title: miR-200 expression regulates epithelial-to-mesenchymal transition in bladder cancer cells and reverses resistance to epidermal growth factor receptor therapy.
abstract: OBJECTIVE
  The epithelial-to-mesenchymal transition (EMT) is a cell development-regulated process in which noncoding RNAs act as crucial modulators. Recent studies have implied that EMT may contribute to resistance to epidermal growth factor receptor (EGFR)-directed therapy. The aims of this study were to determine the potential role of microRNAs (miRNA) in controlling EMT and the role of EMT in inducing the sensitivity of human bladder cancer cells to the inhibitory effects of the anti-EGFR therapy.
  
  
  METHODS
  miRNA array screening and real-time reverse transcription-PCR were used to identify and validate the differential expression of miRNAs involved in EMT in nine bladder cancer cell lines. A list of potential miR-200 direct targets was identified through the TargetScan database. The precursor of miR-200b and miR-200c was expressed in UMUC3 and T24 cells using a retrovirus or a lentivirus construct, respectively. Protein expression and signaling pathway modulation, as well as intracellular distribution of EGFR and ERRFI-1, were validated through Western blot analysis and confocal microscopy, whereas ERRFI-1 direct target of miR-200 members was validated by using the wild-type and mutant 3'-untranslated region/ERRFI-1/luciferse reporters.
  
  
  RESULTS
  We identified a tight association between the expression of miRNAs of the miR-200 family, epithelial phenotype, and sensitivity to EGFR inhibitors-induced growth inhibition in bladder carcinoma cell lines. Stable expression of miR-200 in mesenchymal UMUC3 cells increased E-cadherin levels, decreased expression of ZEB1, ZEB2, ERRFI-1, and cell migration, and increased sensitivity to EGFR-blocking agents. The changes in EGFR sensitivity by silencing or forced expression of ERRFI-1 or by miR-200 expression have also been validated in additional cell lines, UMUC5 and T24. Finally, luciferase assays using 3'-untranslated region/ERRFI-1/luciferase and miR-200 cotransfections showed that the direct down-regulation of ERRFI-1 was miR-200-dependent because mutations in the two putative miR-200-binding sites have rescued the inhibitory effect.
  
  
  CONCLUSIONS
  Members of the miR-200 family appear to control the EMT process and sensitivity to EGFR therapy in bladder cancer cells and the expression of miR-200 is sufficient to restore EGFR dependency at least in some of the mesenchymal bladder cancer cells. The targets of miR-200 include ERRFI-1, which is a novel regulator of EGFR-independent growth.
authors: The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA. ladam@mdanderson.org
authors: Liana
authors: L
authors: Adam
authors: Meng
authors: M
authors: Zhong
authors: Woonyoung
authors: W
authors: Choi
authors: Wei
authors: W
authors: Qi
authors: Milena
authors: M
authors: Nicoloso
authors: Ameeta
authors: A
authors: Arora
authors: George
authors: G
authors: Calin
authors: Hua
authors: H
authors: Wang
authors: Arlene
authors: A
authors: Siefker-Radtke
authors: David
authors: D
authors: McConkey
authors: Menashe
authors: M
authors: Bar-Eli
authors: Colin
authors: C
authors: Dinney
mesh_terms: D000970:Antineoplastic Agents / Q000627:therapeutic use*; D002277:Carcinoma / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D054337:Cell Dedifferentiation / Q000235:genetics; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D004848:Epithelium / Q000378:metabolism / Q000502:physiology*; D066246:ErbB Receptors / Q000037:antagonists & inhibitors*; D015870:Gene Expression / Q000502:physiology; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000378:metabolism / Q000502:physiology*; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D020411:Oligonucleotide Array Sequence Analysis; D014407:Tumor Cells, Cultured; D001749:Urinary Bladder Neoplasms / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology
chemical_list: D000970:Antineoplastic Agents; C533896:MIRN200 microRNA, human; D035683:MicroRNAs; D066246:ErbB Receptors