4.Discussion
Bronchopulmonary dysplasia is one of the most common complications
arising in preterm infants, especially in those born underweight and
those of small gestation weeks. It has been reported that up to 70 % of
babies born before 26 wk of gestation will develop BPD (Durrmeyer et
al., 2012). The progression of BPD is known to be driven by multiple
mechanisms, with the participation of a few important protein and
signaling pathways,
such
as thec vascular endothelial growth factor (VEGF), interleukin (IL), and
phosphatidyl inositol-3-enzyme-serine/threonine kinase (PI3K-AKT)
signaling pathway(Kalikkot Thekkeveedu et al., 2017). Therefore, it is
important to clarify the pathophysiology of BPD and discover means of
early diagnosis and treatment-related biomarkers. Bioinformatics
analysis and efficient microarray might be conducive to our
understanding of the molecular mechanisms of disease occurrence and
development, thus helping the exploration of genetic alternations and
identification of underlying diagnostic biomarkers.
In this study, we screened out 19 significant DEMs, of which 10 were
shown to be upregulated, whereas 9 downregulated. Rcesults of functional
enrichment analysis indicated that these significant DEGs were
associated with the virus infection, antigen processing and
presentation, B-cell receptor, phagosome, hematopoietic cell lineage,
and CAMs signaling pathways in BPD. Among these signaling pathways, the
CAMs pathway was also obtained in the analysis of GSE32472(Pietrzyk et
al., 2013) and GSE125873(Ryan et al., 2019). In these 2 series they both
identified the signaling pathway of T-cell receptor which was not
obtained in our study, maybe due to the use of different grouping
methods and sampling time. Key DEGs, such as CD19, CD22, CD72, CD74,
MS4A1, and FCGR2B were identified as hub genes in PPI networks.
Moreover, through the construction of the PPI network, we could
recognize key genes, with which miRNAs might interplay with. Despite
filtering the genes with the potential targets of the 19 significant
DEMs, we could still identify 140 upregulated and 33 downregulated
genes. Hence, considering the total number of DEMs, the enormous and
complex miRNA-mRNA regulatory network could be unimaginable. The hub
genes of a network are known to always be important, resembling ”seeds”,
that could combine different signal pathways.
Furthermore, some of these DEGs were validated and found to be
correlated with BPD. One of the DEGs, called adrenomedullin (ADM), which
was found to be downregulated in our study, was shown to be regulated by
hsa-miR-423-5p, hsa-miR-3940-5p, hsa-miR-767-5p, and hsa-miR-4301.
Moreover, ADM has been shown to have potent angiogenic,
anti-inflammatory, and antioxidant
properties. Zhang et al. reported that ADM
deficiency
in human pulmonary microvascular endothelial cells (HPMEC) resulted in
significantly increased the generation of hyperoxia-induced reactive
oxygen species and cytotoxicity compared with ADM sufficient HPMEC,
finally causing BPD (Zhang et al., 2015); however this finding remains
to be validated. Likewise, WNT 3/16 were demonstrated to be upregulated
in our study through many miRNAs, such as the underexpressed
hsa-miR-767-5p, hsa-miR-5681b, hsa-miR-423-5p, hsa-miR-3940-5p, and
hsa-miR-3960.The WNT family have also been found to be associated with
the development of BPD. Hyperoxia is known to increase the expression of
WNT2b,
WNT 5a, WNT 9a, and WNT 16, and decrease the expression of WNT 4, WNT
10a, and WNT 11 (Lingappan and Savani, 2020). The WNT family of proteins
includes a large number of members that control a variety of
developmental processes, including cell fate, proliferation, polarity,
and migration (Ota et al., 2016). Li et al. found that patients with BPD
were characterized by an increased activity of Wnt/β-catenin (Li et al.,
2015). Similar to that, we also found an increased expression of WNT 16
in BPD, but the mechanisms by which WNT3 might cause BPD remain to be
explored. Among the identified DEGs, we also found the upregulated
TLR10, which was shown to be regulated by miRNA, such as the
underexpressed hsa-miR-767-5p, hsa-miR-5681b,
hsa-miR-423-5p,hsa-miR-3940-5p, and overexpressed hsa-miR-33a-5p, and
hsa-miR-337-5p, to be enriched in the immune response process term of
the GO-BP category. Toll Like Receptors (TLRs) are known to play an
important role in regulating inflammation, maintaining mucosal
homeostasis and preventing bacterial invasion (Akira et al., 2006).
Rising evidence has implied that the TLR signaling pathway is the
pivotal component of the pulmonary homeostatic program that abrogates
lung inflammation and injury(Sampath et al., 2012). Many studies were
aimed at Toll-interleukin 1 receptor domain-containing adaptor protein
(TIRAP). Researchers have also found that TLR5 and TLR4 were associated
with the occurrence of BPD via the MyD88-dependent pathway(Malash et
al., 2016; Yao et al., 2017), and TLR10 was reported to active the TRL4
signaling pathway. So, TLR10 might also be related to the occurrence of
BPD; another finding that requires confirmation.
In this study, we screened out 19 DEMs, suggested to modulate the
expression of DEGs and contribute in the regulation of many pathways.
Besides, we also found that single miRNA could interplay with many mRNA
species, as well as that a single mRNA could also interplay with many
miRNA species. Although most of them have not been reported in the
mechanisms so far studied in patients with BPD, we could still obtain
some information from existing studies. One such case was the
hsa-miR-15b-5p, one of the identified DEMs in our study. Zhang et al.
found that miR-15b-5p was upregulated in BPD mice(Zhang et al., 2011).
Fu et al. have also reported that it has a protective action against
oxidative stress in HG-stimulated podocytes (Fu et al., 2019), while
Ezzie et al. found that it was increased in patients with chronic
obstructive pulmonary disease (COPD) and could potentiate the
progression of fibrosis in lung tissues (Ezzie et al., 2012).As such,
overexpression of the hsa-miR-15b-5p in the BPD blood samples in our
results, might indicate a similar underlying association. Another DEM,
hsa-miR-301a-3p,
which was overexpressed in our study, was demonstrated to modulate DEGs,
such as TLR10, CD72, and BMP3. This effect has been previously observed
in animal experiments. The study by Dong et al. showed the
overexpression of miR-301a in a murine model of hyperoxia-induced
bronchopulmonary dysplasia(Dong et al., 2012). Therefore,
hsa-miR-301a-3p might also play a role in the mechanism of BPD
development in infants, which remains to be validated.
Although we investigated the miRNA-mRNA regulatory pathway in BPD using
bioinformatics methods, our study had some limitations that should be
clarified. First, the samples were limited and might have led to high
false-positive rates and one-sided results. Therefore, it is required to
improve the detection power by integrating more datasets in future
studies. Second, the source of microarray data was only from blood
samples. Body fluids that could be noninvasively obtained in the clinic,
such as sputum and urine might also contain miRNAs. Third, to confirm
the mechanisms of hub genes related to BPD, it will be helpful to add
some in vitro or in vivo experiments to validate our results. Forth, due
to the absence of clinical data, we were unable to assess the
relationship between DEMs and the severity of BPD. More clinical and
demographic characteristics of infants with BPD are required for further
analysis. Finally, experimental evidence, obtained from wet research,
such as western blot, real-time PCR and immunohistochemistry assays are
required to better delineate the role of hub genes and the potential
mechanisms of BPD.
In this study, we found multiple miRNA-mRNA regulatory pathways and
potential biomarkers of BPD, in line with our current knowledge of the
pathophysiology of this disease. We believe that this
hypothesis-generating study offers a new insight into the molecular
mechanisms of BPD through the and identification of several latent
biomarkers that could be used toward its diagnosis and treatment.