Feng Xiaona, Wang Kaifeng, Yang Ting, Dang Bo, Wang Xiaodong
Department of Obstetrics and Gynecology, First Affiliated Hospital of Jiamusi University, No. 348, Dexiang Street, Xiangyang District, Jiamusi City, Heilongjiang Province, 154002, China.
Vascular surgery, First Affiliated Hospital of Jiamusi University, No. 348, Dexiang Street, Xiangyang District, Jiamusi City, Heilongjiang Province, 154002, China.
Curr Mol Med. 2023 Aug 22. doi: 10.2174/1566524023666230822120453.
This research has investigated the role of miR-382-3p in chronic thromboembolic pulmonary hypertension (CTEPH).
Human pulmonary artery smooth muscle cells (hPASMCs) were treated with PDGF-BB to induce proliferation, and then transfected with miR-382-3p mimic, miR-382-3p inhibitor, ATG7 overexpression plasmid, and siATG7. MiR-382-3p, ATG7, VEGF, PCNA, p62, and LC3-Ⅱ/LC3-I levels were detected by qRT-PCR and Western blotting. Cell viability and migration were tested through CCK-8 and wound healing assays, respectively. Target genes of miR-382-3p were predicted by Targetscan and starBase, and pathway analysis was implemented through WebGestalt. The binding relationship between miR-382-3p and ATG7 was analyzed by the dual-luciferase reporter and RIP assays. A CTEPH model was constructed in rats with the treatment of miR-382-3p antagomir or agomir, and mean pulmonary artery pressure (mPAP) was measured. Lung tissue was observed through the HE staining assay.
MiR-382-3p level in hPASMCs was obviously upregulated with the increasing dose of PDGF-BB. MiR-382-3p mimic promoted yet miR-382-3p inhibitor suppressed hPASMC proliferation. MiR-382-3p directly targeted ATG7. ATG7 overexpression repressed hPASMC proliferation and migration, whereas siATG7 exerted the opposite effects. ATG7 overexpression partly neutralized the effects of miR-382-3p mimic on proliferation, migration, and autophagy-related proteins (ATG7, p62, and LC3-Ⅱ/LC3-I) in hPASMCs, whereas siATG7 partly offset the impacts of miR-382-3p inhibitor. MiR-382-3p antagomir reversed CTEPH-induced mPAP elevation, miR-382-3p upregulation, thickening of the pulmonary artery wall, and increased expressions of VEGF, PCNA, and autophagy-related proteins in rats, while miR-382-3p agomir potentiated these effects induced by CTEPH.
Overexpressed miR-382-3p promotes vascular remodeling via ATG7 inhibition in CTEPH.
本研究调查了miR-382-3p在慢性血栓栓塞性肺动脉高压（CTEPH）中的作用。
用血小板衍生生长因子-BB（PDGF-BB）处理人肺动脉平滑肌细胞（hPASMCs）以诱导增殖，然后分别用miR-382-3p模拟物、miR-382-3p抑制剂、自噬相关蛋白7（ATG7）过表达质粒和小干扰RNA（siATG7）进行转染。通过实时定量聚合酶链反应（qRT-PCR）和蛋白质免疫印迹法检测miR-382-3p、ATG7、血管内皮生长因子（VEGF）、增殖细胞核抗原（PCNA）、p62和微管相关蛋白轻链3Ⅱ/微管相关蛋白轻链3Ⅰ（LC3-Ⅱ/LC3-I）的水平。分别通过细胞计数试剂盒-8（CCK-8）和伤口愈合实验检测细胞活力和迁移能力。通过Targetscan和starBase预测miR-382-3p的靶基因，并通过WebGestalt进行通路分析。通过双荧光素酶报告基因实验和RNA免疫沉淀（RIP）实验分析miR-382-3p与ATG7之间的结合关系。用miR-382-3p拮抗剂或激动剂处理构建大鼠CTEPH模型，并测量平均肺动脉压（mPAP）。通过苏木精-伊红（HE）染色实验观察肺组织。
随着PDGF-BB剂量增加，hPASMCs中miR-382-3p水平明显上调。miR-382-3p模拟物促进而miR-382-3p抑制剂抑制hPASMCs增殖。miR-382-3p直接靶向ATG7。ATG7过表达抑制hPASMCs增殖和迁移，而siATG7则发挥相反作用。ATG7过表达部分抵消了miR-382-3p模拟物对hPASMCs增殖、迁移和自噬相关蛋白（ATG7、p62和LC3-Ⅱ/LC3-I）的影响，而siATG7部分抵消了miR-382-3p抑制剂的作用。miR-382-3p拮抗剂可逆转CTEPH诱导的大鼠mPAP升高、miR-382-3p上调、肺动脉壁增厚以及VEGF、PCNA和自噬相关蛋白表达增加，而miR-382-3p激动剂则增强了CTEPH诱导的这些作用。
在CTEPH中，过表达的miR-382-3p通过抑制ATG7促进血管重塑。
