Fibronectin (FN) is a main component of extracellular matrix (ECM) in most adult tissues. Under pathological conditions, particularly inflammation, wound healing and tumors, an alternatively spliced exon extra domain A (EDA) is included in the FN protein (EDA+FN), which facilitates cellular proliferation, motility, and aggressiveness in different lesions. In this study we investigated the effects of EDA+FN on bone destruction in human radicular cysts and explored the possibility of editing FN gene or blocking the related paracrine signaling pathway to inhibit the osteoclastogenesis. The specimens of radicular cysts were obtained from 20 patients. We showed that the vessel density was positively associated with both the lesion size (R = 0.49, P = 0.001) and EDA+FN staining (R = 0.26, P = 0.022) in the specimens. We isolated fibroblasts from surgical specimens, and used the CRISPR/Cas system to knockout the EDA exon, or used IST-9 antibody and bevacizumab to block EDA+FN and VEGF, respectively. Compared to control fibroblasts, the fibroblasts from radicular cysts exhibited significantly more Trap+MNCs, the relative expression level of VEGF was positively associated with both the ratio of EDA+FN/total FN (R = 0.271, P = 0.019) and with the number of Trap+MNCs (R = 0.331, P = 0.008). The knockout of the EDA exon significantly decreased VEGF expression in the fibroblasts derived from radicular cysts, leading to significantly decreased osteoclastogenesis; similar results were observed using bevacizumab to block VEGF, but block of EDA+FN with IST-9 antibody had no effect. Furthermore, the inhibitory effects of gene editing on Trap+MNC development were restored by exogenous VEGF. These results suggest that EDA+FN facilitates osteoclastogenesis in the fibrous capsule of radicular cysts, through a mechanism mediated by VEGF via an autocrine effect on the fibroblasts. Bevacizumab inhibits osteoclastogenesis in radicular cysts as effectively as the exclusion of the EDA exon by gene editing.

中文翻译：

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贝伐单抗或纤连蛋白基因编辑抑制源自人根性囊肿的成纤维细胞的破骨细胞作用。

纤连蛋白 (FN) 是大多数成人组织中细胞外基质 (ECM) 的主要成分。在病理条件下，特别是炎症、伤口愈合和肿瘤，FN 蛋白 (EDA+FN) 中包含一个选择性剪​​接的外显子额外域 A (EDA)，它促进不同病变中的细胞增殖、运动和侵袭性。在本研究中，我们研究了 EDA+FN 对人根性囊肿骨破坏的影响，并探讨了编辑 FN 基因或阻断相关旁分泌信号通路以抑制破骨细胞生成的可能性。根性囊肿标本取自 20 名患者。我们发现血管密度与标本中的病变大小 (R = 0.49, P = 0.001) 和 EDA+FN 染色 (R = 0.26, P = 0.022) 呈正相关。我们从手术标本中分离成纤维细胞，并使用 CRISPR/Cas 系统敲除 EDA 外显子，或使用 IST-9 抗体和贝伐单抗分别阻断 EDA+FN 和 VEGF。与对照成纤维细胞相比，根性囊肿的成纤维细胞表现出明显更多的 Trap+MNCs，VEGF 的相对表达水平与 EDA+FN/总 FN 的比值（R = 0.271，P = 0.019）和数量呈正相关。 Trap+MNCs (R = 0.331, P = 0.008)。EDA 外显子的敲除显着降低了源自根性囊肿的成纤维细胞中 VEGF 的表达，导致破骨细胞生成显着降低；使用贝伐单抗阻断 VEGF 观察到类似结果，但使用 IST-9 抗体阻断 EDA+FN 没有效果。此外，外源性VEGF恢复了基因编辑对Trap+MNC发育的抑制作用。这些结果表明，EDA+FN 通过 VEGF 介导的机制，通过对成纤维细胞的自分泌作用，促进根性囊肿纤维囊中的破骨细胞生成。贝伐单抗抑制根性囊肿中的破骨细胞生成与通过基因编辑排除 EDA 外显子一样有效。