title: Knockdown of β-Catenin through shRNA cause a reversal of EMT and metastatic phenotypes induced by HIF-1α.
abstract: OBJECTIVE
  Wnt/β-catenin signaling pathway regulates pattern formation during embryogenesis as well as tumor progression. Numbers of studies suggest that this signaling pathway may play an important role in Epithelial-Mesenchymal transition (EMT), however, there was no evidence that Wnt/β-catenin signaling pathway directly controlled the EMT occurrence. Our previous research has successfully proved that overexpression of hypoxia inducible factor-1α (HIF-1α) could induce EMT in LNCaP cells, but not in PC-3. Consistently, the expression of β-catenin protein increased in LNCaP/HIF-1α cells, but not in PC-3/HIF-1α. This study mainly aimed at exploring the essentiality and importance of Wnt/β-catenin signaling pathway in HIF-1α-induced EMT.
  
  
  METHODS
  Human prostate cancer cells (LNCaP) were stably transfected by recombinant plasmid pcDNA3.1(-)/HIF-1α. The positive clones were selected by G418 and confirmed through western blot analysis, reverse transcription-polymerase chain reaction (RT-PCR), and indirect immunofluoesence. Then LNCaP/HIF-1α was transiently transfected with β-catenin shRNA (shRNA1 and shRNA2) and negative shRNA (shRNA-scr). The epithelial markers, mesenchymal markers, and critical proteins in Wnt/β-catenin signaling pathway were separately detected by western blot analysis. Finally, the invasive potency of cells in different transfection group was examined by Matrgel transwell assay.
  
  
  RESULTS
  We successfully established prostate cancer cell line LNCaP/HIF-1α and LNCaP/HIF-1α/β-catenin(-). LNCaP/HIF-1α displayed high expression of mesenchymal markers and low expression of epithelial markers. However, compared with LNCaP/HIF-1α, the epithelial marker E-cadherin was increased in LNCaP/HIF-1α/β-catenin(-), whereas the expression of mesenchymal marker N-cadherin, vimentin, MMP-2 were significantly decreased. Inhibition of Wnt signal activity through β-catenin shRNA cause a reversal of EMT induced by HIF-1α in human prostate cancer.
  
  
  CONCLUSIONS
  Overexpression of HIF-1α stimulates the invasion potency of human prostate carcinoma cells through EMT pathway and Wnt/β-catenin signaling pathway played a vital role in this process. Wnt/β-catenin signaling pathway might be a necessary endogenous signal that directly controlled the EMT occurrence induced by HIF-1α.
authors.affiliation: Department of Urology, Beijing Anzhen Hospital Affiliated to Capital Medical University, China.
authors.forename: Jia-Hui
authors.initials: JH
authors.lastname: Zhao
authors.forename: Yong
authors.initials: Y
authors.lastname: Luo
authors.forename: Yong-Guang
authors.initials: YG
authors.lastname: Jiang
authors.forename: Da-Lin
authors.initials: DL
authors.lastname: He
authors.forename: Chun-Ting
authors.initials: CT
authors.lastname: Wu
mesh_terms: D045744:Cell Line, Tumor; D058750:Epithelial-Mesenchymal Transition; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000502:physiology*; D008297:Male; D009361:Neoplasm Invasiveness; D009362:Neoplasm Metastasis; D010641:Phenotype; D011471:Prostatic Neoplasms / Q000473:pathology*; D034741:RNA, Small Interfering / Q000235:genetics*; D015398:Signal Transduction / Q000502:physiology; D051153:Wnt Proteins / Q000502:physiology; D051176:beta Catenin / Q000037:antagonists & inhibitors / Q000502:physiology*
chemical_list: C495270:CTNNB1 protein, human; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D034741:RNA, Small Interfering; D051153:Wnt Proteins; D051176:beta Catenin