DNA methylation of miR-200 clusters promotes epithelial to mesenchymal transition in human conjunctival epithelial cells

Abstract

Epithelial to mesenchymal transition (EMT) contributes to fibrosis associated pathologies including scarring of different ocular tissues. Recently targeting EMT is seen as an appropriate therapeutic approach for different fibrosis related eye diseases such as macular degeneration or glaucoma surgery related fibrosis. Nevertheless, for ocular surface diseases, target genes specific for particular cell type or condition are still undefined. This study aimed to expose the complex regulatory mechanisms that trigger EMT in human conjunctival epithelial (HCjE) cells. EMT was induced by prolonged treatment with two TGF-β isoforms, TGF-β1 and TGF-β2, and their combination. TGF-β1 showed the strongest potential for initiating EMT in HCjE cells, reflected on morphological changes, cell migration and the levels of mRNA expression of different epithelial (CDH1, OCLN, DSP) and mesenchymal (CDH2, FN1, VIM, SNAI1, ZEB2, TWIST1) marker genes. Co-treatment with the DNA demethylating agent 5-Azacytidine (5-AzaC) was capable of stopping the transition of HCjE cells towards a mesenchymal phenotype, based on morphological features, reduced cell mobility and mRNA and protein expression levels of epithelial and mesenchymal marker genes. An EMT qRT-PCRbased array revealed that EMT induced considerable alterations in gene expression, with downregulation of the majority of epithelial marker genes and upregulation of genes specific for the mesenchymal state. The major effect of 5-AzaC treatment was observed as a suppression of mesenchymal marker genes, suggesting the involvement of upstream negative regulator(s) whose promoter demethylation and subsequent expression will in turn promote EMT switch off. The expression level of miRNAs potentially important for EMT induction was determined using qRT-PCRbased array which pointed at members of miR-200 family as main regulators of EMT process in HCjE cells. 5-AzaC treatment induced increased expression of miR-200a, -200b, -200c and miR-141 towards the control level, indicating important role of DNA methylation in their regulation. The DNA methylation status of both miR-200 family clusters, analyzed with high-resolution melting (HRM) and bisulfite sequencing (Bis-Seq), revealed that TGF-β1-induced EMT was accompanied by increase in promoter CpG methylation of both miR-200 loci, which was reverted after 5-AzaC treatment. In conclusion, our results indicate that DNA demethylation of promoters of miR-200 loci is critically important for stopping and reverting the EMT in human conjunctival epithelial cells, suggesting the potential for the development of novel epigenetic-based therapeutic strategies for treating conjunctival conditions associated with EMT.

Interactions between circRNAs and miR-141 in Cancer: From Pathogenesis to Diagnosis and Therapy

“…Targeting EMT is seen as a novel therapeutic approach for fibrosis-related eye diseases such as the degeneration of the macula or glaucoma-surgery-related fibrosis [51]. The methylation of DNA bases plays an important role in the epigenetic regulation of gene expression, including the modulation of miR-141 expression in normal and cancer cells [52].…”

“…Since it is known that miR-141 is involved in EMT, the aberrant methylation of CpG islands near the start of transcription of miR-141/200c cluster is associated with their inappropriate silencing in neoplastic cells and phenotypic conversion in normal cells [52]. The DNA demethylating agent, 5-Azacytidine (5-AzaC), inhibits the transition of human conjunctival epithelial cells towards a mesenchymal phenotype and induces the expression of the miR-200 cluster, including miR-141, suggesting the potential for the invention of epigenetics-based strategies for the treatment of diseases associated with EMT [51].…”

MicroRNA and Their Potential Role in Conjunctival Disorders

“…The RT-PCR-based array directed at members of miR-200 family as the key regulators of EMT process in human conjunctival epithelial cells. It further indicated that DNA demethylation of promoters of miR-200 loci is very important for reverting the EMT in human conjunctival epithelial cells, suggesting the prospective for the developing new epigenetic-based therapeutic methods for treating conjunctival conditions associated with EMT [ 37 ].…”

Cascade of Inflammatory, Fibrotic Processes, and Stress-Induced Senescence in Chronic GVHD-Related Dry Eye Disease

“…Among the fibrotic processes that could account for cGVHD-related dry eye disease, EMT is a possible candidate for the process because the ocular surface epithelium loses the capability to secrete mucins when the epithelial cells undergo the transition to a mesenchymal phenotype [ 23 ]. Several studies have shown that EMT contributes to various fibrotic diseases of the kidneys, lungs, and liver and the ocular surface epithelium [ 97 , 98 , 99 ] and retinal pigment epithelium [ 100 ] in the eye. For example, 40% of fibroblasts in kidney fibrosis arise from epithelial cells via local EMT triggered by inflammatory stress, and 15% of fibroblasts are derived from the bone marrow under inflammatory stress [ 101 ].…”