In silico and transcriptional analysis of transcription factors involved in the initial phenotype commitment of human periodontal ligament mesenchymal cells

• Grant number: 22/07707-1
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Effective date (Start): November 01, 2022
• Effective date (End): October 31, 2023
• Field of knowledge: Health Sciences - Dentistry - Periodontology
• Principal Investigator: Denise Carleto Andia
• Grantee: Bárbara Jardim Boaro
• Host Institution: Vice-Reitoria de Pesquisa e Pós-Graduação. Universidade Paulista (UNIP). São Paulo , SP, Brazil

Abstract

Multipotent periodontal ligament cells (PDLCs) have shown the ability to differentiate in some lineages, being good candidates for regenerative therapies. Our research group has characterized PDLCs presenting distinct osteogenic potentials, showing high (h-PDLCs) or low (l-PDLCs) capacity of mineral nodule formation, in vitro. Recently, amongst other genomic methodologies, these h- and l-DLCs were submitted to the RNA-seq, followed by bioinformatic analysis, to access the whole transcriptome (FAPESP/University of Birmingham - 2017/07944-5). Interestingly, even at basal (without any differentiation), both h- and l-PDLCs have demonstrated differences in several parameters analyzed, at epigenetic and transcriptional levels. The initial analysis pointed out the adipogenic pathway as active in the l-PDLCs and some osteogenic pathways in the h-PDLCs. These results were the foundation for this current project, which aims to identify some transcription factors (TFs) related to adipogenesis and osteogenesis that could characterize the initial phenotype of PDLCs, confirming the distinct transcriptional profile and the mechanisms involved in the heterogeneity of the differentiation of these cells. After PDLCs were obtained (patients aged 18 to 20), the cell characterization was performed aiming to define their osteogenic potential (flow cytometry - CD105/CD166/CD34/CD45), osteogenic (Alizarin red, 21 days) and adipogenic differentiation (Oil red O, 25 days) in our previous projects with published results. l-PDLCs and h-PDLCs were collected after 10 days in culture (without any differentiation induction) and the RNA was extracted and purified by the TRIzol method. Here, in Part 1, the student will be trained on Bioinformatic tools and programs, followed by the selection of the osteogenic and adipogenic TFs, which were differently expressed through the RNA-seq raw data (previous FAPESP's grant). In Part 2, the transcriptional levels of the selected targets by the in silico analysis will be analyzed by real-time PCR, with GAPDH, B-ACTIN or 16S as reference genes (the more stable ones). The calculations will be performed according to the CT method.(AU)