Circ-USP9X 通过作为 miR-148b-3p 的海绵体和上调 SRC 激酶信号抑制剂 1 加速骨折后深静脉血栓形成。
Circ-USP9X accelerates deep vein thrombosis after fracture by acting as a miR-148b-3p sponge and upregulates SRC kinase signaling inhibitor 1.
机构信息
Department of Joint Sport Medicine, The First Affiliated Hospital of Hunan Medical College, Huaihua City, Hunan Province, PR China.
School of Nursing, Hunan Medical College, Huaihua City, Hunan Province, PR China.
出版信息
Clinics (Sao Paulo). 2024 Jun 14;79:100403. doi: 10.1016/j.clinsp.2024.100403. eCollection 2024.
OBJECTIVES
This study aims to elucidate the role of circUSP9X (Circular RNA Ubiquitin Specific Peptidase 9 X-Linked) in the development of venous thrombosis in the lower extremities.
METHODS
An animal model of Deep Vein Thrombosis (DVT) and a hypoxic model of Human Umbilical Vein Endothelial Cells (HUVECs) treated with Cobalt (II) Chloride (CoCl) were developed. The expression levels of circUSP9X, microRNA-148b-3p (miR-148b-3p), and SRC Kinase Signaling Inhibitor 1 (SRCIN1) were quantified using quantitative reverse transcription Polymerase Chain Reaction and Western blot analysis. Cell cytotoxicity, viability, apoptosis, and inflammation in HUVECs were assessed via Lactate Dehydrogenase (LDH) assay, MTT assay, flow cytometry, Enzyme-Linked Immunosorbent Assay, and Western blot, respectively. Hematoxylin and Eosin staining were employed for histopathological examination of the venous tissues in the animal model. The interaction between circUSP9X, miR-148b-3p, and SRCIN1 was further explored through dual-luciferase reporter assays and RNA Immunoprecipitation experiments.
RESULTS
The present findings reveal a significant upregulation of circUSP9X and SRCIN1 and a concurrent downregulation of miR-148b-3p in DVT cases. Knockdown of circUSP9X or overexpression of miR-148b-3p ameliorated CoCl-induced apoptosis in HUVECs, reduced LDH release, enhanced cellular viability, and mitigated inflammation. Conversely, overexpression of circUSP9X intensified CoCl's cytotoxic effects. The effects of manipulating circUSP9X expression were counteracted by the corresponding modulation of miR-148b-3p and SRCIN1 levels. Additionally, circUSP9X knockdown effectively inhibited the formation of DVT in the mouse model. A competitive binding mechanism of circUSP9X for miR-148b-3p, modulating SRCIN1 expression, was identified.
CONCLUSION
circUSP9X promotes the formation of DVT through the regulation of the miR-148b-3p/SRCIN1 axis.
目的
本研究旨在阐明环状 RNA 泛素特异性肽酶 9 X 连锁（circUSP9X）在下肢静脉血栓形成中的作用。
方法
建立深静脉血栓形成（DVT）动物模型和缺氧人脐静脉内皮细胞（HUVEC）模型，用氯化钴（CoCl）处理。采用定量逆转录聚合酶链反应和 Western blot 分析检测 circUSP9X、微小 RNA-148b-3p（miR-148b-3p）和 SRC 激酶信号抑制剂 1（SRCIN1）的表达水平。通过乳酸脱氢酶（LDH）测定、MTT 测定、流式细胞术、酶联免疫吸附测定和 Western blot 分别评估 HUVEC 细胞的细胞毒性、活力、凋亡和炎症。采用苏木精和伊红染色法对动物模型静脉组织进行组织病理学检查。通过双荧光素酶报告基因检测和 RNA 免疫沉淀实验进一步探讨 circUSP9X、miR-148b-3p 和 SRCIN1 之间的相互作用。
结果
本研究发现 DVT 病例中 circUSP9X 和 SRCIN1 显著上调，miR-148b-3p 下调。circUSP9X 敲低或 miR-148b-3p 过表达可改善 CoCl 诱导的 HUVEC 细胞凋亡，减少 LDH 释放，增强细胞活力，减轻炎症。相反，circUSP9X 过表达增强了 CoCl 的细胞毒性作用。circUSP9X 表达调控的作用可被 miR-148b-3p 和 SRCIN1 水平的相应调节所拮抗。此外，circUSP9X 敲低可有效抑制小鼠模型 DVT 的形成。鉴定出 circUSP9X 通过调节 miR-148b-3p/SRCIN1 轴竞争性结合 miR-148b-3p 的机制。
结论
circUSP9X 通过调节 miR-148b-3p/SRCIN1 轴促进 DVT 的形成。
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