希望的目标。MiR-874-3p通过FER1L4抑制PDLSCs的成骨分化并逆转成骨作用。此外，miR-874-3p靶向VEGFA，这是成骨分化中的关键基因，而FER1L4上调了VEGFA的表达。在体内，与对照组相比，FER1L4的过表达导致更多的骨形成，如微型CT和组织学分析所证实。结论FER1L4通过miR-874-3p和VEGFA积极调节PDLSC的成骨分化。我们的研究为增强PDLSCs的成骨潜能和牙周再生提供了有希望的目标。MiR-874-3p通过FER1L4抑制PDLSCs的成骨分化并逆转成骨作用。此外，miR-874-3p靶向VEGFA，这是成骨分化中的关键基因，而FER1L4上调了VEGFA的表达。在体内，与对照组相比，FER1L4的过表达导致更多的骨形成，如微CT和组织学分析所证实。结论FER1L4通过miR-874-3p和VEGFA积极调节PDLSC的成骨分化。我们的研究为增强PDLSCs的成骨潜能和牙周再生提供了有希望的目标。显微CT和组织学分析表明，与对照组相比，FER1L4的过表达导致更多的骨形成。结论FER1L4通过miR-874-3p和VEGFA积极调节PDLSC的成骨分化。我们的研究为增强PDLSCs的成骨潜能和牙周再生提供了有希望的目标。显微CT和组织学分析表明，与对照组相比，FER1L4的过表达导致更多的骨形成。结论FER1L4通过miR-874-3p和VEGFA积极调节PDLSC的成骨分化。我们的研究为增强PDLSCs的成骨潜能和牙周再生提供了有希望的目标。