Hongbing Chen1, Tianjie Yang1, Zhe Lei1, Longqiang Wang1, Haiping Yang1, Xin Tong1, Wen-Tao Yang2, Jun Zhao3, Yunbin Gu4, Yongbing Chen5, Hong-Tao Zhang6. 1. Soochow University Laboratory of Cancer Molecular Genetics, Medical College of Soochow University, Suzhou 215123, China. 2. Department of Thoracic and Cardiovascular Surgery, The Second Affiliated Hospital of Soochow University, Medical College of Soochow University, Suzhou 215004, China. 3. Department of Thoracic and Cardiovascular Surgery, The First Affiliated Hospital of Soochow University, Medical College of Soochow University, Suzhou 215006, China. 4. Department of Radiology, The First Affiliated Hospital of Soochow University, Medical College of Soochow University, Suzhou 215006, China. 5. 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. 6. Soochow University Laboratory of Cancer Molecular Genetics, Medical College of Soochow University, Suzhou 215123, China. Electronic address: htzhang@suda.edu.cn.
Abstract
OBJECTIVES: 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. MATERIALS AND 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.
OBJECTIVES: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 humancancers 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. MATERIALS AND 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 humanNSCLC 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.