miRNA-122介导血管外膜APLN-AMPK自噬信号调控在高血压血管重构损伤的作用研究

MicroRNA-122 (miR-122) is involved in the onset of hypertension. Apelin (APLN) is one of the potential target genes of miR-122, which may modulate blood pressures and promote the expression of AMPK and its related autophagy signaling. In our former National Natural Science Foundation (No.91339108) study, we found that there were marked increases in levels of miR-122 and cellular apoptosis and a decrease in adventitial APLN expression in hypertension animal model with vascular injury. However, the regulatory roles of miR-122 in autophagy signaling and vascular remodeling remain unclear. This project intends to carry out in vitro and in vivo intervention experiments with pAAV-miR-122, miR-122 antagomiR and Apelin-13 by the use of the APLN gene knockout (KO) and miR-122KO model, spontaneously hypertensive rats and hypertensive vascular injury animal induced by an osmotic minipump with angiotensin II or by transverse aortic constriction (TAC) operation as well as cellular model of primary rat adventitial fibroblasts, and to implore effects of miR-122 and APLN on the vascular adventitial autophagy signal (AMPK/mTOR, Akt/Bcl2/Beclin-1, p62/LC3 II), inflammasome and the vascular function and structural changes. Therefore, this work will provide a scentific basis to establish miR-122 and APLN as new targets for early prevention and treatment of vascular injury in hypertension.

微小RNA-122(miR-122)参与高血压发病。Apelin(APLN)是miR-122的潜在靶基因之一，可调控血压与AMPK自噬信号。我们前一国家自然科学基金(No.91339108)研究发现，高血压血管损伤状态下miR-122和凋亡水平增加，伴血管外膜APLN表达下调，但miR-122对自噬信号与血管重构的调控作用尚不清楚。本课题拟利用miR-122、APLN基因敲除模型、自发性高血压大鼠、TAC术及经Ang II制备高血压血管损伤动物及血管外膜成纤维细胞模型，开展pAAV-miR-122、miR-122抑制剂和Apelin-13体内外干预实验，系统探讨miR-122与APLN对血管外膜自噬信号(AMPK/mTOR、Akt/Bcl2/Beclin-1、p62/LC3 II)、炎症小体及血管结构功能的影响，为确立miR-122与APLN作为高血压血管损伤早期防治新靶点提供科学依据。

业已证实自噬凋亡失衡参与高血压血管重构损伤过程，与微小RNA（miRNA）异常密切相关。本项目研究提示，与正常健康对照相比，高血压患者血浆中miRNA-122水平明显增加，但ELA和APLN水平出现下调。采用高通量RNA测序分析分别筛选了自发性高血压大鼠（SHR）主动脉血管组织中差异表达CircRNA和miRNA。高血压大鼠SHR血管组织miRNA-122-5p明显上调，而APLN、APLNR、FOXO3及ACE2水平明显下调。转染rAAV-miRNA-122-5p过表达后在SHR中加重血管和肾脏纤维化、凋亡和炎症损伤，与FOXO3、Beclin-1、LC3B、ATG5、BNIP3、p-AMPK磷酸化水平下调以及P62、NOX4、CTGF、TGF-β1及p-mTOR磷酸化增加有关。在体外培养的大鼠血管外膜成纤维细胞（AFs）中，与对照组比较，Ang II刺激后氧化应激和炎症反应及迁移能力增强，beclin-1、LC3-II蛋白、p-AMPK磷酸化水平均明显下降，而p62蛋白和p-mTOR磷酸化水平大大增加。给予miRNA-122 mimic干预治疗后，AFs细胞中beclin-1和LC3-II蛋白及p-AMPK磷酸化水平进一步下调，而p62, IL1, IL6, IL18, TNF, MCP-1和p-mTOR磷酸化水平进一步上调，伴有细胞迁移能力、氧化应激和炎症反应进一步增强，提示miRNA-122 mimic具有一定的抗自噬和促迁移、促氧化及促炎症功效。相反，miRNA-122抑制剂、Pyr-Apelin-13及mTOR抑制剂Rapamycine干预治疗后，可明显对抗Ang II的促凋亡及抗自噬效应，伴有p62和p-mTOR水平下调，而AFs细胞中APLN、beclin-1、LC3-II及p-AMPK上调。另外，APLNKO小鼠出现血管重构损伤加重，伴有AMPK和mTOR磷酸化异常改变（Acta Pharm Sin B 2020、Hypertension 2017，2021、Circ Res 2020、Gigascience 2019、Front Cardiovasc Med 2021、J Am Heart Assoc 2019、FASEB J 2021、EJP 2020）。本项目为miRNA-122 及其靶基因APLN调控高血压血管重构损伤防治提供科学依据。