title: RNF111/Arkadia is regulated by DNA methylation and affects TGF-β/Smad signaling associated invasion in NSCLC cells.
abstract: OBJECTIVE
  RNF111/Arkadia is a critical regulator of TGF-β signaling, being required for SMAD3-mediated responses such as TGF-β-induced repression of E-cadherin. Previous studies show that mutations in RNF111 in human cancers are rare and RNF111 promotes lung tumor metastasis. However, the epigenetic mechanisms underlying the role of RNF111 in non-small cell lung cancer (NSCLC) metastasis remain unknown. Here, we mainly focused on low- (95C) and high-metastatic (95D) NSCLC cell lines, which share a similar genetic background, and investigated the methylation-based regulation of RNF111 expression.
  
  
  METHODS
  Clonal bisulfite sequencing, real-time qRT-PCR, western blot analysis, luciferase reporter assays, RNA interference, chromatin immunoprecipitation (ChIP) assay and transwell migration and invasion assays were performed on human NSCLC cell lines 95C and 95D.
  
  
  RESULTS
  RNF111 was high-expressed in 95D cells, which showed low-level methylation at -459CpG site in RNF111 promoter. The opposite results were obtained in 95C cells. Cell-based and biochemical assays revealed that -459CpG methylation can inhibit RNF111 transcriptional expression by interfering with the recruitment of Sp1 to RNF111 promoter. On TGF-β stimulation, siRNA-mediated RNF111 knockdown inhibited TGF-β/Smad signaling activity and Snail (an inducer of metastasis) expression, and enhanced E-cadherin (an epithelial-to-mesenchymal transition marker) expression in 95C and 95D cells. Furthermore, demethylation-induced upregulation of RNF111 enhanced phosphorylation of SMAD3 and Snail expression, and repressed E-cadherin expression in 95C cells expressing low RNF111.
  
  
  CONCLUSIONS
  Our results suggest that -459CpG methylation in Sp1-binding site of RNF111 promoter transcriptionally decreases RNF111 expression, which inhibits TGF-β/Smad signaling associated invasion in NSCLC cells.
authors.affiliation: Soochow University Laboratory of Cancer Molecular Genetics, Medical College of Soochow University, Suzhou 215123, China.
authors.forename: Hongbing
authors.initials: H
authors.lastname: Chen
authors.affiliation: Soochow University Laboratory of Cancer Molecular Genetics, Medical College of Soochow University, Suzhou 215123, China.
authors.forename: Tianjie
authors.initials: T
authors.lastname: Yang
authors.affiliation: Soochow University Laboratory of Cancer Molecular Genetics, Medical College of Soochow University, Suzhou 215123, China.
authors.forename: Zhe
authors.initials: Z
authors.lastname: Lei
authors.affiliation: Soochow University Laboratory of Cancer Molecular Genetics, Medical College of Soochow University, Suzhou 215123, China.
authors.forename: Longqiang
authors.initials: L
authors.lastname: Wang
authors.affiliation: Soochow University Laboratory of Cancer Molecular Genetics, Medical College of Soochow University, Suzhou 215123, China.
authors.forename: Haiping
authors.initials: H
authors.lastname: Yang
authors.affiliation: Soochow University Laboratory of Cancer Molecular Genetics, Medical College of Soochow University, Suzhou 215123, China.
authors.forename: Xin
authors.initials: X
authors.lastname: Tong
authors.affiliation: Department of Thoracic and Cardiovascular Surgery, The Second Affiliated Hospital of Soochow University, Medical College of Soochow University, Suzhou 215004, China.
authors.forename: Wen-Tao
authors.initials: WT
authors.lastname: Yang
authors.affiliation: Department of Thoracic and Cardiovascular Surgery, The First Affiliated Hospital of Soochow University, Medical College of Soochow University, Suzhou 215006, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Zhao
authors.affiliation: Department of Radiology, The First Affiliated Hospital of Soochow University, Medical College of Soochow University, Suzhou 215006, China.
authors.forename: Yunbin
authors.initials: Y
authors.lastname: Gu
authors.affiliation: Department of Thoracic and Cardiovascular Surgery, The Second Affiliated Hospital of Soochow University, Medical College of Soochow University, Suzhou 215004, China. Electronic address: chentongt@sina.com.
authors.forename: Yongbing
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Soochow University Laboratory of Cancer Molecular Genetics, Medical College of Soochow University, Suzhou 215123, China. Electronic address: htzhang@suda.edu.cn.
authors.forename: Hong-Tao
authors.initials: HT
authors.lastname: Zhang
mesh_terms: D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D019175:DNA Methylation; D058750:Epithelial-Mesenchymal Transition; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D009154:Mutation; D009361:Neoplasm Invasiveness; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051900:Smad3 Protein / Q000235:genetics / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation
chemical_list: D009687:Nuclear Proteins; D012333:RNA, Messenger; C497790:SMAD3 protein, human; D051900:Smad3 Protein; D016212:Transforming Growth Factor beta; C486067:RNF111 protein, human; D044767:Ubiquitin-Protein Ligases
keywords: -459CpG methylation; Invasion; NSCLC; RNF111; TGF-β/Smad signaling