Impact of TAZ (WWTR1) depletion on gene expression during primary first-trimester extravillous trophoblast differentiation
Human placental cytotrophoblasts undergo differentiation into extravillous trophoblasts (EVTs) that anchor the placenta to the uterine wall and establish blood flow to the developing fetus. The transcriptional co-activator TAZ (WWTR1) is one of the main effectors of the Hippo signaling pathway, which regulates organ size by controlling important cellular processes such as cell proliferation, apoptosis, stem cell self-renewal and differentiation. Additionally, TAZ is involved in mechano-sensing and cell contact inhibition. In order to gain deeper insights into the function of TAZ in the EVT differentiation process, siRNA (small interfering RNA) treatment was employed to reduce TAZ levels in cytotrophoblasts, which were collected from placentae between 8th and 10th weeks of pregnancy, and underwent EVT differentiation in vitro. The objective of the study was to elucidate the influence of TAZ on the differentiation and function of EVTs by comparing the gene expression profiles of TAZ gene-silenced cells to those with normal TAZ levels. Overall design: Cytotrophoblasts were isolated from first trimester placental tissue (8th-10th weeks of gestation). Differentiated, HLA-G-positive extravillous trophoblasts were removed by HLA-G PE magnetic bead sorting (MACS Miltenyi Biotec). The remaining HLA-G- negative trophoblasts were plated onto fibronectin-coated cell culture dishes and transfected with non-targeting (ntc) or TAZ siRNAs one hour after seeding. RNA was isolated from the differentiated TAZ siRNA- and ntc-treated cells after 72 hours of cultivation, at the time when the majority of cells developed into EVTs. Subsequently, gene expression analysis was conducted using Next Generation Sequencing, encompassing six samples, three replicates each, derived from three independent cell preparations.