摘要： 目的 探讨泛素特异性蛋白酶22 （USP22）与雌激素/雌激素受体（ER）之间的关系，并明确USP22在血管钙化过程中的作用及机制。方法 用高磷培养基处理人动脉平滑肌细胞（HASMCs），建立血管钙化的细胞模型，并通过钙含量比色检测试剂盒及茜素红染色对该模型进行评价；Western blotting观察钙化过程中USP22蛋白水平的变化情况；双荧光素酶报告试验检测USP22对于ERα的转录调控作用；免疫共沉淀试验（co-IP）检测USP22与ERα之间的相互作用；对HASMCs进行过表达/沉默USP22之后再通过钙含量比色检测试剂盒检测钙化的改变。结果 成功建立HASMCs钙化模型，钙含量比色检测试剂盒测定结果显示，细胞钙含量明显升高（P < 0.05），茜素红染色也提示平滑肌细胞钙化阳性。随着钙化培养基处理时间的延长，Western blotting检测发现HASMCs的Runt相关转录因子（Runx2）与USP22蛋白表达量均有所升高。双荧光素酶报告试验结果显示，USP22可以抑制ERα的转录活性（P < 0.05）。co-IP证实USP22与ERα之间可以结合。过表达USP22时，细胞钙化加重，Runx2表达也升高；沉默USP22时，细胞钙化减轻，Runx2表达也下降（P < 0.05）。结论 高磷条件可以诱导HASMCs钙化，HASMCs钙化时USP22表达水平升高，USP22可以通过与ERα直接结合的方式抑制其转录活性，USP22通过ERα促进钙化。

泛素特异性蛋白酶22通过调控雌激素受体α的转录活性抑制磷诱导的人动脉平滑肌细胞钙化

聂涵¹, 周晓煦¹, 赵越², 田文¹

1. 中国医科大学附属第一医院老年医学科, 沈阳 110001;
2. 中国医科大学基础医学院细胞生物学教研室, 沈阳 110122

收稿日期:2020-04-27出版日期:2021-10-30发布日期:2021-10-11
通讯作者:田文E-mail:Dr_wentian@163.com
作者简介:聂涵(1993-),男,硕士研究生.
基金资助:沈阳市科技计划人口与健康专项（19-112-4-063）


关键词: 泛素特异性蛋白酶22, 雌激素受体, 人动脉平滑肌细胞钙化
Abstract: Objective To investigate the relationship between ubiquitin-specific peptidase 22 (USP22) and estrogen/estrogen receptor (ER), and whether USP22 can play a role in the process of vascular calcification through this mechanism. Methods Human aortic smooth muscle cells (HASMCs) were treated with a high-phosphorus medium to establish a cell model of vascular calcification. The model was evaluated using a calcium colorimetric kit and alizarin red staining. The changes in USP22 protein levels during calcification were observed by western blotting. The effect of USP22 on the transcriptional activity of ER was detected using a dual-luciferase assay. The interaction between USP22 and ER was detected by co-immunoprecipitation. After overexpression/silencing of USP22 in HASMCs, the calcium colorimetric assay kit was used to detect calcification. Results The HASMCs calcification model was successfully established and the calcium content was significantly increased (P < 0.05);significantly deepened staining was observed with alizarin red. Western blotting showed that the expression of Runx2 and USP22 increased in the calcification model. The dual-luciferase assay showed that USP22 inhibited the transcriptional activity of ER (P < 0.05). Co-immunoprecipitation confirmed that USP22 could combine with ERα. When USP22 was overexpressed, calcification increased and Runx2 expression increased;when USP22 was silenced, calcification decreased and Runx2 expression decreased (P < 0.05). Conclusion High phosphorus can induce calcification of HASMCs. Calcification increased the expression of USP22 in HASMCs. USP22 inhibits the transcriptional activity of ERα by directly binding to ERα. USP22 promotes the calcification of HASMCs through ERα.
Key words: ubiquitin specific peptidase 22, estrogen receptor, calcification of human aortic smooth muscle cells
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