TY  - JOUR
AU  - Rajić, Jovana
AU  - Dinić, Svetlana
AU  - Uskoković, Aleksandra
AU  - Arambašić Jovanović, Jelena
AU  - Tolić, Anja
AU  - Đorđević, Marija
AU  - Đorđević, Miloš
AU  - Poznanović, Goran
AU  - Mihailović, Mirjana
AU  - Inic-Kanada, Aleksandra
AU  - Barisani-Asenbauer, Talin
AU  - Grdović, Nevena
AU  - Vidaković, Melita
PY  - 2020
UR  - http://www.ncbi.nlm.nih.gov/pubmed/32387379
UR  - https://radar.ibiss.bg.ac.rs/handle/123456789/3687
UR  - https://radar.ibiss.bg.ac.rs/handle/123456789/3695
AB  - 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-PCR-based 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-PCR-based 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.
PB  - Elsevier BV
T2  - Experimental Eye Research
T1  - DNA methylation of miR-200 clusters promotes epithelial to mesenchymal transition in human conjunctival epithelial cells.
VL  - 197
DO  - 10.1016/j.exer.2020.108047
SP  - 108047
ER  - 

@article{
author = "Rajić, Jovana and Dinić, Svetlana and Uskoković, Aleksandra and Arambašić Jovanović, Jelena and Tolić, Anja and Đorđević, Marija and Đorđević, Miloš and Poznanović, Goran and Mihailović, Mirjana and Inic-Kanada, Aleksandra and Barisani-Asenbauer, Talin and Grdović, Nevena and Vidaković, Melita",
year = "2020",
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-PCR-based 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-PCR-based 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.",
publisher = "Elsevier BV",
journal = "Experimental Eye Research",
title = "DNA methylation of miR-200 clusters promotes epithelial to mesenchymal transition in human conjunctival epithelial cells.",
volume = "197",
doi = "10.1016/j.exer.2020.108047",
pages = "108047"
}

Rajić, J., Dinić, S., Uskoković, A., Arambašić Jovanović, J., Tolić, A., Đorđević, M., Đorđević, M., Poznanović, G., Mihailović, M., Inic-Kanada, A., Barisani-Asenbauer, T., Grdović, N.,& Vidaković, M.. (2020). DNA methylation of miR-200 clusters promotes epithelial to mesenchymal transition in human conjunctival epithelial cells.. in Experimental Eye Research
Elsevier BV., 197, 108047.
https://doi.org/10.1016/j.exer.2020.108047

Rajić J, Dinić S, Uskoković A, Arambašić Jovanović J, Tolić A, Đorđević M, Đorđević M, Poznanović G, Mihailović M, Inic-Kanada A, Barisani-Asenbauer T, Grdović N, Vidaković M. DNA methylation of miR-200 clusters promotes epithelial to mesenchymal transition in human conjunctival epithelial cells.. in Experimental Eye Research. 2020;197:108047.
doi:10.1016/j.exer.2020.108047 .

Rajić, Jovana, Dinić, Svetlana, Uskoković, Aleksandra, Arambašić Jovanović, Jelena, Tolić, Anja, Đorđević, Marija, Đorđević, Miloš, Poznanović, Goran, Mihailović, Mirjana, Inic-Kanada, Aleksandra, Barisani-Asenbauer, Talin, Grdović, Nevena, Vidaković, Melita, "DNA methylation of miR-200 clusters promotes epithelial to mesenchymal transition in human conjunctival epithelial cells." in Experimental Eye Research, 197 (2020):108047,
https://doi.org/10.1016/j.exer.2020.108047 . .