miR-328-3p targets TLR2 to ameliorate oxygen-glucose deprivation injury and neutrophil extracellular trap formation in HUVECs via inhibition of the NF-κB signaling pathway

Endothelial cell injury is one of the important pathogenic mechanisms in thrombotic diseases, and also neutrophils are involved. MicroRNAs (miRNAs) have been demonstrated to act as essential players in endothelial cell injury, but the potential molecular processes are unknown. In this study, we used cellular tests to ascertain the protective effect of miR-328-3p on human umbilical vein endothelial cells (HUVECs) treated with oxygen-glucose deprivation (OGD).

miR-328-3p inhibits the NF-κB signaling pathway in OGD-treated HUVECs while inhibiting neutrophil migration and NETs formation, and ameliorating endothelial cell injury, which provides new ideas for the pathogenesis of thrombotic diseases.

In our study, an OGD-induced HUVECs model was established, and we constructed lentiviral vectors to establish stable HUVECs cell lines. miR-328-3p and Toll-like receptor 2 (TLR2) interacted, as demonstrated by the dual luciferase reporter assay. We used the CCK8, LDH release, and EdU assays to evaluate the proliferative capacity of each group of cells. To investigate the expression of TLR2, p-P65 NF-κB, P65 NF-κB, NLRP3, IL-1β, and IL-18, we employed Western blot and ELISA. Following OGD, each group's cell supernatants were gathered and co-cultured with neutrophils. An immunofluorescence assay and Transwell assay have been performed to determine whether miR-328-3p/TLR2 interferes with neutrophil migration and neutrophil extracellular traps (NETs) formation.

In OGD-treated HUVECs, the expression of miR-328-3p is downregulated. miR-328-3p directly targets TLR2, inhibits the NF-κB signaling pathway, and reverses the proliferative capacity of OGD-treated HUVECs, while inhibiting neutrophil migration and neutrophil extracellular trap formation. Conclusions: miR-328-3p inhibits the NF-κB signaling pathway in OGD-treated HUVECs while inhibiting neutrophil migration and NETs formation, and ameliorating endothelial cell injury, which provides new ideas for the pathogenesis of thrombotic diseases.