title: In silico, in vitro and in vivo analysis identifies a potential role for steroid hormone regulation of FOXD3 in endometriosis-associated genes.
abstract: OBJECTIVE
  Can bioinformatics analysis of publically available microarray datasets be utilized in identifying potentially important transcription factors (TF) in the hormonal regulation of the endometrium?
  
  
  CONCLUSIONS
  Systems integration and analysis of existing complex (published) datasets, predicted a role for the novel transcription factor, Forkhead Box D3 (FOXD3) in healthy endometrium and in endometriosis, which was followed by the demonstration of decreased levels of the protein upon decidualisation of normal human endometrial stromal cells in vitro and differential endometrial expression in the stroma in endometriosis.
  
  
  BACKGROUND
  The reported endometriosis-associated endometrial aberrations are most pronounced in the progesterone-dominant secretory phase and progesterone resistance is a proposed causative factor.
  
  
  METHODS
  The study was initially an 'in silico' study, with confirmatory 'wet lab' data from western blotting (WB), qPCR and Immunohistochemistry (IHC) on endometrial biopsies obtained from 142 women undergoing gynaecological surgery.
  
  
  METHODS
  The study was conducted at a University Research Institute. Bioinformatic analysis of selected published microarray datasets identified differentially regulated genes for the early and mid-secretory phases relative to the proliferative phase. Diseases and Functions categories were identified with Ingenuity (IPA) 'core analysis' software. The key transcription factors controlling secretory phase gene changes were revealed with oPOSSUM software. FOXD3 expression levels were examined in human endometrial samples from women aged 18-55 years by WB, IHC, and qPCR. The progesterone regulation of endometrial FOXD3 levels was examined in vivo and in cultured primary human endometrial stromal cells in vitro.
  
  
  RESULTS
  Initial data mining and subsequent bioinformatics analysis of human endometrial microarray datasets identified FOXD3 to be a key regulator of gene expression specific to secretory phase/endometriosis. FOXD3 was dynamically expressed in healthy endometrium and differentially expressed in endometriosis. In vitro decidualisation of primary endometrial stromal cells significantly decreased FOXD3 protein (P = 0.0005) and progestagen (Levonorgestrel) treatment also reduced the high endometrial FOXD3 protein (P = 0.0001) and mRNA levels (P = 0.04) seen in untreated women with endometriosis, with a shift of FOXD3 from the nucleus to the cytoplasm.
  
  
  CONCLUSIONS
  The quality of Bioinformatics analysis and results depends on the published micro-array data.
  
  
  CONCLUSIONS
  An in depth analysis of FOXD3 function and its relationship with estrogen and progesterone might provide insights into its potential deregulation in proliferative disorders of the endometrium including endometrial cancer where its expression is also deregulated. Further, FOX transcription factors are increasingly seen as novel therapeutic targets in disease.
  
  
  BACKGROUND
  We acknowledge the support by Wellbeing of Women project grant RG1073 (D.K.H., A.J.V.). We also acknowledge the support of Liverpool Women's Hospital Foundation Trust (J.A.D.), Institute of Translational Medicine (D.M., A.J.V., D.K.H.) and the Institute of Integrative Biology (O.V.), University of Liverpool. All authors declare no conflict of interest.
  
  
  BACKGROUND
  N/A.
authors.affiliation: Department of Women's and Children's Health, University of Liverpool, Liverpool L8 7SS, UK.
authors.forename: D
authors.initials: D
authors.lastname: Mathew
authors.affiliation: Department of Women's and Children's Health, University of Liverpool, Liverpool L8 7SS, UK.
authors.forename: J A
authors.initials: JA
authors.lastname: Drury
authors.affiliation: Department of Women's and Children's Health, University of Liverpool, Liverpool L8 7SS, UK.
authors.forename: A J
authors.initials: AJ
authors.lastname: Valentijn
authors.affiliation: Institute of Integrative Biology, University of Liverpool, Liverpool L69 7ZB, UK.
authors.forename: O
authors.initials: O
authors.lastname: Vasieva
authors.affiliation: Department of Women's and Children's Health, University of Liverpool, Liverpool L8 7SS, UK dharani.hapangama@liv.ac.uk.
authors.forename: D K
authors.initials: DK
authors.lastname: Hapangama
mesh_terms: D000293:Adolescent; D000328:Adult; D001706:Biopsy; D015153:Blotting, Western; D019295:Computational Biology; D003198:Computer Simulation; D010064:Embryo Implantation; D004715:Endometriosis / Q000235:genetics*; D004717:Endometrium / Q000002:abnormalities; D004847:Epithelial Cells / Q000378:metabolism; D005260:Female; D051858:Forkhead Transcription Factors / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D007150:Immunohistochemistry; D016912:Levonorgestrel / Q000494:pharmacology; D008875:Middle Aged; D020411:Oligonucleotide Array Sequence Analysis; D011374:Progesterone / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D017154:Stromal Cells / Q000378:metabolism; D014591:Uterine Diseases; D055815:Young Adult
chemical_list: C503514:FOXD3 protein, human; D051858:Forkhead Transcription Factors; D012333:RNA, Messenger; D011374:Progesterone; D016912:Levonorgestrel
keywords: FOXD3; FOXO1A; Mirena (LNG-IUS); computational biology; decidualisation; endometriosis; endometrium; progesterone