Circ-USP9X accelerates deep vein thrombosis after fracture by acting as a miR-148b-3p sponge and upregulates SRC kinase signaling inhibitor 1

Abstract

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 (CoCl₂) were developed. The expression levels of cir-cUSP9X, 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 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.

Conclusion:

circUSP9X promotes the formation of DVT through the regulation of the miR-148b-3p/SRCIN1 axis.

Keywords:
CircUSP9X; miR-148b-3p; Deep vein thrombosis; SRCIN1

HIGHLIGHTS

circUSP9X reduction increases cell viability and decreases apoptosis and inflammation in HUVECs

SRCIN1: A Downstream Target of miR-148b-3p in DVT Pathogenesis.

SRC Kinase Signaling Inhibitor 1 (SRCIN1) is controlled by miR-148b-3p.