DNA methylation of miR-200 clusters promotes epithelial to mesenchymal transition in human conjunctival epithelial cells.
2020
Autori:
Rajić, JovanaDinić, 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
Tip dokumenta:
Članak u časopisu (Recenzirana verzija)
Metapodaci
Prikaz svih podataka o dokumentuApstrakt:
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.
Napomena:
This is the peer reviewed version of the following article: 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. Exp Eye Res. 2020;197:108047. http://dx.doi.org/10.1016/j.exer.2020.108047
Ključne reči:
5-Azacytidine (5-AzaC); Conjunctival fibrosis; DNA methylation; Epithelial to mesenchymal transition (EMT); Human conjunctival epithelial cells (HCjE); TGF-β; miR-200 family; miRNAsIzvor:
Experimental Eye Research, 2020, 197, 108047-Finansiranje / projekti:
- Ministarstvo nauke, tehnološkog razvoja i inovacija Republike Srbije, institucionalno finansiranje - 200007 (Univerzitet u Beogradu, Institut za biološka istraživanja 'Siniša Stanković') (RS-MESTD-inst-2020-200007)
- “Laura Bassi Centers of Expertise” program of the Austrian Federal Ministry of Economy through the Austrian Research Promotion Agency (FFG project number 822768)
Povezane informacije:
- Druga verzija
https://radar.ibiss.bg.ac.rs/handle/123456789/3687
DOI: 10.1016/j.exer.2020.108047
ISSN: 0014-4835
PubMed: 32387379
WoS: 000565679200010
Scopus: 2-s2.0-85086110831
URI
http://www.ncbi.nlm.nih.gov/pubmed/32387379https://radar.ibiss.bg.ac.rs/handle/123456789/3687
https://radar.ibiss.bg.ac.rs/handle/123456789/3695