HSP90-Dependent Upregulation of EZH2 Promotes Hypoxia/Reoxygenation-Induced Pyroptosis by Inhibiting miR-22 in Endothelial Cells

Abstract

Endothelial cell pyroptosis induced by hypoxia/reoxygenation (H/R) plays a key role in the pathogenesis of myocardial infarction (MI). However, the underlying mechanism is not clearly elucidated. Methods: Human umbilical vein endothelial cells (HUVECs) exposed to H/R acted as in vitro model to investigate the mechanism of H/R-induced endothelial cell pyroptosis. CCK-8 assays were performed to investigate the viability of HUVECs. Calcein-AM/PI staining was carried out to quantify the death of HUVECs. The expression level of miR-22 was measured by RT-qPCR. The protein expression levels of zeste 2 polycomb repressive complex 2 subunit (EZH2), NLRP3, cleaved caspase-1 (c-caspase-1), GSDMD-N and heat shock protein 90 (HSP90) were measured by Western blot. Levels of IL-1β and IL-18 in culture medium were detected by ELISA. The intracellular localization of EZH2 was detected by immunofluorescence staining. Chromatin immunoprecipitation (ChIP) assay was used to detect the enrichment of EZH2 and H3K27me3 in the miR-22 promoter region. The binding between miR-22 and NLRP3 in HUVECs was confirmed by the dual luciferase assay. Reciprocal coimmunoprecipitation was conducted to detect the direct interaction between HSP90 and EZH2. Results: H/R increased EZH2 expression, and the EZH2 siRNA could inhibit H/R-induced pyroptosis in HUVECs. H/R reduced miR-22 expression, which was reversed by EZH2 siRNA. Silencing of miR-22 by its inhibitor reversed EZH2 siRNA-induced pyroptosis inhibition in H/R-exposed HUVECs. Upregulation of miR-22 by its mimic suppressed EZH2 overexpression-enhanced pyroptosis in H/R-exposed HUVECs. ChIP assay confirmed that EZH2 bound to the miR-22 promoter region and repressed miR-22 expression through H3K27me3. Furthermore, luciferase reporter assay indicated that NLRP3 was a direct target of miR-22 in HUVECs. Finally, HSP90 siRNA inhibited H/R-induced EZH2 expression, miR-22 downregulation, and pyroptosis in HUVECs. Conclusion: H/R induces pyroptosis via the HSP90/EZH2/miR-22/NLRP3 signaling axis in endothelial cells.