We studied DNA methyltransferases (DNMT) implicated in CpG methylation pattern. The aim of our work was to extensively check the presence of DNMT (mRNA and protein levels) and quantify their expressions in the anterior segment.Methods
mRNAs were extracted from human cornea, conjunctiva, capsule, trabeculum and from related cell lines (HCE, HTCE, CHANG and TM). cDNAs were generated by reverse transcription, quantified by amplification with specific primers for DNMT1, 3A, 3B and 3L and normalized to the housekeeping gene 36B4. For immunohistochemistry, normal human corneas were provided by the regional corneal graft bank. All proteins were detected by specific primary antibody for DNMT1, 3A, 3B and 3L (Santa-Cruz®), negative control were done with a rabbit IgG antibody as primary one. Secondary antibody Cy3-coupled was used.Results
All DNMT are expressed in the human eyes but at different levels. DNMT3L looks like to be the weakly expressed. All tested cell lines also expressed DNMTs. Quantitative RT-PCR demonstrated strong differences between members and zones of expression of these DNMTs. Our transcripts results were confirmed by immunohistochemistry. All tested cell lines also expressed DNMTs. Quantitative RT-PCR demonstrated strong differences between members and zones of expression of these DNA methyltransferases. Our transcripts results were confirmed by immunohistochemistry experiments on cornea sections.Conclusion
For the first time, an inventory of the DNMT expression in tissues and cells lines from human ocular anterior segment was performed. Future studies will certainly permit to demonstrate the presence of a direct link between a deregulation of the CpG methylation pattern and the already “well known” eye pathologies.