O papel do Versicam na regulação da transição epitélio-mesenquimal e motilidade de células trofoblásticas

Abstract

Introduction: Versican is a proteoglycan known for activating intracellular signaling pathways involved in inflammation, proliferation, migration, invasion, adhesion, cytoskeleton organization, and epithelial-mesenchymal transition (EMT). EMT is characterized by morphological and functional differentiation, occurring in various physiological and pathological situations, extensively studied for its fundamental role in neoplastic metastases and essential for trophoblastic cells to invade maternal tissues and establish the maternal-fetal interface in the first trimester. Objective: To investigate whether gene silencing of versican influences epithelial-mesenchymal transition processes and trophoblastic cell motility in vitro. Methods: HTR-8/SVneo cells were silenced for versican, and efficiency was tested at mRNA and protein levels. Western Blot, flow cytometry and immunofluorescence were performed for AKT, mTOR, IRF3, and IRF9 after 24 hours of versican silencing. Cell invasion was examined using 3D invasion assays. Cell directionality was evaluated through timelapse microscopy. Atomic Force Microscopy was conducted for cellular topography and membrane elasticity. Different EMT markers were assessed by RT-PCR and immunofluorescence. Results: Gene silencing of versican was efficient in HTR8/SVneo cells, and signaling pathway analyses showed no differences in p-AKT Thr308 and p-mTOR expression, although p-AKT Ser473, p-IRF3, and IRF9 increased after 24 hours of silencing (p<0.05). Silenced cells exhibited unusual migration directional patterns, increased pFAK expression and had reduced invasive capacity in co-culture invasion assays (p<0.05). Silenced cells showed a lower amount of F-actin stress fibers, and their Young's Modulus was higher (p<0.01), indicating loss of elasticity, as well as reduced height. Additionally, different markers of TEM, such as TGF-β2, E-cadherin, β-catenin, Snail, and vimentin, were altered by versican gene silencing. Conclusion: Versican silencing significantly influences HTR-8/SVneo cells at molecular and functional levels, primarily affecting their motility and EMT profile.