title: ADAM12-L is a direct target of the miR-29 and miR-200 families in breast cancer.
abstract: BACKGROUND
  ADAM12-L and ADAM12-S represent two major splice variants of human metalloproteinase-disintegrin 12 mRNA, which differ in their 3'-untranslated regions (3'UTRs). ADAM12-L, but not ADAM12-S, has prognostic and chemopredictive values in breast cancer. Expression levels of the two ADAM12 splice variants in clinical samples are highly discordant, suggesting post-transcriptional regulation of the ADAM12 gene. The miR-29, miR-30, and miR-200 families have potential target sites in the ADAM12-L 3'UTR and they may negatively regulate ADAM12-L expression.
  
  
  METHODS
  miR-29b/c, miR-30b/d, miR-200b/c, or control miRNA mimics were transfected into SUM159PT, BT549, SUM1315MO2, or Hs578T breast cancer cells. ADAM12-L and ADAM12-S mRNA levels were measured by qRT-PCR, and ADAM12-L protein was detected by Western blotting. Direct targeting of the ADAM12-L 3'UTR by miRNAs was tested using an ADAM12-L 3'UTR luciferase reporter. The rate of ADAM12-L translation was evaluated by metabolic labeling of cells with (35)S cysteine/methionine. The roles of endogenous miR-29b and miR-200c were tested by transfecting cells with miRNA hairpin inhibitors.
  
  
  RESULTS
  Transfection of miR-29b/c mimics strongly decreased ADAM12-L mRNA levels in SUM159PT and BT549 cells, whereas ADAM12-S levels were not changed. ADAM12-L, but not ADAM12-S, levels were also significantly diminished by miR-200b/c in SUM1315MO2 cells. In Hs578T cells, miR-200b/c mimics impeded translation of ADAM12-L mRNA. Importantly, both miR-29b/c and miR-200b/c strongly decreased steady state levels of ADAM12-L protein in all breast cancer cell lines tested. miR-29b/c and miR-200b/c also significantly decreased the activity of an ADAM12-L 3'UTR reporter, and this effect was abolished when miR-29b/c and miR-200b/c target sequences were mutated. In contrast, miR-30b/d did not elicit consistent and significant effects on ADAM12-L expression. Analysis of a publicly available gene expression dataset for 100 breast tumors revealed a statistically significant negative correlation between ADAM12-L and both miR-29b and miR-200c. Inhibition of endogenous miR-29b and miR-200c in SUM149PT and SUM102PT cells led to increased ADAM12-L expression.
  
  
  CONCLUSIONS
  The ADAM12-L 3'UTR is a direct target of miR-29 and miR-200 family members. Since the miR-29 and miR-200 families play important roles in breast cancer progression, these results may help explain the different prognostic and chemopredictive values of ADAM12-L and ADAM12-S in breast cancer.
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Kansas State University, 141 Chalmers Hall, Manhattan, KS, 66506, USA. sduhach1@ksu.edu.
authors.forename: Sara
authors.initials: S
authors.lastname: Duhachek-Muggy
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Kansas State University, 141 Chalmers Hall, Manhattan, KS, 66506, USA. zolkiea@ksu.edu.
authors.forename: Anna
authors.initials: A
authors.lastname: Zolkiewska
mesh_terms: D020413:3' Untranslated Regions; D051722:ADAM Proteins / Q000737:chemistry / Q000235:genetics*; D000072199:ADAM12 Protein; D017398:Alternative Splicing; D001483:Base Sequence; D001665:Binding Sites; D001943:Breast Neoplasms / Q000235:genetics*; D045744:Cell Line, Tumor; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics*; D035683:MicroRNAs / Q000737:chemistry / Q000235:genetics*; D034622:RNA Interference; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics*
chemical_list: D020413:3' Untranslated Regions; C533896:MIRN200 microRNA, human; C532045:MIRN29a microRNA, human; D008565:Membrane Proteins; D035683:MicroRNAs; D012333:RNA, Messenger; D051722:ADAM Proteins; D000072199:ADAM12 Protein; C000606480:ADAM12 protein, human