Análise funcional do silenciamento gênico de versicam em células trofoblásticas e sua implicação em doenças gestacionais

Abstract

Introduction: Versican is a proteoglycan responsible for several functions in different cells, being especially upregulated in invasive tumors. Versican V0 and V1 isoforms are produced only by invading trophoblast cells from the first trimester of pregnancy and from abnormally invasive placentas, although versican possible functions remains elusive. As such, the verification of versican roles in trophoblast cells is essential to future research on invasive diseases as the abnormally invasive placenta. Aim: To analyze cellular and functional effects of versican gene silencing in trophoblast cells. Methods: First trimester-derived HTR-8/SVneo cells were silenced for versican and its efficiency tested at both mRNA and protein levels. Cell death, proliferation, cell cycle phases, focal adhesion kinase (FAK) phosphorilation and different motility parameters, as well as Th1/Th2 cytokines production were analyzed. Lastly, Rho/ROCK signaling pathway phosphorylation was verified. Results: Versican silencing was efficient, as versican mRNA and protein levels were utterly reduced (p <0,001). Proliferation was slightly reduced, and in vitro wound closure time was greatly delayed (p < 0,001). Cell speed was analyzed through time-lapse microscopy and it was also reduced (p < 0,0001), as well as the invasion through fibronectin-coated transwell chambers (p< 0,05). The analysis of pFAK showed decreased phosphorylation after silencing. Several cytokines were also affected, with reduced production of IL-1β, IL-5, IL-6 and IL-10, while the increased production of IL-2 and IL-13 (p < 0,05). The analysis of Rho/ROCK pathway showed decreased phosphorylation after silencing, confirming its involvement in versican signaling. Conclusions: Versican proved to be an important key molecule upregulator of trophoblast motility and with relevant immune modulation properties in the placenta microenvironment. Therefore, it could be a pharmacological target in future therapies focusing on the modulation of trophoblast motility.