PDF(Pubmed)
Abstract：
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.
An animal model of Deep Vein Thrombosis (DVT) and a hypoxic model of Human Umbilical Vein Endothelial Cells (HUVECs) treated with Cobalt (II) Chloride (CoCl2) 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.
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 CoCl2-induced apoptosis in HUVECs, reduced LDH release, enhanced cellular viability, and mitigated inflammation. Conversely, overexpression of circUSP9X intensified CoCl2\'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.
circUSP9X promotes the formation of DVT through the regulation of the miR-148b-3p/SRCIN1 axis.
An animal model of Deep Vein Thrombosis (DVT) and a hypoxic model of Human Umbilical Vein Endothelial Cells (HUVECs) treated with Cobalt (II) Chloride (CoCl2) 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.
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 CoCl2-induced apoptosis in HUVECs, reduced LDH release, enhanced cellular viability, and mitigated inflammation. Conversely, overexpression of circUSP9X intensified CoCl2\'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.
circUSP9X promotes the formation of DVT through the regulation of the miR-148b-3p/SRCIN1 axis.
摘要：
目的：本研究旨在阐明circusp9X（环状RNA泛素特异性肽酶9X连锁）在下肢静脉血栓形成发展中的作用。
方法:建立深静脉血栓形成(DVT)的动物模型和用氯化钴(II)(CoCl2)处理的人脐静脉内皮细胞(HUVECs)的低氧模型。circUSP9X的表达水平，microRNA-148b-3p（miR-148b-3p），和SRC激酶信号传导抑制剂1(SRCIN1)使用定量逆转录聚合酶链反应和Western印迹分析进行定量。细胞毒性,生存能力，凋亡，通过乳酸脱氢酶（LDH）测定评估HUVECs的炎症，MTT测定，流式细胞术,酶联免疫吸附测定，和蛋白质印迹,分别。苏木精和伊红染色用于动物模型中的静脉组织的组织病理学检查。circUSP9X之间的相互作用，miR-148b-3p,通过双荧光素酶报告基因测定和RNA免疫沉淀实验进一步探索SRCIN1。
结果：目前的发现揭示了在DVT病例中circusp9X和SRCIN1的显著上调和miR-148b-3p的同时下调。敲除circUSP9X或miR-148b-3p过表达改善CoCl2诱导的HUVECs细胞凋亡,减少LDH释放，增强细胞活力，减轻炎症。相反，circusp9X的过表达增强了CoCl2的细胞毒性作用。操纵circUSP9X表达的作用被miR-148b-3p和SRCIN1水平的相应调节所抵消。此外,circusp9X敲低可有效抑制小鼠模型中DVT的形成。circusp9X与miR-148b-3p的竞争性结合机制，调节SRCIN1表达，已确定。
结论：circUSP9X通过调节miR-148b-3p/SRCIN1轴促进DVT的形成。
方法:建立深静脉血栓形成(DVT)的动物模型和用氯化钴(II)(CoCl2)处理的人脐静脉内皮细胞(HUVECs)的低氧模型。circUSP9X的表达水平，microRNA-148b-3p（miR-148b-3p），和SRC激酶信号传导抑制剂1(SRCIN1)使用定量逆转录聚合酶链反应和Western印迹分析进行定量。细胞毒性,生存能力，凋亡，通过乳酸脱氢酶（LDH）测定评估HUVECs的炎症，MTT测定，流式细胞术,酶联免疫吸附测定，和蛋白质印迹,分别。苏木精和伊红染色用于动物模型中的静脉组织的组织病理学检查。circUSP9X之间的相互作用，miR-148b-3p,通过双荧光素酶报告基因测定和RNA免疫沉淀实验进一步探索SRCIN1。
结果：目前的发现揭示了在DVT病例中circusp9X和SRCIN1的显著上调和miR-148b-3p的同时下调。敲除circUSP9X或miR-148b-3p过表达改善CoCl2诱导的HUVECs细胞凋亡,减少LDH释放，增强细胞活力，减轻炎症。相反，circusp9X的过表达增强了CoCl2的细胞毒性作用。操纵circUSP9X表达的作用被miR-148b-3p和SRCIN1水平的相应调节所抵消。此外,circusp9X敲低可有效抑制小鼠模型中DVT的形成。circusp9X与miR-148b-3p的竞争性结合机制，调节SRCIN1表达，已确定。
结论：circUSP9X通过调节miR-148b-3p/SRCIN1轴促进DVT的形成。
