School of First Clinical Medicine, Wenzhou Medical University, Wenzhou, PR China.
School of Second Clinical Medicine, Wenzhou Medical University, Wenzhou, PR China.
Vascul Pharmacol. 2021 Feb;136:106820. doi: 10.1016/j.vph.2020.106820. Epub 2020 Nov 22.
Exsomes play a significant role in increasing pathophysiological processes by delivering their content. Recently, a variety of studies have showed exosomal microRNAs (miRNAs) are involved in pulmonary hypertension (PH) notably. In this study, we found that exosomal miR-211 was overexpressed in hypoxia-induced PH rats but its intrinsic regulation was unclear. Therefore, our aim was to reveal the underlying mechanism which overexpressed exosomal miR-211 targeted in the development of PH.
18 male SD rats were randomly divided into normoxia and hypoxia group, housed in normal or hypoxic chamber for 3 weeks respectively. Then, mean pulmonary arterial pressure (mPAP), pulmonary vascular resistance(PVR), right ventricular hypertrophy index(RV/(LV + S)), the percentage of medial wall area (WA%) and the percentage of medial wall thickness (WT%) were measured. Expression of miR-211 in exosomes was detected by qRT-PCR. Expression of Ca/calmodulin-dependent kinase1(CaMK1)and peroxisome proliferator-activated receptors-γ(PPAR-γ)in lung tissue were detected by Western blot(WB); After miR-211 overexpressed exosomes were injected to rats through caudal vein, mPAP, PVR, RV/(LV + S), WA% and WT% were also measured. Sequentially, hypoxia rats were injected with lentivirus riched in miR-211 inhibitor via tail vein, and PH-related indicators were measured. In vitro, after miR-211 was positively or negatively regulated in pulmonary arterial smooth muscle cell (PASMC) by plasmid transfection, proliferation of PASMC was detected by CCK8, as well as the expression of CaMK1 and PPAR- γ. Further, the relationship between CaMK1 and miR-211 was verified by Dual-Luciferase assay. And the regulatory relationship of CaMK1/PPAR- γ aixs was demonstrated in PASMC.
Evident increases of mPAP, PVR, RVHI, WT% and WA% were observed with hypoxia administration. And the concentration of plasma exosomes in hypoxia rats was increased and positively correlated with the above indexes. miR-211 in exosomes of PH was upregulated while the expression of CaMK1 and PPAR-γ decreased in lung tissues. Further, injection of exosomes overexpressed with miR-211 demonstrated that exosomal miR-211 aggravated PH while inhibition of miR-211 attenuated PH in rats. In vitro, overexpression of miR-211 promoted the proliferation of PASMC and inhibited expression of CaMK1 and PPAR-γ in PASMC. And Dual-luciferase assay demonstrated that CaMK1 was a downstream gene of miR-211. Plasmid transfection experiments indicated that CaMK1 can promote PPAR-γ expression.
Exosomal miR-211 promoted PH via inhibiting CaMK1/PPAR-γ axis, promoting PASMC proliferation in rats.
外体通过传递其内容在增加病理生理过程中发挥重要作用。最近，多种研究表明外体 microRNAs（miRNAs）在肺动脉高压（PH）中显著参与。在这项研究中，我们发现缺氧诱导的 PH 大鼠中过表达了外体 miR-211，但其内在调节机制尚不清楚。因此，我们的目的是揭示外体 miR-211 靶向 PH 发展的潜在机制。
18 只雄性 SD 大鼠随机分为常氧和缺氧组，分别在常氧或缺氧室中饲养 3 周。然后，测量平均肺动脉压（mPAP）、肺血管阻力（PVR）、右心室肥厚指数（RV/（LV+S））、中膜面积百分比（WA%）和中膜厚度百分比（WT%）。通过 qRT-PCR 检测外体中 miR-211 的表达。通过 Western blot（WB）检测肺组织中钙/钙调蛋白依赖性激酶 1（CaMK1）和过氧化物酶体增殖物激活受体-γ（PPAR-γ）的表达；通过尾静脉注射 miR-211 过表达外体后，也测量 mPAP、PVR、RV/（LV+S）、WA%和 WT%。随后，通过尾静脉注射富含 miR-211 抑制剂的慢病毒，测量 PH 相关指标。体外通过质粒转染正向或负向调节肺动脉平滑肌细胞（PASMC）中的 miR-211，通过 CCK8 检测 PASMC 的增殖，以及 CaMK1 和 PPAR-γ 的表达。进一步通过双荧光素酶报告实验验证 CaMK1 与 miR-211 的关系，并在 PASMC 中证明 CaMK1/PPAR-γ 轴的调节关系。
缺氧给药后，mPAP、PVR、RVHI、WT%和 WA%明显升高。并且缺氧大鼠血浆外体浓度升高，与上述指标呈正相关。PH 肺组织中外体 miR-211 上调，而 CaMK1 和 PPAR-γ 表达下调。进一步，注射 miR-211 过表达外体表明外体 miR-211 加重 PH，而大鼠 miR-211 抑制减轻 PH。体外，miR-211 过表达促进 PASMC 增殖，抑制 PASMC 中 CaMK1 和 PPAR-γ 的表达。双荧光素酶报告实验表明 CaMK1 是 miR-211 的下游基因。质粒转染实验表明 CaMK1 可以促进 PPAR-γ 的表达。
外体 miR-211 通过抑制 CaMK1/PPAR-γ 轴促进 PH，促进大鼠 PASMC 增殖。
