Feng Xiaona, Wang Kaifeng, Yang Ting, Liu Yanhui, Wang Xiaodong
Department of Obstetrics and Gynecology, First Affiliated Hospital of Jiamusi University, No. 348, Dexiang Street, Xiangyang District, Jiamusi, 154002, Heilongjiang, China.
Vascular Surgery, First Affiliated Hospital of Jiamusi University, No. 348, Dexiang Street, Xiangyang District, Jiamusi, 154002, Heilongjiang, China.
Genes Genomics. 2022 Apr;44(4):395-404. doi: 10.1007/s13258-021-01202-z. Epub 2022 Jan 23.
We have clarified the role of miR-382-3p in chronic thromboembolic pulmonary hypertension (CTEPH), but what is less clear lies in its upstream regulatory mechanism.
To explore the regulation mechanism of GAS5/miR-382-3p axis on CTEPH.
In vitro, we constructed cell models by treating Pulmonary Artery Smooth Muscle Cells (PASMCs) with platelet-derived growth factor-BB (PDGF-BB). The effects of different concentrations of PDGF-BB on the activity of PASMCs were tested by cell counting kit-8 (CCK-8). The upstream lncRNA of miR-382-3p was screened and confirmed through bioinformatics analysis, RNA pull-down, quantitative reverse transcription polymerase chain reaction (qRT-PCR), dual luciferase reporter gene and RNA immunoprecipitation assays. The effects of GAS5/miR-382-3p axis on the viability, migration, and expressions of autophagy- and angiogenesis-related proteins were confirmed by rescue experiments (CCK-8, wound healing and western blot). In vivo, animal models by perfusing autologous blood vessels, the effects of GAS5 overexpression or silencing on the expressions of miR-382-3p, angiogenesis- and autophagy-related genes, mean pulmonary arterial pressure (mPAP) and pulmonary artery wall were determined by biological signal acquisition system, hematoxylin-eosin staining, qRT-PCR and western blot.
PDGF-BB dose-dependently promoted PASMCs viability. XIST and GAS5 expressions in PASMCs were affected by the concentration of PDGF-BB, but only GAS5 can be pulled down by miR-382-3p probe. GAS5 targeted miR-382-3p to inhibit the viability and migration of PAMSCs, mPAP in CTEPH rats, pulmonary artery wall thickening and angiogenesis, and promote autophagy.
GAS5/miR-382-3p axis is involved in the regulation of pulmonary artery remodeling and autophagy in CTEPH.
我们已经阐明了miR-382-3p在慢性血栓栓塞性肺动脉高压（CTEPH）中的作用，但其上游调控机制尚不清楚。
探讨GAS5/miR-382-3p轴对CTEPH的调控机制。
在体外，我们通过用血小板衍生生长因子-BB（PDGF-BB）处理肺动脉平滑肌细胞（PASMCs）构建细胞模型。用细胞计数试剂盒-8（CCK-8）检测不同浓度的PDGF-BB对PASMCs活性的影响。通过生物信息学分析、RNA下拉、定量逆转录聚合酶链反应（qRT-PCR）、双荧光素酶报告基因和RNA免疫沉淀试验筛选并确认miR-382-3p的上游lncRNA。通过拯救实验（CCK-8、伤口愈合和蛋白质印迹法）证实GAS5/miR-382-3p轴对自噬和血管生成相关蛋白的活力、迁移及表达的影响。在体内，通过灌注自体血管建立动物模型，用生物信号采集系统、苏木精-伊红染色、qRT-PCR和蛋白质印迹法测定GAS5过表达或沉默对miR-382-3p、血管生成和自噬相关基因的表达、平均肺动脉压（mPAP）和肺动脉壁的影响。
PDGF-BB剂量依赖性地促进PASMCs活力。PASMCs中XIST和GAS5的表达受PDGF-BB浓度影响，但只有GAS5能被miR-382-3p探针下拉。GAS5靶向miR-382-3p抑制PAMSCs的活力和迁移、CTEPH大鼠的mPAP、肺动脉壁增厚和血管生成，并促进自噬。
GAS5/miR-382-3p轴参与CTEPH中肺动脉重塑和自噬的调控。
