Diabetes incidence is rising worldwide year by year, causing multiorgan damage. Unlike diabetic retinopathy, in patients with DK, hyperglycemia does not cause detectable and obvious clinical symptoms unless CECs are damaged or eyes are injured.
43 Patients with DK present with delayed wound healing of the corneal epithelium and decreased corneal sensitivity.
38 Our limited knowledge of the pathogenesis of delayed epithelial wound closure leads to the importance of identifying its pathogenesis. LncRNAs are important and pervasive genes that can act as ceRNAs and are involved in the development of diseases.
15,18 However, the role of lncRNAs and lncRNA-associated ceRNAs in diabetic CECs remains unclear. Over the years, this is the first work to explain the lncRNA-miRNA-mRNA regulatory network in diabetic CECs. Buried in other kinds of RNAs, lncRNAs generally exhibit low abundance.
44 There are many errors and clear limitations in RNA-Seq, exon detection, or RNA quantification.
45,46 The microarray is rich, precise, and comprehensive and therefore remains the preferred platform for lncRNA expression profiling.
47 In this work, an lncRNA microarray was performed to detect lncRNA expression. A total of 228 (111 lncRNAs, upregulated; and 117 lncRNAs, downregulated) significantly DELs were detected in the DM versus Crtl groups. The qRT–PCR analyses of some lncRNAs demonstrated the high credibility of the microarray in this study. Moreover, subcellular localization of lncRNAs is the basis of the ceRNA mechanism,
48 but it has rarely been mentioned in the previous literature. In this work, lncRNAs localized to the nucleus exclusively identified by the lncATLAS database were expelled. The subcellular localization of lncRNAs must be demonstrated in further research. Further in-depth studies would likely advance our knowledge on the ceRNA regulatory networks in corneal epithelial wound closure of DM mice.
In research on noncoding RNAs (ncRNAs) in diabetic CECs, miRNAs were most widely studied. Nine miRNAs were found to be significantly dysregulated in diabetic CECs, six miRNAs were upregulated, and three miRNAs were downregulated.
22 Among them, miR-204-5p-targeted SIRT delayed TKE2 cell cycle traversal and then postponed corneal epithelial wound healing upon high-glucose challenge. Additionally, our previous research found that an miR-181a-5p antagomir clearly contributed to corneal epithelium healing in type 1 DM mice.
21 Apart from these DEmiRs, miR-146a, miR-424, and miR-10b have also been found to be altered and perform a function in human diabetic corneas.
13,49 To further improve the accuracy of this study, only experimentally verified miRNAs in CECs of type 1 DM mice were included. To date, there have been no reports about other ncRNAs in diabetic CECs.
According to previous reports, 17 DEGs were regarded as regulating epithelial homeostasis in type 1 DM mice. Then, the TargetScan and miRanda algorithms were used to build the ceRNA network. Nevertheless, some genes (Bcl-2, Hgf, and Smad2, etc.),
38 which have not been validated in CECs of type 1 DM mice, were also not included in this work.
To further elucidate the ceRNA network of diabetic CECs, a subnetwork was extracted from the global ceRNA network. Our previous study showed that the inhibition of miR-181a-5p expression has a protective effect on corneal epithelium healing in type 1 DM mice. To further explore the ceRNA mechanism in the pathogenesis of delayed corneal epithelial wound healing, we focused on lncRNAs that were included in the GENCODE database and capable of binding miR-181a-5p based on databases (TargetScan and miRanda). Then, a novel lncRNA, Rik, with high expression abundance was selected as a biomarker for further investigation. Using the TargetScan and miRanda algorithms, five DEGs were found to be regulated by miR-181a-5p. For these genes, their differential expression changes and mechanisms have been well elaborated in previous studies. For example, Serpine1 was correlated with Plau levels and the rates of diabetic epithelial wound closure.
40 Reduced expression of Sirt1 could delay diabetic corneal epithelial wound healing through the IGFBP3/IGF-1R/AKT pathway via P53.
8 Hyperglycemia-suppressed Sema3c postponed diabetic corneal wound healing.
39 Hmgb1 is highly involved in diabetic CECs, and its blockade contributes to diabetic corneal epithelial wound healing.
42 Given the classic role of lncRNA as a ceRNA, we hypothesize that Rik may accelerate epithelial wound closure in diabetic corneas by sponging miR-181a-5p.
Figure 7 briefly displays how Rik functions.
To further elucidate the ceRNA mechanism, the potential mechanism and function of Rik were explored. The TKE2 cell line has been widely used in the research of corneal epithelial lesions.
7,22,34–36,41,50,51 Naturally, the expression of miR-181a-5p and Rik was verified in both CECs and TKE2 cell lines by qRT–PCR, and the luciferase reporter assay confirmed the interaction between Rik and miR-181a-5p. RNA FISH experiments targeting Rik showed both cytoplasmic and nuclear dots, further suggesting that Rik might perform important functions through the ceRNA mechanism. In addition, the scratch assay experiment revealed that overexpression of Rik could significantly promote TKE2 cell migration in a hyperglycemic environment. It is worth noting that there was a significant migration of TKE2 cells after scratching at 24 hours, and the healing area of the cells was larger at 48 hours in our study, consistent with studies in numerous diabetic corneal epithelial lesions.
34–36 TKE2 cells were cultured in keratinocyte serum-free medium (KSFM) supplemented with human recombinant epidermal growth factor and bovine pituitary extract.
22,34 The complete medium contains no serum, which may result in cell growth not as fast as expected, warranting our further investigation.
Our study also presents some limitations. The sample size of the gene expression profile was modest, and no microarray or RNA-Seq data of DEmiRs or DEGs in CECs of type 1 DM mice were found in the GEO and Array Express databases. Moreover, some genes or miRNAs related to the physiopathology of diabetic CECs have not been verified by experiments in type 1 DM mice, although with high conservation across several animal species, none of them has been included in our study. All of these factors might hinder us from establishing a relevant and comprehensive regulatory ceRNA network. Given the low conservation of Rik (76%), its role in humans deserves further investigation. Finally, the function and mechanism of a large number of lncRNAs located in the nucleus deserve further investigation.
To our knowledge, this is the first work revealing lncRNA profiling and the ceRNA network of diabetic corneal epithelial wound closure. A significantly downregulated lncRNA, Rik, was revealed to potentially activate diabetic corneal epithelial wound healing by sponging miR-181a-5p. Other lncRNAs aside from Rik might also rescue DM mice from corneal injury, warranting further studies.