Candidate genes and pathogenesis investigation for sepsis-related acute respiratory distress syndrome based on gene expression profile

Acute respiratory distress syndrome (ARDS) is a potentially devastating form of acute inflammatory lung injury as well as a major cause of acute respiratory failure [1]. Its development leads to a high short-term mortality rate and significant long-term consequences among survivors, such as physical and cognitive impairment [2]. This disease has represented an important and costly public health problem. The predisposing factors of ARDS are numerous and assorted, including sepsis, pneumonia, multiple blood transfusions, lung contusion, aspiration of stomach contents, and drug abuse or overdose [3]. Although researchers have made significant progresses in elucidating the pathophysiology of this complex syndrome over the years [4], the absence of a universal detail disease mechanism up until now has led to a series of practical problems for a definitive treatment [5].

Previous study has indicated that ARDS has a close relation with severe sepsis [6], but little is known about the detail differences between sepsis-related and non-sepsis-related ARDS. Clinical research reveals that sepsis-related ARDS has poorer recovery from lung injury, higher overall disease severity and higher mortality than non-sepsis-related ARDS [7], however, the mechanism leading to the development of ARDS is still unclear.

Over the past few years, considerable work has been done to test the contribution of genetic factors that may increase the risk of developing ARDS. A genome-wide association study by Wang et al. [8] have suggested that the BCL2-associated agonist of cell death (BAD) gene is a candidate gene associated with the development of ARDS. Besides, variants in more than 30 genes have been associated with ARDS [9]. For instance, common genetic variation in the angiopoietin-2 (Ang-2) gene is proved to be associated with increased risk of ARDS [10]. Gong et al. [11] suggested that the mannose binding lectin-2 (MBL-2) deficiency was associated with increased susceptibility to sepsis and ARDS. Additionally, based on the whole genome expression analysis, Kangelaris et al. [12] believe that the exploration of gene expression differences occurring early in the development of sepsis-related ARDS may further reveal the mechanisms of ARDS. Furthermore, based on the gene expression profiling on peripheral blood from ARDS patients, Dolinay et al. [13] found that the inflammasome pathway and its downstream cytokines play critical roles in ARDS development; Wang et al. [14] found that peptidase inhibitor 3 (PI3) may be a useful clinical marker for monitoring the early development of ARDS. Thus, the analysis of potential ARDS related genes and pathways based on gene expression profile may be a breakthrough for the further understanding of ARDS pathological mechanism.

In the present study, a bioinformatics analysis was performed based on a previous mRNA expression profile from patients with sepsis or sepsis-related ARDS, which was provided by Kangelaris et al. [12]. Via the investigation of disease related differentially expressed genes (DEGs) and pathways, we tried to explore the mechanism of the ARDS and to provide valid biological information for further investigation of this devastating disease.

Although intensively and continuously studies have been conducted in ARDS, its mortality is still as high as 30–40 % [33]. In the present study, a bioinformatics analysis between sepsis-related ARDS mRNA samples and human sepsis mRNA samples was performed to explore the mechanism of the ARDS. A total of 122 up-regulated and 91 down-regulated DEGs were obtained. The up- and down-regulated differentially expressed mRNAs were mainly involved in functions like mitotic cell cycle and pathway like cell cycle. PPI network of ARDS analysis revealed 20 hub genes such as CCNB1, CCNB2 and TOP2A. In addition, seven transcription factors were revealed in the transcription factor-target gene regulation network. Furthermore, 14 co-cited genes including CCNB2–CCNB1 were revealed in literature mining analysis. These findings may contribute to understanding the development of sepsis-related ARDS.

In the present study, GO functional analysis showed that the up-regulated DEGs, such as CCNB1, CCNB2 and TOP2A, were most significantly assembled in BP related to mitotic cell cycle (GO: 0000278, P = 1.70E−09) (Table 1). Meanwhile, KEGG analysis also showed that the most significant pathway enriched by up-regulated DEGs was cell cycle (04110, count = 5, P = 0.0035) (Table 2). Study has reported that errors in mitosis can either kill a cell through apoptosis or cause mutations which may lead to disease [34]. Therefore, we speculated that some key factors might play important roles in the development of ARDS via taking part in mitosis cell cycle. When and how did these DEGs regulate the process of ARDS? The Reactome pathways analysis showed that mitotic prometaphase (68877, count = 8, P = 4.95E−06) (Table 2) was the most significant pathway enriched by the DEGs. GO analysis showed that both the up- and down-regulated DEGs were assembled in same function like spindle (GO: 0005819). Thus, we speculated that the DEGs might involve in the progression of ARDS via interfering the progress of spindle in premetaphase of mitotic. However, a further investigation is needed to clarify this speculation.

In this study, CCNB1 and CCNB2 were two outstanding ARDS-related genes based on the calculation of subgraph centrality in PPI network. Furthermore, the literature mining analyses showed that CCNB2–CCNB1 had the highest co-cited times. As a mitotic cyclin, cyclin B is necessary for the progression of the cells into and out of M phase of the cell cycle [35]. An abnormal cytoplasmic cyclin B1 expression has been found to be associated with a specific T-cell response and cyclin B1-specific immune responses [36]. Importantly, increasing evidences indicate that the immune system plays a key role in lung diseases, including acute lung injury [37]. Activation of the innate immune response by binding of cell injury-associated endogenous molecules to pattern recognition receptors such as the Toll-like receptors on the lung epithelium and alveolar macrophages is now recognized as a potent driving force for ARDS [1]. Taken together, although there is no direct evidence that CCNB1 and CCNB2 are involved in ARDS, we speculated that CCNB1 and CCNB1 might have close relations in the development of ARDS.

Furthermore, TOP2A was also a hub gene had the highest score in betweenness centrality and degree centrality. TOP2A encodes a DNA topoisomerase that controls and alters the topologic states of DNA during transcription. Actually, the enzyme of TOP2A gene is an essential nuclear enzyme involved in processes such as chromosome condensation and chromatid separation during DNA transcription and replication [38]. Recent study reported that its encoding protein TOP2α is responsible for causing genomic DNA damage [39]. Interestingly, DNA damage is implicated in diverse pulmonary disorders, including acute lung injury [40]. Therefore, we speculated that the up-regulation of TOP2A in our study might have a potential relation in the development of ARDS.

The transcription factor-target gene regulation network analysis in this study revealed seven transcription factors including FOXM1. FOXM1 regulates the expression of a large array of G2/M-specific genes including CCNB2, and plays an important role in maintenance of chromosomal segregation and genomic stability [41]. In this study, HMMR was a target gene of FOXM1. HMMR (also identified as CD168) was originally discovered as a soluble protein that altered migratory cell behavior and bound to hyaluronan [42]. HMMR is less well studied than the main hyaluronan receptor of CD44 that has been examined in ARDS secondary to bleomycin injury. Priit et al. [43] have suggested that CD44 plays a role in resolving lung inflammation during the process of ARDS. Although the role of HMMR (CD168) in ARDS has not been studied before, we speculate that HMMR may be related with the progress of ARDS.

Despite of the results obtained above, there were some limitations in this study. Firstly, no verification experiments based on cells or tissues or joint analysis of expression profile data were performed to confirm our results, besides, the sample size was small. Secondly, due to the data themselves, there was no correction for multiple comparisons in the DEGs identification. Thirdly, the mRNA expression profile used in this study was extracted from blood leukocytes, and another important cells in ARDS, such as epithelial and endothelial cells of the lung had not been studied. Therefore, more investigations related to another cells in ARDS with experimental verification and diverse samples are needed in the further study.

In conclusion, the pathways like mitotic cell cycle were closed related with the development of sepsis related ARDS. Genes including CCNB1, CCNB2 and TOP2A, as well as transcription factors FOXM1 may be used as the novel gene therapy targets for ARDS.