Pulmonary veno-occlusive disease (PVOD) is sometimes misdiagnosed with idiopathic pulmonary arterial hypertension (PAH) in the clinic and at least 3–12% of patients with PAH should be diagnosed as PVOD. In addition, PVOD is a highly fatal disease with a mortality rate of 72% within 1 year of diagnosis. Currently, there is a lack of effective treatment except for lung transplantation (1, 2). PVOD is related to a variety of risk factors, such as genetics, smoking, infection, and drugs [mitomycin-C (MMC)], but its pathogenesis is less studied (3). Therefore, in view of its high-fatality rate and lack of effective therapeutic drugs, it is very important to determine the pathogenesis and therapeutic targets.

Perros et al. (4) analyzed the clinical characteristics in seven patients of MMC-induced PVOD and found that all patients had severe hypoxemia and a low-diffusing capacity of the lung for carbon monoxide. Furthermore, right-sided heart catheterization confirmed that the mean pulmonary artery pressure (mPAP) was significantly increased, while a decreased of cardiac index. In addition, high-resolution CT of the chest identified septal lines, centrilobular ground-glass opacities, and lymph enlargement in all patients. What is more, a rat model of MMC-induced PVOD was successfully established by Perros et al. and found that the mPAP, total pulmonary resistance, and the fulton index were increased, and also the severe pulmonary vascular remodeling including smooth muscle cell hypertrophy/hyperplasia in pulmonary arteries, vasculitis of the pulmonary arteries and veins, foci of pulmonary edema and capillaritis, and foci of alveolar wall thickening. In addition, the expression of general control non-derepressible 2 kinase (GCN2) protein was decreased, whose gene is the major one linked to PVOD development and associated with heritability of PVOD.

microRNA (miRNA, miR) is a type of non-coding RNA with regulatory functions and a length of about 22–25 nucleotides. miRNA can regulate genes expression by incomplete or complete direct binding to the mRNA 3'-untranslated region to participate in genes regulation. It plays an important role in regulating genes expression, growth, proliferation and differentiation of cells, and participating in disease development (5, 6). miRNAs have been confirmed as being related to PAH. A study showed that miRNA-124 is down-regulated in a hypoxic PAH mouse model. However, miRNA-21 is upregulated in pulmonary artery smooth muscle cells, which leads to abnormal proliferation and pulmonary vascular remodeling (7–9). However, it is unclear whether miRNA is involved in the development of PVOD.

The tight junction is an important structure through which interactions between multiple-related proteins, maintains endothelial barrier function, and vascular permeability (10). Occludin, claudins, and zonula occludens (ZO) are key members of tight junction proteins. The ZO is an important component of the tight junction which includes ZO-1, ZO-2, and ZO-3 proteins. As a protein complex, the tight junction connects with F-actin through the ZO-1 protein (11). Pulmonary edema is a typical pathological change of PVOD. Studies have shown that the expression of occludin and ZO-1 is significantly decreased in lipopolysaccharide (LPS)-induced mouse, while piceatannol can recover the expression of occludin and ZO-1 and alleviate LPS-induced damage of the air–blood barrier and pulmonary edema (12). In PAH, tight junction destruction can lead to barrier dysfunction of pulmonary artery endothelial cells and promote the progression of the disease (13–15). So, based on the pathological changes of PVOD, we speculated that tight junction might be involved in the pathogenesis of PVOD.

Therefore, the purpose of this study is to investigate the differential expression profile of miRNAs and tight junction in the lung tissues of rats with MMC-induced PVOD.

Pulmonary veno-occlusive disease is disease with difficult to diagnosis and treatment, of the pathogenesis remains unclear. In this study, we found that the levels of mPAP and RVHI were increased, and there were severe pathological changes including pulmonary artery hypertrophy/hyperplasia, thickening, and occlusion in small pulmonary veins, pulmonary edema, dilated capillaries, and inflammatory cell infiltrate in MMC-induced rat. This was consistent with the study of Perros et al. (4). Therefore, the rat PVOD model was successfully established according to the typical pathological changes, mPAP and RVHI.

GCN2 is a serine-threonine kinase responsible for the phosphorylation of eukaryotic translation initiation factor to regulate the cell cycle and participate in the development of disease. Studies have shown that the mutation of GCN2 is linked to the development of PVOD and the expression of GCN2 is significantly decreased in patients with PVOD and MMC-induced rat pulmonary microvascular endothelial cells (20–22). In addition, Chen et al. (23) found that GCN2 deficiency decreased ATF3-dependent p38 phosphorylation inhibition in development of PVOD. This implies that GCN2 plays an important role in the pathogenesis of PVOD. We also found the same result, that the expression of GCN2 was decreased in rats with PVOD.

miRNA is a type of non-coding RNA with regulatory functions and plays an important role in regulating genes expression and disease development. In addition, miRNA is involved in the regulation of vascular proliferation and remodeling, as well as vascular endothelial cells apoptosis (24). However, whether miRNA is involved in the development of PVOD has not been studied. In this study, we used high-throughput sequencing which is an efficient, rapid method to analyze the differential expression profile of miRNAs between PVOD and normal rats, and screened out a total of 106 miRNAs among 1,030 transcripts that are differentially expressed in the lung tissues. This implies that the profile of these significantly differentially expressed miRNAs might be involved in the development of PVOD. As described in the results, the top upregulated including miRNA-495, miRNA-214-5p, and miRNA-190a-3p and the downregulated miRNAs including miRNA-141-3p, miRNA-509-5p, and miRNA-509-3p. In addition, RT-PCR confirmed that miRNA-214-5p was upregulated, while miRNA-141-3p was downregulated that were consistent with our results of high-throughput sequencing. PVOD is a rare form of PAH. Studies have shown that participates of miRNA in the development of PAH. Stevens et al. (25) found that miRNA-214 is shown to be significantly upregulated in rat models of PAH. In addition, miRNA-214 can promote smooth muscle cell phenotype changes and proliferation and participate in PAH vascular dysfunction (26). This is consistent with our results of sequencing analysis and validation. Fu et al. (27) found that attenuation of miRNA-495 derepresses phosphatase and tensin homolog to effectively protect right ventricular hypertrophy in rats with PAH. Another study showed that miRNA-190 plays an important role in hypoxic pulmonary vascular constriction which participates in the development of PAH (28). Lei et al. (29) found that the expression of miRNA-141 is downregulated in patients with PAH, while enhanced miRNA-141 expression can suppress the RhoA/ROCK pathway to regulate vascular remodeling of PAH. Tang (30) found that the expression of miRNA-509-3p expression decreases in the serum of patients with PAH. This is consistent with our results of high-throughput sequencing. However, the mechanisms by which these differentially expressed miRNAs participated in the development of PVOD need to be further researched.

Studies have shown that miRNAs are involved in the regulation of multiple-signaling pathways and functional analysis of the target genes is helpful for understanding the functional mechanisms of miRNAs (31). In this study, the GO and the KEGG pathway analyses showed that miRNAs are involved in various signaling pathways and metabolic processes, such as the fatty acid biosynthesis, tight junctions, mammalian target of rapamycin (mTOR) signaling pathway, PI3K-AKT signaling pathway, notch signaling pathway, hedgehog signaling pathway, AMPK signaling pathway, and cAMP signaling pathway. Therefore, we have performed a comprehensive literature search using the online databases PubMed and Embase up to October 2021 to determine whether multiple genes are regulated by miRNA-214-5p (Supplementary Table 6) and miRNA-141-3p (Supplementary Table 7).

Studies have shown that tight junctions are involved in the development of PAH (32). The study of Dalvi et al. (33) showed that Tat/cocaine-mediated production of reactive oxygen species activates the Ras/Raf/ERK1/2 pathway that contributes to disruption of the tight junction-related protein ZO-1, leading to pulmonary vascular remodeling and promoting the development of PAH. In our study, we also found significantly decreased expression of the tight junction-related protein of ZO-1, occludin and claudin-5 which might be involved in the development of PVOD. In addition, Lv et al. (34) study showed that miRNA-214 linked with ZO-1 to promote epithelial-mesenchymal transition process that was consistent with our finding.

Early studies have shown that fatty acid biosynthesis plays an important role in PVOD. Ogawa et al. (35) found that epoprostenol, as a metabolite of fatty acids, can be considered as a therapeutic option in patients with PVOD. Another study showed that a remarkable improvement of hemodynamics and of the clinical course of PVOD was produced by adjunctive use of oral sildenafil in association with intravenous high-dose epoprostenol (36). Brittain et al. (37) also found that abnormalities in fatty acid metabolism can be detected in the blood and myocardium in patients with PAH. This is consistent with our pathway prediction analysis results that it is indeed involved in the development of PVOD.

Although these pathways have not been studied in PVOD, they have been shown to be involved in the development of PAH. mTOR is a member of the serine/threonine protein kinase family that regulates cell proliferation, protein synthesis, and actin cytoskeleton; it is the catalytic subunit of two structurally distinct kinase complexes termed TOR complex 1 (TORC1) and TORC2 (38). Studies have shown that mTOR is involved in the occurrence and development of PAH. Inhibition of mTOR can reduce hypoxia-induced right ventricular hypertrophy and remodeling in animals (39) and improve PAH in patients (40). In addition, AKT as the substrate of mTORC2, can activate mTORC1 to involve in the development of disease (41). Jia et al. (42) found that osteoprotegerin induces pulmonary arterial smooth muscle cell proliferation by interacting with integrin αvβ3 to elicit downstream focal adhesion kinase and AKT pathway activation to facilitate PAH pathogenesis. Notch signaling is critically involved in the vascular morphogenesis and function including Notch 1–4. It has been determined that Notch 3 is associated with PAH. The expression of Notch 3 is increased in the lung tissues of patients with PAH and hypoxia-induced mouse with PAH (43). mTOR has previously been identified as a positive regulator of Notch 3 (44).

Hedgehog is a proangiogenic factor involved in the regulation of endothelial cell proliferation. Ghouleh et al. (45) found that the expression of hedgehog is increased in hypoxia-induced pulmonary artery endothelial cells and involved in the progression of PAH. Adenosine monophosphate-activated protein kinase (AMPK), a sensor of cellular energy, has been found to regulate cell proliferation (46). Studies have suggested that activation of AMPK by metformin prevents the development of PAH in animal models and activation of AMPK prevents the development of PAH by targeting nuclear factor-kappa B (NF-κB) to suppress autophagy and vascular remodeling (47, 48). cAMP signaling is involved in extracellular matrix metabolism as well as in proliferation control. The expression of p21(Waf1/Cip1) is regulated by C/EBP-α, which in turn is controlled by cAMP. Similarly, treprostinil, through cAMP-C/EBP-α p42-p21 (WAf1/Cip1) signaling, reduces arterial wall remodeling which benefits patients with PAH (49–51). So, the molecular mechanisms in PVOD are worth further exploration.

This study still has potential limitations. First, all the RNA and protein were extracted using the lung tissues, not pulmonary veins. In fact, it is also very difficult to separate small pulmonary veins and the capillaries also had serious pathological changes in rats with PVOD. In addition, in this study, we used lung tissues to perform high-throughput sequencing and validate the differentially expressed miRNAs so that it is unclear whether the results were from endothelial cells or smooth muscle cells. Then, not all of the differentially expressed miRNAs in rat with PVOD were validated by RT-PCR that might decrease the accuracy. Finally, we found that various signaling pathways might be involved in the development of PVOD by enrichment analysis, but we just detected the expression of tight junction protein of ZO-1, occludin, and claudin-5 which is consistent with the result of pathways enrichment analysis. Surely, we will further validate other differentially expressed miRNAs and molecular pathways to reveal their specific mechanisms of PVOD in the future.

In summary, we screened the expression profile of miRNAs closely related to PVOD by using high-throughput sequencing. In addition, the GO and the KEGG pathway analyses could help us to explored and predicted the function of certain miRNAs, which would provide a new experimental basis and ideas for further research on the effect of miRNAs on the occurrence and development of PVOD in future. In addition, the tight junction protein ZO-1, occludin, and claudin-5 might be related to be PVOD, which confirmed the GO and KEGG pathway analyses, but the underlying mechanisms need to be explored further.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE189080.

The animal study was reviewed and approved by Qinghai Red Cross Hospital. Written informed consent was obtained from the owners for the participation of their animals in this study.

QS performed the data analysis, drafted manuscript, and drew the figures. PC, YC, and S-JW coordinated the study and performed the experiment. YZ conceived the study, performed the experiment, and revised the manuscript. All the authors read and approved the final version of the manuscript.

This study was supported by the Major Scientific and Technological Achievement Transformation Project of Qinghai Province, China [2017-SF-122].

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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