In this study, to detect circRNAs in an HPMEC model of ARDS, isolated HPMECs were assigned to the experimental group (LPS challenge) or the control group (exposure to normal medium). Differentially expressed circRNAs were successfully detected in treated HPMECs but not in controls.
We performed whole-genome sequencing of HPMECs cultured in LPS medium to identify changes in the expression profiles of circRNAs. A total of 379 significantly up-regulated and 448 significantly down-regulated circRNAs were obtained by sequencing. We performed GO and KEGG analyses on the differentially expressed circRNAs’ target genes. The results of GO analysis showed that circRNA target genes were mainly enriched as part of the cellular response to DNA damage, including DNA repair. Previous studies have shown that during the development of ARDS, the inflammatory response leads to cellular oxidative stress[30], resulting in DNA molecular damage[31]. DNA repair in damaged areas facilitates the recovery of lung tissue in patients with ARDS[31]. However, additional research on the role of circRNA in DNA repair is necessary.
The results of KEGG analysis showed that circRNA is mainly involved in cell signaling, such as the MAPK pathway. MAPK is involved in the process of alveolar damage and repair[32]. Studying the role of circRNA in this pathway will inform the development of therapeutic strategies for patients with ARDS.
The observed correlations in the differential expression of circRNA revealed by RT-qPCR analysis were consistent with the trends observed in the sequencing data. These circRNAs may therefore serve as diagnostic biomarkers for ARDS.
Many studies are showing that circRNAs and miRNAs play important roles in the development of lung diseases through interaction. For example, circ-CPA4 regulates non-small cell lung cancer (NSCLC) cells in the immune microenvironment of NSCLC tumors through the let-7 miRNA/PD-L1 axis[33]. Guo et al. showed that circRNA BBS9 contributes to the development of chronic obstructive pulmonary disease by inhibiting miRNA function[34]. It has been shown that the effects of circRNA-WDR27 on the development of tuberculosis are mediated by the miRNA-370-3p/fstl1 axis[35].
To further clarify the role of circRNA and miRNA in regulating target gene expression, we established a ceRNA network. We found there may be a mutual regulatory relationship among 6 miRNAs and 49 competitive circRNAs. In addition, GO enrichment analysis of regulated genes in the ceRNA network showed that differential gene expression was primarily a response to fluid shear stress or a means to regulate angiogenesis, vasculature development, and cell adhesion. The results of in vivo and in vitro studies have shown that vascular endothelium cell permeability varies according to the degree of shear stress[36, 37]. Vascular endothelial growth factor/vascular permeability factor acts to increase the permeability of microvascular endothelial cells and is one of the most potent vascular permeability agents known [38]. It has been reported that junctional adhesion molecule-C regulates vascular permeability by modulating vascular endothelial contractility and VE-calmodulin–mediated adhesion[38].
In the current study, the circRNAs found to have the greatest changes in expression are likely to play an essential role in the regulation of vascular permeability[39, 40]. Furthermore, the corresponding target genes in the ceRNA network were closely associated with human lung microvascular endothelial cell injury and altered permeability, pathognomonic characteristics of ARDS. These circRNAs are likely to affect the altered pulmonary microvascular permeability observed in ARDS.