Affiliations: Department of Cardiology, Xi’an International Medical Center Hospital, Xi’an, China
Correspondence:
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Corresponding author: Zhao Gao, Department of Cardiology, Xi’an International Medical Center Hospital, 777 Xitai Road, Chang’an District, Xi’an City, Shaanxi Province, 710100, China, E-mail: drgaozhao@163.com.
Abstract: OBJECTIVE:The present study probes into the role and mechanism of ubiquitin specific peptidase 14 (USP14) in coronary heart disease (CHD)-triggered endothelial cell pyroptosis. METHODS:An in vitro CHD model was established by inducing human coronary artery endothelial cells (HCAECs) with oxidized low-density lipoprotein (ox-LDL). HCAECs were transfected with si-USP14, followed by evaluation of cell viability by CCK-8 assay, detection of lactate dehydrogenase (LDH) activity by assay kit, detection of USP14, miR-15b-5p, NLRP3, GSDMD-N, and Cleaved-Caspase-1 expressions by qRT-PCR or Western blot, as well as IL-1β and IL-18 concentrations by ELISA. Co-IP confirmed the binding between USP14 and NLRP3. The ubiquitination level of NLRP3 in cells was measured after protease inhibitor MG132 treatment. Dual-luciferase reporter assay verified the targeting relationship between miR-15b-5p and USP14. RESULTS:USP14 and NLRP3 were highly expressed but miR-15b-5p was poorly expressed in ox-LDL-exposed HCAECs. USP14 silencing strengthened the viability of ox-LDL-exposed HCAECs, reduced the intracellular LDH activity, and diminished the NLRP3, GSDMD-N, Cleaved-Caspase-1, IL-1β, and IL-18 expressions. USP14 bound to NLRP3 protein and curbed its ubiquitination. Repression of NLRP3 ubiquitination counteracted the inhibitory effect of USP14 silencing on HCAEC pyroptosis. miR-15b-5p restrained USP14 transcription and protein expression. miR-15b-5p overexpression alleviated HCAEC pyroptosis by suppressing USP14/NLRP3. CONCLUSION:USP14 stabilizes NLRP3 protein expression through deubiquitination, thereby facilitating endothelial cell pyroptosis in CHD. miR-15b-5p restrains endothelial cell pyroptosis by targeting USP14 expression.
