Abstract
During implantation, cytotrophoblasts undergo epithelial-to-mesenchymal transition (EMT) as they differentiate into invasive extravillous trophoblasts (EVTs). The primate-specific microRNA cluster on chromosome 19 (C19MC) is exclusively expressed in the placenta, embryonic stem cells and certain cancers however, its role in EMT gene regulation is unknown. In situ hybridization for miR-517a/c, a C19MC cistron microRNA, in first trimester human placentas displayed strong expression in villous trophoblasts and a gradual decrease from proximal to distal cell columns as cytotrophoblasts differentiate into invasive EVTs. To investigate the role of C19MC in the regulation of EMT genes, we employed the CRISPR/dCas9 Synergistic Activation Mediator (SAM) system, which induced robust transcriptional activation of the entire C19MC cistron and resulted in suppression of EMT associated genes. Exposure of human iPSCs to hypoxia or differentiation of iPSCs into either cytotrophoblast-stem-like cells or EVT-like cells under hypoxia reduced C19MC expression and increased EMT genes. Furthermore, transcriptional activation of the C19MC cistron induced the expression of OCT4 and FGF4 and accelerated cellular reprogramming. This study establishes the CRISPR/dCas9 SAM as a powerful tool that enables activation of the entire C19MC cistron and uncovers its novel role in suppressing EMT genes critical for maintaining the epithelial cytotrophoblasts stem cell phenotype.
Highlights
During implantation, cytotrophoblasts undergo epithelial-to-mesenchymal transition (EMT) as they differentiate into invasive extravillous trophoblasts (EVTs)
IPSC-derived CTs, or induced pluripotent stem cells (iPSCs)-derived CTs cultured in normoxia or hypoxia showed a significant increase in the EMT genes CDH2 and SERPINE1 and loss of miR-518c and miR-519d (C19MC miRNAs) compared to undifferentiated iPSCs (Fig. 4h,i). These findings indicate that EMT induced either by hypoxia or by BMP4 used for iPSC-to-CT differentiation is associated with a significant decrease in expression of C19MC
The current study demonstrates that C19MC is primarily expressed in the non-invasive villous trophoblasts and is lost as trophoblasts differentiate into EVTs
Summary
Cytotrophoblasts undergo epithelial-to-mesenchymal transition (EMT) as they differentiate into invasive extravillous trophoblasts (EVTs). The primate-specific microRNA cluster on chromosome 19 (C19MC) is exclusively expressed in the placenta, embryonic stem cells and certain cancers its role in EMT gene regulation is unknown. In situ hybridization for miR517a/c, a C19MC cistron microRNA, in first trimester human placentas displayed strong expression in villous trophoblasts and a gradual decrease from proximal to distal cell columns as cytotrophoblasts differentiate into invasive EVTs. To investigate the role of C19MC in the regulation of EMT genes, we employed the CRISPR/dCas[9] Synergistic Activation Mediator (SAM) system, which induced robust transcriptional activation of the entire C19MC cistron and resulted in suppression of EMT associated genes. EVT differentiation involves extensive changes during which cells lose both their intercellular junctions and apical-basal polarity, while acquire the www.nature.com/scientificreports capacity for migration and invasion These changes represent a prototypical example of the trans-differentiation process known as epithelial-to-mesenchymal transition (EMT)[7,8]. We sought to investigate the role of the entire C19MC on EMT, which is critical for trophoblast cell differentiation and invasion and somatic cell reprogramming
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