4. Discussion
We found that DE miRNAs are involved in IR injury following LT. A total
of 2,476 target genes were predicted with the majority of them
corresponding to cell cycle regulation (Cyclin M3), cell signaling (G
protein-coupled receptor 107), apoptosis (-DanJ heat shock protein C5),
cell adhesion (cadherin 4), transcription regulation (even-skipped
homeobox 2), and phosphatase activity (acid phosphatase 2, lysosomes).
These findings indicate that miRNAs exert their effects via multiple
target genes controlling different aspects of lung ischemic
pathogenesis.
GO annotates the significantly enriched gene terms to three components:
molecular functions, cellular components and biological processes. The
GO analysis of miRNA target genes showed that these corresponded to
metabolic enzymes, protein kinases involved in signal transduction, and
transcription and translation regulators. GO enrichment analysis
demonstrated that the majority of the predicted target genes are
corresponded to biological processes. In addition, apoptosis-related
factors, inflammatory factors, and receptors involved in ischemia, as
well as factors associated with hypoxia response were enriched in
related targets, suggesting the pivotal functions of that these target
genes in the pathological process of IR injury following LT.
The KEGG pathway analysis of target genes revealed that certain enriched
pathways were associated with inflammation and cellular damage and
apoptosis. These pathways included TGF-β signaling pathway, PI3K-Akt
signaling pathway, NF-κB signaling pathway, Ras signaling pathway, and
TLR signaling pathway. Studies on NSCLC metastasis have demonstrated
that, TGF-β1 can induce tumor cell invasion and epithelial-mesenchymal
transition. In addition, lung injury can lead to alveolar epithelial
cell death, basement membrane destruction, and TGF -β activation,
activated TGF-β can promote lung fibrosis (17). Other studies have
reported that the activation of the NF-κB signaling pathway promotes the
development of pulmonary inflammation and increased permeability of the
endothelial barrier. Moreover, inhibition of the NF-κB signaling pathway
can protect against lung injury after LT(18).
Using an early-stage liver injury model, we showed that a
cytokine-mediated systemic inflammatory response is triggered following
transplantation that impairs graft function and disrupts the dynamic
balance of distal organs (19). MiRNA-146a, miRNA-155, and miRNA-451
families, whose functions are related to immunity and inflammation, are
known to be involved in occlusive bronchiolitis after transplantation
(20). Certain studies have shown that primary graft dysfunction could be
alleviated by the treatment of depleting or inhibiting non-classical
monocytes in the donor lungs (21). The secretion of pro-inflammatory
factors induces pulmonary parenchymal and vascular inflammation in the
lung tissue. Increasing evidence suggests that miRNAs regulate tissue
ischemic inflammation (5, 22), and are associated with rejection after
transplantation (20).
We have previously shown a relationship between early injury after LT
and miRNAs and signaling pathways. To further study this association we
simulated allogeneic LT in mice. This design is closer to clinical
applications, and the experimental results are more representative.
Certain studies have shown that miRNA-24 antagonism can prevent renal IR
injury (23). Similarly, miRNA-106b-5p ameliorates cerebral IR injury in
rats by inhibiting apoptosis and oxidative stress (24). MiRNA-122 exerts
a variety of biological effects, for example, it can serve as a
tumor-suppressor in NSCLC (25). In addition, miRNA-122 is involved in
resveratrol-induced liver steatosis (26). In our study, miRNA-122
promoted the expression of inflammatory mediators following LT. After
pretreatment with antagomir-122, ischemia triggered expression of
pro-inflammatory TNF-α and the activity of MPO enzyme were significantly
down-regulated, whereas the expression of IL10, an inflammatory
inhibitor, was significantly increased. This finding revealed that
miRNA-122 acted as a pro-apoptotic / pro-inflammatory factor in lung
tissues and provides insights into the mechanism of IR injury following
LT. In addition, the inhibition of miRNA-122 plays an essential role in
suppressing the inflammatory response. These findings indicated that,
miRNA-122 could serve as a potential target for the treatment of IR
injury following LT.
MiRNAs are known to promote primary graft dysfunction through several
signal transduction pathways (27). For instance, the TLR signaling
pathway contributes to the pathogenesis following organ transplantation
(28, 29). As molecules that play an important role in innate immunity
and inflammatory responses, TLRs can initiate cellular innate immunity
by identifying specific structures of pathogenic microorganisms.
Sufficient evidence demonstrates the association of the TLR signaling
pathway with IR injury. Furthermore, lung IR injury can be alleviated by
inhibiting the activation of TLR 2/4 activity (30). Certain studies have
shown that miRNA-146a reduces lung injury via the TLR4 / NFκB signaling
pathway (31). Similarly, other studies have reported the function of
miR-21 in the development of severe primary graft dysfunction by
activating the key components of the TLR pathway (27). In the present
study, we showed that the expression of TLR 2/4 decreased following
inhibition of miRNA-122, whereas the expression of other damage-related
factors decreased, and that of anti-inflammatory factors increased
simultaneously. These findings suggest that the inhibition of miRNA-122
simultaneously inhibits the activation of TLR signaling pathways,
thereby reduce the IR injury following LT to exert a protective effect
on the donor’s lungs.