The regulatory role and mechanism of USP14 in endothelial cell pyroptosis induced by coronary heart disease.

Clinical hemorheology and microcirculation Pub Date : 2024-01-01 DOI:10.3233/CH-232003

Jie Gao, Zhao Gao

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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.

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USP14 在冠心病诱导的内皮细胞热解过程中的调控作用和机制。

研究目的本研究探讨了泛素特异性肽酶 14（USP14）在冠心病（CHD）诱导的内皮细胞热解中的作用和机制：方法：通过用氧化低密度脂蛋白（ox-LDL）诱导人冠状动脉内皮细胞（HCAECs），建立了体外冠心病模型。用 si-USP14 转染 HCAECs，然后用 CCK-8 检测法评估细胞活力，用检测试剂盒检测乳酸脱氢酶（LDH）活性，用 qRT-PCR 或 Western 印迹法检测 USP14、miR-15b-5p、NLRP3、GSDMD-N 和裂解-Caspase-1 的表达，用 ELISA 检测 IL-1β 和 IL-18 的浓度。Co-IP证实了USP14和NLRP3之间的结合。蛋白酶抑制剂 MG132 处理后，测定了细胞中 NLRP3 的泛素化水平。双荧光素酶报告实验验证了 miR-15b-5p 与 USP14 之间的靶向关系：结果：在暴露于 ox-LDL 的 HCAECs 中，USP14 和 NLRP3 高表达，但 miR-15b-5p 低表达。USP14 沉默增强了暴露于 ox-LDL 的 HCAECs 的活力，降低了细胞内 LDH 活性，减少了 NLRP3、GSDMD-N、Cleaved-Caspase-1、IL-1β 和 IL-18 的表达。USP14 与 NLRP3 蛋白结合并抑制其泛素化。miR-15b-5p抑制了USP14的转录和蛋白表达，miR-15b-5p过表达通过抑制USP14/NLRP3减轻了HCAEC的脓毒症：结论：USP14通过去泛素化稳定NLRP3蛋白的表达，从而促进了CHD患者内皮细胞的脓毒症。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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Clinical hemorheology and microcirculation

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