Primary human bronchial epithelial cells expressing EGFP or DN-GRHL2
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Xia Gao
PROVIDER: E-GEOD-44809 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:The airways of the human lung are lined by an epithelium made up of ciliated and secretory luminal cells and undifferentiated p63+ Krt5+ basal cells. The integrity of this epithelium and its ability to act as a selective barrier are critical for normal lung function. In other epithelia there is evidence that transcription factors of the evolutionarily conserved grainyheadlike (GRHL) family play key roles in co-ordinating the expression of numerous proteins required for epithelial morphogenesis, differentiation, remodeling and repair. However, little is known about their function in the adult lung. We use Affymetrix microarray analysis to compare transcripts in lentivirus transfected primary human bronchial epithelial (HBE) cells expressing either EGFP or DN-GRHL2 for 48h when the transepithelial electrical resistance (TER) reached a threshold level. The goal is to identify direct target genes of GRHL2 and early events in the uncoupling of junctional interactions, including those regulating transepithelial resistance. Primary HBE cells from three donors were infected with EGFP or DN-GRHL2 expression lentivirus. Dox was added for 48h to induce the expression of either EGFP or DN-GRHL2 when TER reached a threshold level in ALI culture.
2013-05-08 | E-GEOD-44808 | biostudies-arrayexpress
Project description:The airways of the human lung are lined by an epithelium made up of ciliated and secretory luminal cells and undifferentiated p63+ Krt5+ progenitors. The integrity of this epithelium and its ability to act as a selective barrier are critical for normal lung function. In other epithelia there is evidence that transcription factors of the evolutionarily conserved grainyheadlike (GRHL) family play key roles in co-ordinating the expression of numerous proteins required for epithelial morphogenesis, differentiation, remodeling and repair. However, little is known about their function in the adult lung. We used Affymetrix microarray analysis to compare transcripts in lentivirus transfected primary human bronchial epithelial (HBE) cells expressing either EGFP or DN-GRHL2 to help identify GRHL2 target genes and their functions in HBE cells. Primary HBE cells from three donors were infected with modified TripZ lentivirus designed to express EGFP or DN-GRHL2 in response to Dox. Dox was added to cells cultured at the air-liquid interface from day 7 to 14.
2013-05-08 | E-GEOD-44807 | biostudies-arrayexpress
Project description:The airways of the human lung are lined by an epithelium made up of ciliated and secretory luminal cells and undifferentiated p63+ Krt5+ basal cells. The integrity of this epithelium and its ability to act as a selective barrier are critical for normal lung function. In other epithelia there is evidence that transcription factors of the evolutionarily conserved grainyheadlike (GRHL) family play key roles in co-ordinating the expression of numerous proteins required for epithelial morphogenesis, differentiation, remodeling and repair. However, little is known about their function in the adult lung. Here, we focus on the role of GRHL2 in primary human bronchial epithelial (HBE) cells, using either shRNA or a dominant negative protein (DN-GRHL2) to inhibit its function. We follow changes in epithelial phenotype, and in gene transcription using RNA-seq or microarray analysis, both in undifferentiated basal cells and in cells differentiating in air-liquid interface culture into a mucociliary epithelium with transepithelial electrical resistance. We identify several hundreds of genes that are directly or indirectly regulated by GRHL2. Using ChIP-seq to map sites of GRHL2 binding in the basal cells we identify 7,687 potential primary targets, and confirm that GRHL2 binding is strongly enriched near GRHL-regulated genes. Different subsets of the large cohort of potential GRHL2 targets appear to be active in basal and differentiated cells. Taken together, the results strongly support the hypothesis that GRHL2 plays a key role in regulating many physiological functions of human airway epithelium, including those involving cell adhesion, polarity and morphogenesis. Frozen primary human bronchial epithelial (HBE) cells were obtained from three donors. Passage 2 cells at 40% confluence were infected with H2B-GFP or DN-GRHL2 lentivirus and 1 mg/ml puromycin added 48 h later. At confluence, Doxycycline 0.5 mg/ml was added for 24 h. RNA-seq was performed on all six samples, as well as samples from two donors that were not infected.
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Project description:Epithelial ovarian cancer (EOC) is clinically heterogeneous, comprising different histological and biological subtypes. Multiple studies have implicated epithelial-mesenchymal transition (EMT), a biological process by which polarized epithelial cells convert into a mesenchymal phenotype, to contribute significantly to this molecular heterogeneity of EOC. From gene expression analyses of a collection of EMT-characterized EOC cell lines, we found that the expression of the transcription factor Grainyhead-like 2 (GRHL2) correlates with E-cadherin expression and the epithelial phenotype. EOC tumors with lower levels of GRHL2 are associated with the Mes (mesenchymal) molecular subtype and show poorer overall survival in patients. Here, we demonstrate that shRNA-mediated knockdown of GRHL2 in EOC cells with an epithelial phenotype resulted in EMT changes, with increased cell migration, invasion and motility. By ChIP-sequencing and gene expression microarray, we identified a variety of target genes regulated by GRHL2, including protein-coding and non-coding genes. Our data suggest that GRHL2 maintains the epithelial phenotype of EOC cells through the regulatory networks of miR-200b/a, ZEB1 and E-cadherin. These findings support GRHL2 as a crucial player in the molecular heterogeneity of EOC. 7 samples were analyzed (shNon control in duplicates; shGRHL2 #10 in duplicates, shGRHL2 #12 in triplicates)
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