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Early growth response 1 transcription factor is essential for the pathogenic properties of human endometriotic epithelial cells
in ReproductionAlthough a non-malignant gynecological disorder, endometriosis displays some pathogenic features of malignancy, such as cell proliferation, migration, invasion and adaptation to hypoxia. Current treatments of endometriosis include pharmacotherapy and/or surgery, which are of limited efficacy and often associated with adverse side effects. Therefore, to develop more effective therapies to treat this disease, a broader understanding of the underlying molecular mechanisms that underpin endometriosis needs to be attained. Using immortalized human endometriotic epithelial and stromal cell lines, we demonstrate that the early growth response 1 (EGR1) transcription factor is essential for cell proliferation, migration and invasion, which represent some of the pathogenic properties of endometriotic cells. Genome-wide transcriptomics identified an EGR1-dependent transcriptome in human endometriotic epithelial cells that potentially encodes a diverse spectrum of proteins that are known to be involved in tissue pathologies. To underscore the utility of this transcriptomic data set, we demonstrate that carbonic anhydrase 9 (CA9), a homeostatic regulator of intracellular pH, is not only a molecular target of EGR1 but is also important for maintaining many of the cellular properties of human endometriotic epithelial cells that are also ascribed to EGR1. Considering therapeutic intervention strategies are actively being developed for EGR1 and CAIX in the treatment of other pathologies, we believe EGR1 and its transcriptome (which includes CA9) will offer not only a new conceptual framework to advance our understanding of endometriosis but will also furnish new molecular vulnerabilities to be leveraged as potential therapeutic options in the future treatment of endometriosis.
Supplementary Materials
- Supplementary Figure 1 The level of EGR1 transcript is significantly increased in human ovarian endometriomas. Expression data were obtained from the Gene Expression Omnibus datasets: GSE7305 and GSE25628 (Crispi et al., 2013, Hever et al., 2007). (A) The relative raw abundance of EGR1 transcripts in human ovarian endometrioma and matched control endometrium (A.U. denotes arbitrary units). Human ovarian endometrioma and matched control endometrium were biopsied during the proliferative (n=2) and secretory (n=8) phases of the menstrual cycle for the GSE7305 dataset. (B) Relative raw abundance of EGR1 transcripts in human ovarian endometrioma and matched control endometrium. Human eutopic, ectopic and control endometrium was obtained during the proliferative phase for the dataset obtained from GSE25628. Data are presented as mean ± SE (n= 6-10); *p-value<0.05, **p-value<0.01, ****p-value<0.0001.
- Supplementary Figure 2 Reduction in EGR1 levels causes S phase cell cycle arrest in iHEECs. Forty-eight hours following transfection with NT or EGR1 siRNAs, iHEECs were sorted by flow cytometry to assess their stage in the cell cycle. (A-B) Histograms show the percentage of cells in each phase of the cell cycle. Data are presented as mean ± SE (*p-value<0.05) and are representative of three independent experiments.
- Supplementary Figure 3 Knockdown of EGR1 expression does not alter EGR2 or EGR3 expression levels in the iHEEC line. (A-B) Transcript levels as measured by qPCR of EGR2 and EGR3 in iHEECs forty-eight hours following transfection with NT siRNAs or siRNAs targeting EGR1.
- Supplementary Figure 4 Human endometriotic stromal cells require EGR1 to maintain their pathogenic cellular properties. (A) Knockdown of EGR1 in iEc-ESCs results in a significantly decreased capacity to proliferate. (B) Colony formation ability is markedly attenuated following EGR1 knockdown in iEc-ESCs; histogram in (C) shows the quantitation of crystal violet staining as a measure of colony number and size. (D) The wound-healing assay demonstrates that the migration ability of iEc-ESCs is significantly decreased with EGR1 knockdown; histogram displays the percentage area migrated for iEc-ESCs transfected with either NT or EGR1 siRNAs. The scale bar shown in bottom left panel applies to all four panels in (G). Results are represented as mean ± SE and representative of three independent experiments; *p-value<0.05, and **p-value<0.01.
- Supplementary Folder 1 List of differential expressed genes (EGR1 siRNA versus NT siRNA groups). The first Excel sheet contains the log2 fold change (log2FC) between the two groups with the p-value, the false discovery rate corrected p-value (FDR) and the individual read counts by gene for each sample. Note the yellow highlighted row for EGR1 (log2FC: -2.8), confirming significant EGR1 knockdown in this group. The second Excel sheet lists the respective GO terms by DAVID analyses. The third Excel sheet displays the PCA result for the two treatment groups in this study.
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Online ISSN: 1741-7899
Print ISSN: 1470-1626
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