Regulação Epigenética de Fatores de Transcrição Osteogênicos em Células do Ligamento Periodontal – um Estudo In Vitro e In Silico.

Resumo (EN)

The aim of this in vitro and in silico was to investigate the epigenetic mechanisms that regulate the (post)transcriptional profile of the osteogenic transcription factors (TFs) Sp7 Transcription Factor (SP7) and Distal-Less Homeobox 4 (DLX4), in addition to the correlation of gene expression between the histone demethylase JARID1B and the osteogenic TF Runt-related transcription factor 2 (RUNX2), in periodontal ligament stem-cells with high (h-PDLCs) and low (l-PDLCs) capacity for mineral deposition. The populations were cultivated in culture media with and without osteogenic induction (OM and DMEM, respectively), during periods of 3 and 10 days. After each culture period, DNA, RNA and protein were extracted from these cells, and this material obtained was submitted to the ATAC-seq, Methylome, RNA-seq, PCR and Western-Blotting techniques. Data were processed and analyzed by bioinformatics programs and tools based on R, Python and webserver environments. ATAC-seq analyzes indicated the genomic regions of SP7 and DLX4 more accessible in l-PDLCs when compared to h-PDLCs. In the Methylome analyses, these TFs showed different methylation patterns among themselves, but without differentially methylated probes (DMPs) between l- and h-PDLCs. The JARID1B methylation pattern was also analyzed, which was less methylated in l-PDLCs, in both culture periods (3 and 10 days). RNA-seq analyzes showed SP7 and DLX4 differentially less expressed in both culture conditions (DMEM and OM) on l-PDLCs. The long non-coding RNAs (lncRNAs) MIR31HG and LINC00939 were found differentially more expressed in both l-PDLC conditions. The RIblast prediction program, based on the LncRRIsearch webserver, predicted base-pairing (RNA:RNA) interactions between SP7, DLX4, MIR31HG and LINC00939 transcripts. The TriplexFPP machine learning program predicted potential for triplex formation (DNA:RNA) for SP7 and for LINC00939. PCR and Western-Blotting results showed reduced levels of JARID1B in h-PDLCs compared to l-PDLCs, and inversely proportional expression correlations with RUNX2(p57). Together, these results indicate the histone demethylase JARID1B and the lncRNAs MIR31HG and LINC00939 as possible epigenetic regulators in the osteogenic commitment of the l-PDLCs.