3.4 Immune and inflammation-related miRNA-mRNA regulatory
network
The empirically validated mouse PPI interactions in the IntAct, MIPS,
and MINT databases were collected, and a total of 4,853 PPI interactions
were obtained after the union of these three sets. A total of 544
verified miRNA-mRNA pairs data were collected from TarBase. Furthermore,
the 3827 miRNA-mRNA pairs obtained in Section 3.3 were integrated with
the 4853 and 544 regulatory relationships to obtain a global miRNA-mRNA
regulatory network (Figure 4A). Remarkably, among the differentially
expressed genes, there were 117 genes for which the GO function or the
GO function of the parent node was related to the immune system process.
Then, based on the global miRNA-mRNA regulatory network, we further
selected edges that contained at least one edge of these 117 genes from
the global network (Figure 4A) to construct an immune and
inflammation-related network. This network had 595 edges and was further
divided into three subnetworks. The first subnetwork had 479 edges
(Figure 4B), the second subnetwork had 115 edges (Figure 4C), and the
third subnetwork had only 1 edge (Protein interaction between H2-Aa and
H2 Ab1).
Interestingly, most miRNAs in the first subnetwork were upregulated,
whereas most miRNAs in the second subnetwork were downregulated. Further
enrichment analysis was performed on 106 genes in the first subnetwork.
These 106 immune-related genes were largely involved in positively
regulating the immune system: position regulation of NF kappaB
transcription, position regulation of natural killer cell mediated
cytotoxicity, position regulation of T cell promotion, and position
regulation of interleukin-6 production (E-Table 9). There were 51 miRNAs
involved in this subnetwork, including miR-21, let-7f, mir-431, and
mir-205.
Functional enrichment analysis of 38 genes in the second subnetwork
revealed that these 38 immune-related genes were involved in negatively
regulating the immune system: negative regulation of cell promotion,
negative regulation of plasma activation, negative regulation of
response to stimulus, and negative regulation of immune system process
(E-Table 10). However, these genes played a positive role in vascular
proliferation and oxygen metabolism: positive regulation of angiogenesis
and positive regulation of oxygen and reactive oxygen species metabolic
process (E-Table 10). There were 30 miRNAs involved in this subnetwork,
including mir-29b, mir-295 and mir-290-5p.
DISCUSSION
Neonatal hyperoxia not only disrupts lung development, but also
reprograms key immunoregulatory molecules in the
lung.29 However, at present, neonatal immunology is a
poorly researched field, with only a handful of studies devoted to the
pulmonary immune response;3 thus, our report
considerably expands knowledge in this field. In this study, we
performed traditional bioinformatics methods to analyze the raw data and
to identify miRNA-mRNA regulatory networks associated with immune and
inflammation processes, which play key roles in the development of BPD.
The downregulated genes were mainly related with immune response and
inflammatory processes, which emphasized their roles in BPD
pathogenesis. There are studies indicate that the classic
proinflammatory cytokines, including Il1α, Il8, Il1β and TNF-α are
overexpressed during the saccular stage in neonates developing BPD, and
their overexpression significantly disrupts lung development, both
structurally and functionally.3,4,8,30,31 While there
are also reports demonstrate that some parameters of the early
inflammatory response (neutrophils, cytokines such as Il1, TNF-α) may
not be detectable after days to weeks of exposure to noxious stimuli;
they have already initiated the signaling pathways of immune and
inflammatory processes that can affect the BPD lung
development.30,32 The seemingly contradictory results
may be due to the different stages of lung development or related with
the complex inflammatory signal pathways during BPD pathogenesis.
Therefore, a better understanding of the mechanisms underlying the
dynamic immune and inflammatory responses during BPD development could
reveal potential therapeutic targets to attenuate lung injury.
The upregulated genes were mainly related with ECM remodeling, and these
data indicated that abnormal ECM remodeling was implicated in the
pathogenesis of BPD, which is consistent with other
reports.33,34 Our previous studies also found that
excessive production and accumulation of collagen appeared around the
airways and blood vessels and in the alveolar regions of BPD mice, and a
number of genes associated with ECM remodeling and fibrosis, such as
fibronectin 1(FN1) and collagen 1α, were significantly dysregulated with
the development of BPD,33,34 these observations were
also consistent with those of our current study. Moreover, these ECM
components are believed to be involved in inflammation and
immune-related signal pathways,33-35 which supports
the notion that the ECM process is central to the development of BPD.
Based on the miRNA-mRNA co-expression networks associated with immune
and inflammation processes, we have found many important and interesting
interactions between miRNAs and mRNAs. Some targeted genes are regulated
by a single miRNA, whereas others can be regulated by multiple miRNAs
which can interact with each other and form an interesting regulatory
network. Of note, miR-205 was the most highly upregulated miRNA with 39
candidate targeted genes. These genes (such as H2-D1, Sh2d1a, Fcer2a,
Lbp, Il18) are mostly enriched in immunity-related functions: positive
regulation of lymphocyte mediated immunity, positive regulation of
lymphocyte mediated immunity, positive regulation of immune response,
positive regulation of lymphocyte mediated immunity, and induction of
apoptosis. For example, Lbp has been investigated as a biomarker for
inflammation.36 It is an acute-phase protein essential
for the response to bacterial lipopolysaccharides or endotoxins in
gram-negative bacteria, and can also increase its expression in response
to subclinical infections.36 Taking miR-21 as another
example, we found that it had 42 candidate targeted genes, and the
functions of these genes were also related to immunity and inflammation,
such as Il12a,37 Cr2,38Lbp,36 and Fasl.39 Furthermore,
these targeted genes may also be regulated by other miRNAs, such as
miR-205 and miR-155,18,40 or may be involved in other
inflammatory pathways through signal interaction. Thus, these miRNAs and
mRNAs construct regulatory networks associated with immune and
inflammation processes by interacting with each other directly or
indirectly. Further research will be needed to validate these miRNA-mRNA
regulatory networks in detail, and we believe this will lead to
additional significant findings.
In summary, this study characterized the miRNA and mRNA expression
profiles of lungs under normoxic and hyperoxic conditions, providing
insights into the immune and inflammatory responses to hyperoxia
exposure during late BPD stages. Our findings further emphasize that the
immune and inflammation processes play a significant role in BPD
development and may provide relevant information for the development of
new biomarkers and therapeutic targets not only to treat BPD, but also
to facilitate normal adaptive immune responses, which will have broader
implications in the management of premature infants.