Identification of Key Players Involved in CoCl2 Hypoxia Induced Pulmonary Artery Hypertension in vitro.

Shu Chen,Fen Hu,Hui Xu,Tao Wang

doi:10.3389/fgene.2020.00232

Abstract

BackgroundThe proliferation of human pulmonary artery smooth muscle cells (HPASMCs) induced by hypoxia was considered as the main cause of pulmonary arterial hypertension (PAH). This study aimed to explore potential genes and long non-coding RNAs (lncRNAs) involved in the mechanism of hypoxia-induced PAH.MethodsCoCl2 was utilized to induce hypoxia in HPASMCs, and then cell proliferation, apoptosis, and expression of hypoxia-inducible factors (HIF)-1α were determined. Meanwhile, the RNA isolated from CoCl2-treated cells and control cells were sequenced and differentially expressed genes/lncRNA (DEGs/DELs) were screened, followed by protein-protein interaction (PPI) construction, functional enrichment analyses, and lncRNA-target prediction. Finally, the expression of key genes and lncRNAs were validated using quantitative real-time PCR and western blotting.ResultsCoCl2 treatment could significantly increase the expression of HIF-1α and the proliferation of HPASMCs. A total of 360 DEGs and 57 DELs were identified between CoCl2 treated and control cells. Functional enrichment analysis showed that up-regulated DEGs and DELs’ targets, including LDHA, PFKP, and VEGFA, were significantly enriched in biological processes related to hypoxia or oxygen levels, and the downregulated DEGs and DELs’ targets were significantly enriched in extracellular-matrix-related biological processes. In addition, LDHA, PFKP, and VEGFA exhibited a strong relationship with miR-100HG and TSPEAR-AS2 in lncRNA-target network. The protein level of LDHA, PFKP, and VEGFA were all increased.ConclusionLDHA, PFKP, VEGFA, and lncRNA miR-100HG and TSPEAR-AS2 probably played crucial roles in the pathogenesis of CoCl2 hypoxia-induced-HAP, which might serve as promising therapeutic targets for PAH.

Highlights

Pulmonary hypertension (PH) is a common complicated disorder characterized by a rest mean pulmonary artery pressure of >25 mm Hg
After being treated with CoCl2, the expression of Hypoxia inducible factors (HIFs)-1α in human pulmonary artery smooth muscle cells (HPASMCs) was determined using RT-PCR, and the results showed that the expression levels of HIF-1α were significantly increased in HPASMCs with a dose-dependent manner at 24 and 48 h (Figure 1A)
The results showed that the proliferation of HPASMCs was significantly increased after treatment with 200 μM CoCl2 both determined by CCK-8 assay (Figure 1B) and by BrdU

Summary

Introduction

Pulmonary hypertension (PH) is a common complicated disorder characterized by a rest mean pulmonary artery pressure of >25 mm Hg. Mechanisms of Hypoxia-Induced PAH belongs to the WHO Group I characterized by an increased vasomotor tone, vascular resistance from abnormal narrowing, and precapillary vasculature remolding associated with abnormalities of proliferation and apoptosis (Simonneau et al, 2013; Ramchandran et al, 2014; Yun et al, 2016). Li et al (2015) have documented that KLF5 mediates hypoxia-induced vascular remodeling via HIF-1α. Jiang et al (2015) have reported that SUMO-1 can interact with HIF-1α to regulate sumoylation of HIF-1α responding to hypoxia. Despite these findings, the mechanism of HIF-1α in PH is still not clearly understood. The proliferation of human pulmonary artery smooth muscle cells (HPASMCs) induced by hypoxia was considered as the main cause of pulmonary arterial hypertension (PAH). This study aimed to explore potential genes and long non-coding RNAs (lncRNAs) involved in the mechanism of hypoxia-induced PAH

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