EZH2 通过 H3K27me3 调控 ANXA6 的表达，并参与血管平滑肌细胞衰老。

EZH2 Regulates ANXA6 Expression via H3K27me3 and Is Involved in Angiotensin II-Induced Vascular Smooth Muscle Cell Senescence.

机构信息

Department of General Surgery, The First People's Hospital of Yunnan Province (The Affiliated Hospital of Kunming University of Science and Technology), Kunming, Yunnan 650032, China.

Department of Hepatic-Biliary-Pancreatic Surgery, The First Hospital of Kunming (The Calmette Hospital), Kunming, Yunnan 650224, China.

出版信息

Oxid Med Cell Longev. 2022 Sep 14;2022:4838760. doi: 10.1155/2022/4838760. eCollection 2022.

DOI:10.1155/2022/4838760

PMID:36160712

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC9492406/

Abstract

OBJECTIVES

Abdominal aortic aneurysm (AAA) has a high risk of rupture of the aorta and is one of the leading causes of death in older adults. This study is aimed at confirming the influence and mechanism of the abnormally expressed ANXA6 gene in AAA.

METHODS

Clinical samples were collected for proteome sequencing to screen for differentially expressed proteins. An Ang II-induced vascular smooth muscle cell (VSMC) aging model as well as an AAA animal model was used. Using RT-qPCR to detect the mRNA levels of EZH2, ANXA6, IK-6, and IL-8 in cells and tissues were assessed. Western blotting and immunohistochemistry staining were used apply for the expression of associated proteins in cells and tissues. SA--gal staining, flow cytometry, and DHE staining were used to detect senescent cells and the level of ROS. The cell cycle was assessed by flow cytometry. Arterial pathology was observed by HE staining. The aging of VSMCs in arterial tissue was assessed by coimmunofluorescence for -SMA and p53.

RESULTS

There were 24 differentially expressed proteins in the AAA clinical samples, including 10 upregulated protein and 14 downregulated protein, and the differential expression of ANXA6 was associated with vascular disease. Our study found that ANXA6 was highly expressed and EZH2 was lowly expressed in an Ang II-induced VSMC aging model. Knockdown of ANXA6 or overexpression of EZH2 inhibited Ang II-induced ROS, inhibited cell senescence, decreased Ang II evoked G1 arrest, and increased cells in G2 phase, while overexpression of ANXA6 played the opposite role. Overexpression of EZH2 inhibited ANXA6 expression by increasing H3K27me3 modification at the ANXA6 promoter. Simultaneous overexpression of EZH2 and the protective effect of EZH2 on cell senescence were partially reversed by ANXA6. Similarly, ANXA6 was highly expressed and EZH2 was lowly expressed in an Ang II-induced AAA animal model. Knockdown of ANXA6 and overexpression of EZH2 alleviated Ang II-induced VSMC senescence and inhibited AAA progression, while simultaneous overexpression of EZH2 and ANXA6 partially reversed the protective effect of EZH2 on AAA.

CONCLUSION

EZH2 regulates the ANXA6 promoter H3K27me3 modification, inhibits ANXA6 expression, alleviates Ang II-induced VSMC senescence, and inhibits AAA progression.

摘要

目的

腹主动脉瘤（AAA）有很高的主动脉破裂风险，是老年人死亡的主要原因之一。本研究旨在证实异常表达的 ANXA6 基因在 AAA 中的影响和机制。

方法

收集临床样本进行蛋白质组学测序，筛选差异表达蛋白。建立 Ang II 诱导的血管平滑肌细胞（VSMC）衰老模型和 AAA 动物模型。采用 RT-qPCR 检测细胞和组织中 EZH2、ANXA6、IK-6 和 IL-8 的 mRNA 水平。应用 Western blot 和免疫组化染色检测细胞和组织中相关蛋白的表达。SA-β-gal 染色、流式细胞术和 DHE 染色检测衰老细胞和 ROS 水平。采用流式细胞术评估细胞周期。通过 HE 染色观察动脉病理学变化。通过 -SMA 和 p53 共免疫荧光评估动脉组织中 VSMC 的衰老。

结果

AAA 临床样本中有 24 个差异表达蛋白，包括 10 个上调蛋白和 14 个下调蛋白，ANXA6 的差异表达与血管疾病有关。我们的研究发现，在 Ang II 诱导的 VSMC 衰老模型中，ANXA6 高表达，EZH2 低表达。敲低 ANXA6 或过表达 EZH2 抑制 Ang II 诱导的 ROS，抑制细胞衰老，减少 Ang II 诱导的 G1 期阻滞，增加 G2 期细胞，而过表达 ANXA6 则起到相反的作用。EZH2 通过增加 ANXA6 启动子上的 H3K27me3 修饰来抑制 ANXA6 的表达。同时过表达 EZH2 和 EZH2 对细胞衰老的保护作用部分被 ANXA6 逆转。同样，在 Ang II 诱导的 AAA 动物模型中，ANXA6 高表达，EZH2 低表达。敲低 ANXA6 和过表达 EZH2 减轻 Ang II 诱导的 VSMC 衰老，抑制 AAA 进展，而过表达 EZH2 和 ANXA6 则部分逆转了 EZH2 对 AAA 的保护作用。