探索和验证缺氧诱导系统性硬化症内皮-间充质转化相关的枢纽基因。
Exploration and validation of the hub genes involved in hypoxia-induced endothelial-mesenchymal transition of systemic sclerosis.
机构信息
Zhang Zhongjing College of Chinese Medicine, Nanyang Institute of Technology, Nanyang, Henan, and Henan Key Laboratory of Zhang Zhongjing Formulae and Herbs for Immunoregulation, Nanyang Institute of Technology, Nanyang, Henan, China.
Traditional Chinese Medical College, North China University of Science and Technology, Tangshan, Hebei, China.
出版信息
Clin Exp Rheumatol. 2023 Aug;41(8):1618-1631. doi: 10.55563/clinexprheumatol/j7ema8. Epub 2023 May 25.
OBJECTIVES
During the development of systemic sclerosis (SSc), endothelial-mesenchymal transition (EndoMT) has been shown to be one of the mechanisms leading to pulmonary fibrosis. However, the correlation between hypoxia and EndoMT was mostly unknown.
METHODS
R software was used to analyse differentially expressed genes (DEGs) in vascular endothelial cells under hypoxic conditions, and fibroblasts derived from SSc-related pulmonary fibrotic tissues, respectively. Using a web-based online Venn diagram tool, we analysed overlapping genes of DEGs between endothelial cells and fibroblasts. Finally, the protein-protein interaction network of EndoMT hub genes were constructed using the STRING database. The hub genes were knockdown by transfection of siRNAs in the hypoxia model of HULEC-5a cells constructed by liquid paraffin closure and then used to detect the effect on EndoMT-related biomarkers by western blot.
RESULTS
In this study, we found that INHBA, DUSP1, NOX4, PLOD2, BHLHE40 were upregulated in SSc fibroblasts and hypoxic-treated endothelial cells, while VCAM1, RND3, CCL2, and TXNIP were downregulated. In the hypoxia model of HULEC-5a cells, the expression of these 9 hub genes was confirmed by western blot. In addition, through Spearman's correlation analysis and Western blot, we confirmed that these hub genes were closely related to the EndoMT-related markers. The mechanisms of these hypoxia-induced EndoMT hub genes may be related to TGF-β, Notch, Wnt, NF-κ B, TNF and mTOR signalling pathways.
CONCLUSIONS
Our study provides new insights into the occurrence and development of SSc-related pulmonary fibrosis resulting from hypoxia-induced EndoMT.
目的
在系统性硬化症（SSc）的发展过程中，内皮-间充质转化（EndoMT）已被证明是导致肺纤维化的机制之一。然而，缺氧与 EndoMT 之间的相关性大多未知。
方法
使用 R 软件分别分析缺氧条件下血管内皮细胞和 SSc 相关肺纤维化组织来源的成纤维细胞中的差异表达基因（DEGs）。使用基于网络的在线 Venn 图工具，分析内皮细胞和成纤维细胞之间 DEGs 的重叠基因。最后，使用 STRING 数据库构建 EndoMT 枢纽基因的蛋白质-蛋白质相互作用网络。通过液体石蜡封闭构建的 HULEC-5a 细胞缺氧模型中转染 siRNA 敲低枢纽基因，然后通过 Western blot 检测对 EndoMT 相关生物标志物的影响。
结果
本研究发现，INHBA、DUSP1、NOX4、PLOD2 和 BHLHE40 在 SSc 成纤维细胞和缺氧处理的内皮细胞中上调，而 VCAM1、RND3、CCL2 和 TXNIP 下调。在 HULEC-5a 细胞的缺氧模型中，通过 Western blot 验证了这 9 个枢纽基因的表达。此外，通过 Spearman 相关性分析和 Western blot，我们证实这些枢纽基因与 EndoMT 相关标志物密切相关。这些缺氧诱导的 EndoMT 枢纽基因的机制可能与 TGF-β、Notch、Wnt、NF-κB、TNF 和 mTOR 信号通路有关。
结论
我们的研究为缺氧诱导的 EndoMT 导致的 SSc 相关肺纤维化的发生和发展提供了新的见解。
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