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.