Angiogenesis is an important part of cerebral ischemia repair, and promoting angiogenesis is considered a promising strategy for the treatment of ischemic stroke (Sun et al. 2020; Wlodarczyk et al. 2021). Numerous studies have shown that non-coding RNAs, such as miRNAs and lncRNAs, play a critical role in IS angiogenesis (Gao et al. 2020; Heydari et al. 2020). Nowadays, increasing evidence also suggests a complex and close relationship between miRNAs and lncRNAs, based on Salmena's hypothesis that lncRNAs may attract miRNAs as molecular sponges and participate in IS angiogenesis by regulating the expression of downstream genes through miRNAs (Mengqi Zhang and Xinyi Lv 2020; Yan et al. 2020). For example, Yan et al. (2020) showed that LncRNA MACC1-AS1 acts as a ceRNA for miR-6867-5p to regulate TWIST1 to promote cell proliferation, migration and angiogenesis. Zhang et al. (2020) found that overexpression of lncRNA DANCR enhanced survival and angiogenesis in OGD-treated BMECs through the miR-33a-5p/XBP1s axis. However, studies on ceRNA networks in angiogenesis are scarce, so we used WGCNA and ceRNA networks to identify biomarkers in peripheral blood of ischemic stroke patients and healthy control patients for a comprehensive study.
To our knowledge, the present study is the first to apply WGCNA to construct a ceRNA network for IS, linking the ceRNA network to angiogenesis for the first time. WGCNA analysis takes the correlation coefficients of gene expression values to the Nth power, thus making the distribution of correlation coefficients more consistent with scale-free network analysis and biological rules (Kakati et al. 2019). In this study, NR4A1, PTGS2, ERG3 and VEGFA were identified as hub genes for IS angiogenesis by screening and enrichment analysis of the modules. Subsequently, by constructing a ceRNA network, we suggest that the upregulated LncRNA HCG18 may be critical for angiogenesis, as it may act as a ceRNA to downregulate the expression of has-let-7i-5p and has-miR-148a-3p, leading to the upregulation of NR4A1, PTGS2, ERG3, and VEGFA, respectively. GSEA analysis suggests that PTGS2 and ERG3 may affect angiogenesis by influencing NFKB-TNFA signaling pathway; NR4A1 is involved in angiogenesis through hypoxia, oxidative phosphorylation, while VEGFA may be associated with DNA repair, apoptosis.
NR4A1 (also known as TR3, Nur77, NGFI-B, TIS1 and NAK-1), nuclear receptor subfamily 4 group A member 1, is involved in a variety of biological processes including apoptosis, proliferation, inflammation and metabolism (Crean and Murphy 2021; Nie et al. 2016). Therefore, NR4A1 overexpression may have the potential to promote IS angiogenesis. This has been confirmed in several previous studies. Using a middle cerebral artery occlusion (MCAO) model，Ling et al. (2020) found that NR4A1 expression was downregulated, but miR-224-5p inhibitor ameliorated OGD-induced neuronal apoptosis by targeting the 3'-UTR of NR4A1. In addition, Nur77 overexpression in mouse endothelial cells upregulates integrin β4 to promote vascular neogenesis (Bourbon et al. 2015). The present study suggests that has-let-7i-5p may regulate NR4A1, while HCG18 may interact with has-let-7i-5p as ceRNA. To our knowledge, no studies have explored the role of HCG18 in IS, but its role in other diseases may indirectly explain its role in IS. Notably, Zou et al. (2020) demonstrated that lncRNA HCG18 promotes the proliferation and migration of hepatocellular carcinoma cells. Li et al. (2020) revealed that the HCG18 sponge miR-34a-5p mediates HMMR expression and promotes cell proliferation, migration and invasion in lung adenocarcinoma, thereby aggravating the progression of lung adenocarcinoma. Furthermore, Xiang et al. (2017) reported that let-7i overexpression significantly alleviates cell death and increases the survival of OGD-treated human brain microvascular endothelial cells. Jickling et al. (2016) found that let-7i is decreased in circulating leukocytes of IS patients and is involved in leukocyte activation, recruitment and proliferation pathways. It is hypothesized that HCG18 may be upregulated in IS, further sequester has-let-7i-5p prevent it from inhibiting the expression of NR4A1, ultimately leading to upregulation of NR4A1 in IS. This hypothesis was confirmed in the results of this study.
VEGFA (also known as VPF, VEGF, MVCD1), a member of the VEGF family, is an important regulator of angiogenesis, neuroprotection and neurogenesis (Geiseler and Morland 2018). Therefore, upregulation of VEGFA is a protective strategy for IS. Ren et al. (2018) confirmed that lncRNA-MALAT1 promotes angiogenesis by sponging miR-145 and blocking its inhibition of VEGFA expression. In this study, VEGFA expression was significantly upregulated in IS patients, and HCG18 may sponge has-miR-148a-3p to regulate VEGFA expression. The role of has-miR-148a-3p in IS has not been analyzed, but its role in other diseases has been demonstrated. For example, Wang et al. (2019) demonstrated by real-time quantitative PCR analysis that has-miR-148a-3p was lowly expressed in esophageal cancer samples, promoted cell proliferation and invasion, and was associated with a poorer prognosis. In addition, Wang et al. (2020) also found that miR-148a-3p overexpression could cause damage to the blood retinal barrier and inhibit angiogenesis. Thus, HCG18 may also be upregulated to sponge has-miR-148a-3p, thus facilitating the upregulation of VEGFA by reducing the expression of has-miR-148a-3p, and subsequently promoting angiogenesis.
Early growth response proteins belong to the immediate - early transcription factor family and are expressed in many types of tumor cells and are induced by a variety of stimulus (O'Donovan et al. 2000). Among the four EGR members (EGR1-4), EGR3 is mainly involved in neurodevelopmental processes such as myotome development (Tourtellotte and Milbrandt 1998) and sympathetic neuron differentiation (Jackson et al. 2014). Therefore, it is speculated that ERG3 upregulation may be one of the protective factors for IS. This hypothesis was confirmed, Liu et al. (2008) reveal that ERG3 upregulation promotes VEGF-mediated endothelial cell proliferation, migration, and angiogenesis. Chen et al. (2020) also confirmed that ERG3 promotes angiogenesis both in vivo and in vitro. Consistent with these studies, ERG3 was highly expressed in the IS samples in this study. Moreover, this study also demonstrated that both has-miR-148a-3p and has-let-7i-5p could regulate ERG3 expression, whereas HCG18 could sponge has-miR-148a-3p and has-let-7i-5p, respectively.
PTGS2 (aka C0X2), regulated by specific stimulus events, is involved in the biosynthesis of prostaglandins related to inflammation and mitosis (Hashemi Goradel et al. 2019). Experimental studies have shown that the specific knockdown of PTGS2 expression could repress the NF‐κB signaling pathway, thereby inhibits apoptosis and promotes proliferation, migration and angiogenesis of EPCs, providing protective effect on mice with ischemic stroke (Zhou et al. 2019). Meanwhile, Fan et al. (2018) concluded that PTGS2 expression downregulation not only promoted angiogenesis but also reduced brain infarct volume and inhibited inflammation in MCAO rats. However, PTGS2 was significantly upregulated in the peripheral blood of IS patients in this study, and this inconsistent result may be due to different sample selection. Similar to ERG3, HCG18 regulates PTGS2 expression by sponging has-miR-148a-3p and has-let-7i-5p.
The present study still has limitations. Based on bioinformatics techniques, the study preliminarily explores the mechanism of IS angiogenesis and identifies the interactions among lncRNA, miRNA and mRNA in angiogenesis. Further experimental studies are needed to verify the interactions among identified ceRNA axis in IS.
In conclusion, this study identifies the lncRNA -miRNA-mRNA interaction axis (HCG18-has-let-7i-5p-NR4A1/PTGS2/ERG3、HCG18-miR-148a-3p-PTGS2/ERG3/ VEGFA)，which provides a new perspective for the study of IS angiogenesis mechanism and a potential target for IS therapy. In the future, we will also conduct clinical, vitro and vivo experiments to validate the expression of IS angiogenesis-related genes identified in this study and their function relationships.