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Evaluation of thyroid hormone action upon encoding RNAs expression in osteoblastic cells derived from adipose tissue.

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Bruna Moretto Rodrigues
Total Authors: 1
Document type: Master's Dissertation
Press: Botucatu. 2018-04-06.
Institution: Universidade Estadual Paulista (Unesp). Faculdade de Medicina. Botucatu
Defense date:
Advisor: Célia Regina Nogueira

The skeletal system is complex and have an intense metabolism compound with cells, proteins and minerals. The osteoblasts are fundamental cells to the tissue, executing two mainly functions: bone formation and regulation of reabsorption through osteoclastogenesis modulation. Therefore, these cells perform primordial functions to the bone development and maintenance. Several systemic molecules act in the tissue and the thyroid hormones are one of them. They are fundamental to the bone metabolism, once hormone alterations result in bone disorders. Osteoblasts have T3 nuclear receptors, and although not well elucidated it affects many aspects of the cell development so as pathways that modulates the bone remodeling. Several cells lineages have been used in osteoblasts studies, and human adipose-derived Mesenchymal stem-cells (hA-MSCs) are a promising model to osteoinduction. Thus, the aim of this study was to investigate the supraphysiological T3 (T3S) influence in the gene differential expression in osteoblasts differentiated from hA-MSCs. The cells obtained from three donators were submitted to osteoinduction for 16 days with a differentiation cocktail (dexamethasone, ascorbic acid, β-glycerophosphate) and characterized by the presence of osteocalcin, alkaline phosphate and mineralized matrix. The cells were treated with T3 (10-8M) for 72h and the RNA was extracted to mRNA library prepare and sequenced by Illumina platform. The bioinformatic analysis included the software: FastQC to quality control, Kallisto to alignment (Hg19), Deseq2 in R to differential gene expression (DE) analysis, enrichment analysis to Gene Ontology (GO) and REVIGO to graphs construction. The osteoblastic differentiation was confirmed by the presence of their markers as well as was identified the gene expression of other markers, such as: fibronectin 1 (FN1), osteonectin (SPARC) and collagen type 1 (COL1A1/2). The RNAs, mRNA library and sequencing reads quality were confirmed, providing the identification of 659 genes DE. The majority (55,5%) were upregulated by T3S condition with 30 GO terms identified. Between them, the T3S modulated several bone terms: bone development, cell morphogenesis, osteoblastic differentiation, negative regulation of cell proliferation and ossification; Mechanical pathway terms (cell-substrate adhesion, focal adhesion and response to fluid shear stress); Biochemical pathways (prostaglandins, calmodulins, TGF- β/BMPs); Circadian rhythm; And diverse terms related to molecular action. Also, the T3S increased the expression of the genes SMAD6/7 and NOG, exercising negative action on BMPs and SMADs pathways and promoting the regulation of TGF-β and MAPK pathways. The information obtained in this study confirm the T3 action in osteoblasts from hA-MSCs, demonstrating their modulatory role in several biologic levels. Furthermore, the biochemical pathways identified, are not related only to osteoblasts, but with bone osteoclastic reabsorption, highlighting to the complexity of T3 action in the bone metabolism. Finally, it was proposed a diagram of the signaling pathways regulated by T3S, which can be a guiding study to more specific investigations of this hormonal action in osteoblasts and in the bone tissue. (AU)

FAPESP's process: 15/26747-0 - Evaluation of thyroid hormone action upon noncoding RNA expression in osteoblastic cells derived of adipose tissue
Grantee:Bruna Moretto Rodrigues
Support Opportunities: Scholarships in Brazil - Master