title: Regulation of HBEGF by Micro-RNA for Survival of Developing Human Trophoblast Cells.
abstract: BACKGROUND
  The growth factor HBEGF is upregulated post-transcriptionally in the low O2 environment of the human placenta during the first 10 weeks of pregnancy. We have examined the possible roles of HBEGF turnover and micro-RNA (miRNA) in its regulation by O2 in human first trimester trophoblast.
  
  
  METHODS
  HTR-8/SVneo trophoblast cells were cultured at 2% or 20% O2. The cells were transfected with a dual luciferase reporter construct (psiCHECK-2) containing no insert (control), the HBEGF 3' untranslated region (3'UTR), or sub-regions of the 3'UTR, as well as with siRNA for DGCR8. RNA was extracted from trophoblast cells cultured at 2% O2 for 0-4 h for next-generation sequencing. HBEGF was quantified by ELISA. HBEGF, DGCR8, and β-actin were examined by western blotting.
  
  
  RESULTS
  Protein turnover studies, using 10 μg/ml cyclohexamide, 1 μg/ml lactocystin, or 100 μg/ml MG132, demonstrated faster HBEGF degradation at 20% O2 than 2% O2, mediated by the proteasome. However, proteasome inhibition failed to initiate HBEGF accumulation at 20% O2. Reporter assays, comparing to empty vector, demonstrated that the intact HBEGF 3' UTR inhibited expression (0.26), while fragments containing only its flanking regions increased reporter activity (3.15; 3.43). No differential expression of miRNAs was found in trophoblast cells cultured at 2% and 20% O2. Nevertheless, HBEGF upregulation at 2% O2 was blocked when the miRNA-processing protein DGCR8 was silenced, suggesting a role for miRNA.
  
  
  CONCLUSIONS
  Our findings suggest involvement of flanking regions of the 3'UTR in activating HBEGF protein synthesis in response to 2% O2, possibly through a miRNA-mediated mechanism.
authors.affiliation: Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan, United States of America.|Department of Obstetrics and Gynecology, Wayne State University School of Medicine, Detroit, Michigan, United States of America.
authors.forename: Chandni V
authors.initials: CV
authors.lastname: Jain
authors.affiliation: Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan, United States of America.|Department of Anatomy and Cell Biology, Wayne State University School of Medicine, Detroit, Michigan, United States of America.
authors.forename: Philip
authors.initials: P
authors.lastname: Jessmon
authors.affiliation: Department of Obstetrics and Gynecology, Wayne State University School of Medicine, Detroit, Michigan, United States of America.
authors.forename: Brian A
authors.initials: BA
authors.lastname: Kilburn
authors.affiliation: Department of Obstetrics and Gynecology, Wayne State University School of Medicine, Detroit, Michigan, United States of America.|Department of Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, Michigan, United States of America.
authors.forename: Meritxell
authors.initials: M
authors.lastname: Jodar
authors.affiliation: Department of Obstetrics and Gynecology, Wayne State University School of Medicine, Detroit, Michigan, United States of America.|Department of Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, Michigan, United States of America.
authors.forename: Edward
authors.initials: E
authors.lastname: Sendler
authors.affiliation: Department of Obstetrics and Gynecology, Wayne State University School of Medicine, Detroit, Michigan, United States of America.|Department of Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, Michigan, United States of America.
authors.forename: Stephen A
authors.initials: SA
authors.lastname: Krawetz
authors.affiliation: Department of Obstetrics and Gynecology, Wayne State University School of Medicine, Detroit, Michigan, United States of America.|Department of Anatomy and Cell Biology, Wayne State University School of Medicine, Detroit, Michigan, United States of America.|Program in Reproductive and Adult Endocrinology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, NIH, DHHS, Bethesda, MD, United States of America.
authors.forename: D Randall
authors.initials: DR
authors.lastname: Armant
mesh_terms: D000199:Actins / Q000378:metabolism; D002478:Cells, Cultured; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects; D066257:Heparin-binding EGF-like Growth Factor / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D035683:MicroRNAs; D010100:Oxygen / Q000494:pharmacology*; D011247:Pregnancy; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D017423:Sequence Analysis, RNA / Q000379:methods*; D014327:Trophoblasts / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism
chemical_list: D000199:Actins; C474043:DGCR8 protein, human; C587883:HBEGF protein, human; D066257:Heparin-binding EGF-like Growth Factor; D035683:MicroRNAs; D016601:RNA-Binding Proteins; D010100:Oxygen