Haiping Yang1, Longqiang Wang1, Jun Zhao2, Yongbing Chen3, Zhe Lei1, Xia Liu1, Wei Xia4, Lingling Guo4, Hong-Tao Zhang5. 1. Soochow University Laboratory of Cancer Molecular Genetics, Medical College of Soochow University, Suzhou 215123, China; Suzhou Key Laboratory for Molecular Cancer Genetics, Suzhou 215123, China. 2. Department of Thoracic and Cardiovascular Surgery, The First Affiliated Hospital of Soochow University, Medical College of Soochow University, Suzhou 215006, China. 3. Department of Thoracic and Cardiovascular Surgery, The Second Affiliated Hospital of Soochow University, Medical College of Soochow University, Suzhou 215004, China. 4. Department of Pathology, The Second Affiliated Hospital of Soochow University, Medical College of Soochow University, Suzhou 215004, China. 5. Soochow University Laboratory of Cancer Molecular Genetics, Medical College of Soochow University, Suzhou 215123, China; Suzhou Key Laboratory for Molecular Cancer Genetics, Suzhou 215123, China. Electronic address: htzhang@suda.edu.cn.
Abstract
OBJECTIVES: Epithelial-mesenchymal transition (EMT) is a key process in early stage of cancer metastasis. TGF-β-mediated EMT is characterized by repression of E-cadherin and induction of N-cadherin (CDH2) in various cancers. Although many investigations have focused on the regulation of E-cadherin expression, the transcription-mediated events that directly induce N-cadherin expression in TGF-β-induced EMT are not fully clear. Here, we mainly focus on non-small cell lung cancer (NSCLC) cells, in which expression of CDH2 can be activated upon TGF-β stimulation, to investigate the underlying mechanisms of CDH2 expression regulation. MATERIALS AND METHODS: Western blot analysis, real-time quantitative reverse transcriptase PCR, luciferase reporter gene assays, RNA interference and in vivo chromatin immunoprecipitation (ChIP) assay were performed on human NSCLC cell lines A549 and SPC-A1. Twenty-six paired NSCLC tissues and adjacent noncancerous lung tissues were collected. RESULTS: Luciferase reporter assay revealed that a functional TGF-β-response element was located at position -1078 to -891 in the CDH2 promoter region. Furthermore, in vivo ChIP experiment indicated that TGF-β-activated SMAD3/4 complex was directly recruited to CDH2 promoter region (-1078 to -891). Upon TGF-β1 stimulation, knockdown of SMAD3 or/and SMAD4 led to a significant reduction in CDH2 promoter activity, and silencing of SMAD3 or SMAD4 significantly inhibited CDH2 mRNA and protein expression in A549 and SPC-A1 cells. In human NSCLC tissues, SMAD3 or SMAD4 mRNA level was positively correlated with CDH2 mRNA level, respectively. CONCLUSIONS: We found that TGF-β-activated SMAD3/4 complex may upregulate CDH2 expression by directly interacting with a specific SMAD-binding element in CDH2 promoter. Our findings provide insights into mechanisms underlying the transcriptional regulation of CDH2 expression in TGF-β-induced EMT and SMADs-based therapeutic strategies for NSCLCs.
OBJECTIVES: Epithelial-mesenchymal transition (EMT) is a key process in early stage of cancer metastasis. TGF-β-mediated EMT is characterized by repression of E-cadherin and induction of N-cadherin (CDH2) in various cancers. Although many investigations have focused on the regulation of E-cadherin expression, the transcription-mediated events that directly induce N-cadherin expression in TGF-β-induced EMT are not fully clear. Here, we mainly focus on non-small cell lung cancer (NSCLC) cells, in which expression of CDH2 can be activated upon TGF-β stimulation, to investigate the underlying mechanisms of CDH2 expression regulation. MATERIALS AND METHODS: Western blot analysis, real-time quantitative reverse transcriptase PCR, luciferase reporter gene assays, RNA interference and in vivo chromatin immunoprecipitation (ChIP) assay were performed on humanNSCLC cell lines A549 and SPC-A1. Twenty-six paired NSCLC tissues and adjacent noncancerous lung tissues were collected. RESULTS: Luciferase reporter assay revealed that a functional TGF-β-response element was located at position -1078 to -891 in the CDH2 promoter region. Furthermore, in vivo ChIP experiment indicated that TGF-β-activated SMAD3/4 complex was directly recruited to CDH2 promoter region (-1078 to -891). Upon TGF-β1 stimulation, knockdown of SMAD3 or/and SMAD4 led to a significant reduction in CDH2 promoter activity, and silencing of SMAD3 or SMAD4 significantly inhibited CDH2 mRNA and protein expression in A549 and SPC-A1 cells. In humanNSCLC tissues, SMAD3 or SMAD4 mRNA level was positively correlated with CDH2 mRNA level, respectively. CONCLUSIONS: We found that TGF-β-activated SMAD3/4 complex may upregulate CDH2 expression by directly interacting with a specific SMAD-binding element in CDH2 promoter. Our findings provide insights into mechanisms underlying the transcriptional regulation of CDH2 expression in TGF-β-induced EMT and SMADs-based therapeutic strategies for NSCLCs.
Authors: Joseph R Iacona; Nicholas J Monteleone; Alexander D Lemenze; Ashley L Cornett; Carol S Lutz Journal: RNA Biol Date: 2019-08-23 Impact factor: 4.652