Keratitis is an inflammatory disease that can progress rapidly with corneal ulceration through pathologic wound healing.
4 Corneal infection by filamentous fungus is accompanied by a localized inflammatory response with the secretion of pro-inflammatory cytokines and recruitment of neutrophils to the site of infection.
36 Normally, this is followed by an active wound-healing phase, when the inflammation resolves and the cornea undergoes fibrotic scarring. However, in many patients the inflammation does not resolve completely, and this culminates in corneal opacification and loss of vision.
38 Recent reports on altered miRNA expression in human corneal diseases
10–12 suggest their regulatory role in pathogenesis. However, the miRNA expression profile in fungal keratitis has not yet been studied. Here, we identified the corneal miRNA expression profile in fungal keratitis patients versus normal donors using the small RNA deep sequencing method. We also depicted the regulation network of significant differentially expressed miRNAs and investigated their possible role in wound inflammation. Such a study could help us to better understand the miRNA regulatory role in pathogenesis and to identify new therapeutic strategies.
Overall, several miRNAs were identified in normal and infected corneas (see results). Of these, only 75 miRNAs showed significant DE in keratitis (see volcano plot). Many corneal miRNAs not reported earlier were identified by deep sequencing, demonstrating a more highly sensitive detection than that of microarray studies.
39 Importantly, several miRNAs with profound changes in expression were found in infected corneas. Notably, expression of miR-184 and miR-204-5p, which were abundant in the normal cornea (as shown in earlier studies
40), was dysregulated in keratitis since they may be involved in corneal epithelial cell proliferation and migration during wound repair.
40 Moreover, several less-abundant miRNAs were also dysregulated, especially miR-511, miR-451a, and miR-223-3p, with significantly altered expressions in keratitis may have a regulatory role in disease pathogenesis. This divergence in expression profiles could possibly be due to the presence of infiltrating immune cells during infection compared to a normal condition with sparse resident immune cells like macrophages and dendritic cells.
41 The neutrophils that comprise 65% to 75% of the infiltrating cells in fungal-infected posttransplant corneas
36 have been shown to be a predominant source for miR-223.
42 Furthermore, using comprehensive next generation sequencing (NGS) data, we discovered a bona fide novel miRNA that was differentially upregulated in infected corneas. We further validated our findings by qPCR using independent corneal samples.
Our earlier study reported that the cellular inflammatory response between
A. flavus– and
Fusarium-infected corneas were comparable during early and late stages of the disease.
36 In this regard, we attempted to perform a comparative analysis of miRNA expression (select differentially expressed miRNAs) between
A. flavus– and
Fusarium-infected corneas by qPCR. Corneas from bullous keratopathy patients were used as noninfectious inflammatory controls. Interestingly, we found a significant variation in the magnitude of miRNA expression between the two fungal infections, especially with miR-204-5p and miR-cornea-3p (Hemadevi B, Vidyarani M, Prajna L, Bharanidharan D, unpublished data, 2015). Notably, expression of miR-223-3p was several-fold higher in fungal keratitis corneas compared to bullous keratopathy (Hemadevi B, Vidyarani M, Prajna L, Bharanidharan D, unpublished data, 2015), which further indicates the infiltrating neutrophils as a major source of this miRNA during corneal infections.
The function of a miRNA is ultimately defined by its effect on the expression of those genes that it targets. Though putative targets of miRNAs can be achieved by various databases, the false-positive rate is unacceptably high because of poor sequence complementarity of miRNA-target interaction in humans. In this report, we used an efficient bioinformatics approach to analyze the regulatory role of differentially expressed miRNAs systematically. With this method, we could identify cornea-specific targets, thereby reducing a large number of relatively speculative targets. Based on the targets, we then predicted pathways and biological processes (using DAVID) controlling wound healing (e.g., focal adhesion, proliferation, and migration) and inflammation (e.g., TLR) that may be disrupted in keratitis. Interestingly, the neurotrophin-signaling pathway was significantly regulated and may be involved in corneal wound repair as reported previously in bronchial epithelial cells and dermal fibroblasts.
43,44
We also developed a miRNA target gene regulation network in wound healing and inflammation. Such an approach helped us to identify a subset of miRNA expression profiles and their targets in wound inflammation, an important step in wound healing.
28 The validation of individual protein and miRNA changes will be required in subsequent studies to confirm their regulatory role in wound healing. For the subset of expression profiles, we predicted that the highly dysregulated miRNAs (miR-511-5p miR-142-3p, and miR-155-5p) involved in the TLR-pathway, among other pathways, may be involved in wound healing, as they target only wound inflammatory genes (
Fig. 4). Specifically, we predicted a role for miR-451a in wound inflammation by targeting MIF. Macrophage migration inhibitory factor has been proven to be a potential gene target of miR-451 in cancer cells,
45 where overexpression of miR-451 -downregulates mRNA and protein levels of MIF. Moreover, MIF has been shown to play a central role in wound healing by regulating both the inflammatory and proliferation/migration phases of wound healing.
33 Furthermore, inhibition of MIF has been shown to reduce the consequences of bacterial keratitis in mice, suggesting that it may have therapeutic effects.
46 Here, we report an inverse correlation in the expression of miR-451a and MIF in infected corneas, which may have physiological relevance in wound healing. However, further studies are required to prove the direct effect of miR-451a on downregulating corneal MIF expression at the protein level.
In summary, we report for the first time (to the best of our knowledge) a comprehensive human corneal miRNA expression profile in fungal keratitis using a deep sequencing approach. We identified several differentially expressed miRNAs in infected corneas, which may lay groundwork for new therapeutic strategies. Furthermore, we developed a bioinformatics approach to identify a set of miRNA gene targets involved in the control of inflammatory responses as well as wound healing. Our work indicates that miRNAs play important regulatory roles in corneal wound inflammation, and specifically, miR-451a can be considered a potential target for further investigation.