Thyroid cancer incidence more than tripled in last four decades. Among the different thyroid cancer histologic types, the differentiated thyroid cancer (DTC) represents 90% of cases, being classified as: papillary thyroid cancer (PTC) and follicular thyroid cancer (FTC). Although the DTC has an excellent prognostic, about 30% of cases show metastasis in regional lymph nodes and 2-13% of patients present distant metastasis, which reduced the survival rate. Thus, to recognize metastatic-associated molecular markers can help in identification of prognostic markers. In this sense, our laboratory identified the LIMD2 gene expression in metastatic lymph nodes, but not in normal lymph nodes, neither in primary tumor, suggesting the participation of this gene in metastasis. Thus, this study has as objective to analyze the LIMD2 gene participation on epithelial-mesenchymal transition (EMT), process in which epithelial cells undergo reversible genetic, metabolic and morphological alterations, leading to the acquisition of mesenchymal phenotype, which confers invasive and migratory capability. BCPAP, carrying the BRAF V600E mutation and TPC1 cell lines, carrying the RET/PTC1 fusion will be employed in this study. The LIMD2 expression in these cell lines was previous verified. Nthy-ori 3-1 cell line (normal thyroid) will be used as control. The LIMD2 gene knock-out in BCPAP and TPC1 cell lines will perform using the CRISPR/Cas9 tool. The EMT evaluation will be performed by metabolic analysis, including the instigation of mitochondrial membrane potential and reactive oxygen species productions by immunofluorescence (IF) and flow cytometry (FC) using commercial probes. The levels of clastogenicity will be evaluated trough the comet assay. The stem-cell phenotype acquisition will be analyzed by tumor-sphere test, as well as the ALDH1 and Oct-3/4 expression levels. The expression levels of different EMT markers, including the E- to N-cadherin genetic switch, STAT3, SLUG, TWIST (transcription factors) and vimentin (mesenchymal marker) will be analyzed by IF, FC and Western blot. Cell morphology will be analyzed by phase contrast, electron transmission and scanning microscopy. The migratory phenotype acquisition will be determinate by video time-lapse microscopy. Wild-type and genetically edited cell lines will inject in nude mice to confirm in vivo the LIMD2 gene participation in EMT. Neoplastic growth will be monitored using pachymeter and PET-SCAN (metastasis investigation) for 4-12 weeks. The mice will be euthanized and the primary tumors, as well as eventual metastasis will remove surgically. The material will embed in paraffin, being destined to: (1) histopathological analysis, (2) evaluation of collagen matrix and (3) immunohistochemistry, being analyzed the expression of EMT markers.
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