生物钟蛋白CRY1抑制Ang Ⅱ诱导的小鼠主动脉平滑肌细胞表型蛋白标志物变化

doi:10.16352/j.issn.1001-6325.2023.04.0588

基础医学与临床 ›› 2023, Vol. 43 ›› Issue (4): 588-595.doi: 10.16352/j.issn.1001-6325.2023.04.0588

生物钟蛋白CRY1抑制Ang Ⅱ诱导的小鼠主动脉平滑肌细胞表型蛋白标志物变化

岳秀青¹, 宋其泰¹, 刘超利¹, 李桑柔¹, 杨帆¹, 陈睦虎¹, 钟武^1,2,3*

1.西南医科大学附属医院急诊医学部,四川泸州 646000;
2.泸州市人民医院,四川泸州 646000;
3.四川省康复医院,四川成都 610000

收稿日期:2022-09-29 修回日期:2023-02-15 出版日期:2023-04-05 发布日期:2023-04-03
通讯作者: ^*zhongwu2876@sina.com
基金资助:
西南医科大学附属医院博士科研启动基金(19085);四川省卫生健康委员会医学科技项目(21PJ199)

Circadian clock protein CRY1 inhibits Ang Ⅱ-induced phenotypic transformation of mouse aorta smooth muscular cells

YUE Xiuqing¹, SONG Qitai¹, LIU Chaoli¹, LI Sangrou¹, YANG Fan¹, CHEN Muhu¹, ZHONG Wu^{1, 2, 3*}

1. Emergency Intensive Care Unit, the Affiliated Hospital of Southwest Medical University, Luzhou 646000;
2. Luzhou People's Hospital, Luzhou 646000;
3. Sichuan Provincial Rehabilitation Hospital, Chengdu 610000, China

Received:2022-09-29 Revised:2023-02-15 Online:2023-04-05 Published:2023-04-03
Contact: ^*zhongwu2876@sina.com

摘要/Abstract

摘要： 目的探讨生物钟(circadian clock)相关蛋白隐花色素1(CRY1)在血管紧张素Ⅱ(Ang Ⅱ)诱导的小鼠主动脉血管平滑肌细胞(VSMCs)表型转化中的作用。方法将VSMCs分为对照(control)组和实验组,用不同浓度Ang Ⅱ处理VSMCs不同时间,RT-PCR和Western blot检测收缩型标志物α-平滑肌肌动蛋白(α-SMA)、合成型标志物骨桥蛋白(OPN)及CRY1 mRNA和蛋白的表达;分别构建CRY1的干扰RNA(siCRY1)、YAP1的干扰RNA(siYAP1)进行基因沉默,构建CRY1的过表达质粒(pcDNA-CRY1)进行过表达,RT-PCR、Western blot双重验证各基因组mRNA和蛋白的表达;将VSMCs分为4组,对照组、Ang Ⅱ组、Ang Ⅱ+siCRY1组、Ang Ⅱ+pcDNA-CRY1组,将siCRY1、siYAP1、pcDNA-CRY1转染至经Ang Ⅱ处理VSMCs中,RT-PCR和Western blot检测α-SMA、OPN、CRY1、YAP1 mRNA和蛋白的表达及CCK-8法检测细胞增殖情况。结果不同浓度Ang Ⅱ刺激VSMCs不同时间后,VSMCs收缩型标志物表达减少,VSMCs合成型标志物表达增加,CRY1表达减少;转染siCRY1、siYAP1、pcDNA-CRY1处理VSMCs后,CRY1在siCRY1组中表达下降,在pcDNA-CRY1组中表达升高,YAP1在siYAP1组中表达下降,在siCRY1组中表达升高;转染siCRY1进一步减少Ang Ⅱ诱导VSMC收缩型标志物的表达(P＜0.05),增加合成型标志物、YAP1的表达,以及促进VSMCs的增殖(P＜0.05);转染pcDNA-CRY1质粒增加Ang Ⅱ诱导VSMCs收缩型标志物的表达(P＜0.05),减弱合成型标志物YAP1的表达(P＜0.05)及抑制VSMCs的增殖(P＜0.05)。结论生物钟蛋白CRY1抑制Ang Ⅱ诱导的小鼠主动脉VSMCs表型蛋白标志物变化和增殖,该过程可能与Hippo-YAP信号通路有关。

关键词: 隐花色素1, Yes相关蛋白1, 血管紧张素Ⅱ, 表型转化

Abstract: Objective To investigate the effect of circadian clock protein cryptochrome 1 (CRY1) on angiotensin Ⅱ(Ang Ⅱ)-induced phenotypic transformation of mouse aorta smooth muscular cells. Methods VSMCs were divided into control group and experimental group. VSMCs were treated with different concentrations of Ang Ⅱ for various time, α-smooth muscle actin (α-SMA), osteopontin (OPN) and CRY1 expressions in VSMCs were detected via RT-qPCR and Western blot. VSMCs were transfected with knockdown vector siCRY1 and siYAP1 or overexpression vector, and the corresponding gene expression was detected with RT-PCR and Western blot. VSMCs were divided into 4 groups: Control group, Ang Ⅱ group, Ang Ⅱ+siCRY1 group and Ang Ⅱ+pcDNA-CRY1 group. siCRY1, siYAP1, pcDNA-CRY1 were transfected into VSMCs induced by Ang Ⅱ for observation on α-SMA, CRY1, OPN and YAP1 at mRNA and protein levels detected by RT-PCR and Western blot. CCK-8 method were used to measure proliferation capacity. Results VSMCs were treated with different concentrations of Ang Ⅱ for various time, the expression of CRY1 was decreased in a model of Ang Ⅱ-induced VSMC phenotypic transformation, which contractile markers down-regulated and synthetic markers up-regulated at mRNA and protein levels. After VSMCs were transfected with siCRY1, siYAP1 and pcDNA-CRY1, the expression of CRY1 decreased in siCRY1 group and increased in pcDNA-CRY1 group, while the expression of YAP1 decreased in siYAP1 group and increased in siCRY1 group.Knockdown of CRY1 could stimulate the transformation of VSMCs from contractile phenotype into synthetic phenotypes by further activating expression of synthetic markers and CRY1(P＜0.05), inhibiting expression of contractile markers and YAP1(P＜0.05), the proliferation of VSMCs increased significantly(P＜0.05). While overexpressed CRY1 could block the phenotypic transformation, the expression of synthetic markers and CRY1 was reduced and contractile markers and YAP1 was increased both at mRNA and protein levels(P＜0.05), the proliferation of VSMCs inhibited(P＜0.05). Conclusions CRY1 may inhibit switch of mouse VSMCs phenotypes induced by Ang Ⅱ and inhibit VSMCs proliferation, which is potentially related to the Hippo-YAP signaling pathway.

Key words: CRY1, YAP1, angiotensin Ⅱ, phenotypic transformation

中图分类号:

R329.2
R363

岳秀青, 宋其泰, 刘超利, 李桑柔, 杨帆, 陈睦虎, 钟武. 生物钟蛋白CRY1抑制Ang Ⅱ诱导的小鼠主动脉平滑肌细胞表型蛋白标志物变化[J]. 基础医学与临床, 2023, 43(4): 588-595.

YUE Xiuqing, SONG Qitai, LIU Chaoli, LI Sangrou, YANG Fan, CHEN Muhu, ZHONG Wu. Circadian clock protein CRY1 inhibits Ang Ⅱ-induced phenotypic transformation of mouse aorta smooth muscular cells[J]. Basic & Clinical Medicine, 2023, 43(4): 588-595.

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生物钟蛋白CRY1抑制Ang Ⅱ诱导的小鼠主动脉平滑肌细胞表型蛋白标志物变化

Circadian clock protein CRY1 inhibits Ang Ⅱ-induced phenotypic transformation of mouse aorta smooth muscular cells

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