title: Comparative study of microRNA regulation on FOXL2 between adult-type and juvenile-type granulosa cell tumours in vitro.
abstract: OBJECTIVE
  Despite their distinct biology, granulosa cell tumours (GCTs) are treated similarly to other ovarian tumours. Predominantly expressed in granulosa cells, the transcription factor Forkhead Box L2 (FOXL2) is near absent in juvenile-type GCTs. This research aimed to investigate miRNAs as a mechanism of suppression of FOXL2 expression in juvenile-type GCTs.
  
  
  METHODS
  The miRNA abundance of two GCT cell lines COV434 and KGN was profiled using Affymetrix miRNA GeneChip arrays. Luciferase assays were used to confirm miRNA binding to the 3'UTR of FOXL2. Identified as promising candidates, the miR-17 miRNA family was targeted for knockdown with a miRNA sponge. Additionally, individual family members miR-17, miR-20b and miR-106a were knocked down using Anti-miR™ inhibitors. Subsequently, FOXL2 expression was analysed using RT-qPCR and Western blotting.
  
  
  RESULTS
  The profiling of COV434 and KGN cells revealed unique miRNA signatures, with COV434 expressing miR-17 family miRNAs whilst KGN expressed members of the let-7 miRNA gene family. Luciferase assays confirmed miRNA binding to FOXL2's 3'UTR. Reduction of miR-17 family miRNAs increased FOXL2 mRNA expression, however luciferase assays performed in combination with the sponge suggested this is an indirect effect. As no changes in protein were observed, we propose another miRNA is repressing the translation of FOXL2 mRNA.
  
  
  CONCLUSIONS
  Through miRNA profiling we have begun to unravel the profiles of GCTs, showing that juvenile and adult derived-cell lines are biologically distinct. By expanding on this discovery we may further elucidate the miRNA-mRNA pathways involved in GCT initiation and progression with potential for novel therapeutics for these cancers.
authors.affiliation: Department of Obstetrics and Gynaecology, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand. r.rosario@auckland.ac.nz
authors.forename: Roseanne
authors.initials: R
authors.lastname: Rosario
authors.forename: Cherie
authors.initials: C
authors.lastname: Blenkiron
authors.forename: Andrew Neil
authors.initials: AN
authors.lastname: Shelling
mesh_terms: D020413:3' Untranslated Regions / Q000502:physiology; D001483:Base Sequence; D045744:Cell Line, Tumor; D005260:Female; D000074842:Forkhead Box Protein L2; D051858:Forkhead Transcription Factors / Q000235:genetics*; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D006106:Granulosa Cell Tumor / Q000209:etiology / Q000235:genetics*; D006801:Humans; D035683:MicroRNAs / Q000502:physiology*; D008969:Molecular Sequence Data; D010051:Ovarian Neoplasms / Q000235:genetics*
chemical_list: D020413:3' Untranslated Regions; C421604:FOXL2 protein, human; D000074842:Forkhead Box Protein L2; D051858:Forkhead Transcription Factors; D035683:MicroRNAs