title: Restin suppressed epithelial-mesenchymal transition and tumor metastasis in breast cancer cells through upregulating mir-200a/b expression via association with p73.
abstract: BACKGROUND
  Restin belongs to MAGE superfamily and is known as MAGE H1. Restin was firstly cloned from HL-60 cells treated with all-trans retinoic acid (ATRA). Previous studies showed a pro-apoptotic role of Restin in several cell lines. However, little information is available on its expression patterns and functions in vivo. Our study was performed to detect if Restin plays a role in breast cancer cells in vitro and in vivo.
  
  
  RESULTS
  Real-time PCR and western blot were conducted to detect Restin expression in multiple breast cancer cell lines and Restin level was negatively related with cell motility. Restin overexpression and knockdown stable cell lines were established by transducing lentivirus into MCF-7 and MDA-MB-231 cells. Cell morphology, wound closure assay, transwell migration and invasion assays were performed to detect if Restin inhibited EMT. Our data showed that Restin overexpressed cells exhibited classical epithelial cell morphology, and Restin overexpression resulted in activation of epithelial markers and suppression of mesenchymal markers, and inhibition of cell migration and invasion. Tumor xenograft model was used to characterize the biological functions of Restin in vivo. We found that Restin overexpression led to reduced lung metastasis. Real-time PCR, western blot, luciferase assay and ChIP assay were performed to identify the potential targets of Restin and the underlying molecular mechanisms. Among several master regulators of EMT, only ZEB1/2 levels were dramatically inhibited by Restin. Unexpectedly, Restin indirectly regulated ZEB1/2 expression at post-transcriptional level. We further identified mir-200a/b, well-characterized mediators controlling ZEB1/2 expression, were transcriptionally activated by Restin and the regulation was dependent on the p53 binding site in mir-200b/a/429 promoter. Further mechanical studies demonstrated Restin interacted with p73, one of p53 family members, which contributed to Restin-mediated activation of mir-200a/b and suppression of ZEB1/2.
  
  
  CONCLUSIONS
  Taken together, our results suggest that Restin inhibits EMT and tumor metastasis by controlling the expression of the tumor metastasis suppressor mir-200a/b via association with p73. Our findings not only establish a mechanistic link between Restin, EMT and tumor metastasis, but also provide strong evidence supporting the notion that MAGE Group II proteins may exert a tumor suppressive effect in vivo.
authors.affiliation: Department of Breast surgery, Breast Cancer Center, Affiliated Cancer Hospital of Zhengzhou University, Henan Cancer Hospital, #127 Dongming Road, Zhengzhou, Henan, 450008, People's Republic of China. zhend.lu@163.com.
authors.forename: Zhenduo
authors.initials: Z
authors.lastname: Lu
authors.affiliation: Department of Breast surgery, Breast Cancer Center, Affiliated Cancer Hospital of Zhengzhou University, Henan Cancer Hospital, #127 Dongming Road, Zhengzhou, Henan, 450008, People's Republic of China. dehuang_jiao2008@126.com.
authors.forename: Dechuang
authors.initials: D
authors.lastname: Jiao
authors.affiliation: Department of Breast surgery, Breast Cancer Center, Affiliated Cancer Hospital of Zhengzhou University, Henan Cancer Hospital, #127 Dongming Road, Zhengzhou, Henan, 450008, People's Republic of China. jhqiaojhqiao00@gmail.com.
authors.forename: Jianghua
authors.initials: J
authors.lastname: Qiao
authors.affiliation: Department of Pathogen Biology, Basic Medical College of Zhengzhou University, #100 Science Road, Zhengzhou, 450001, People's Republic of China. yangsensen20140721@126.com.
authors.forename: Sen
authors.initials: S
authors.lastname: Yang
authors.affiliation: Department of Breast surgery, Breast Cancer Center, Affiliated Cancer Hospital of Zhengzhou University, Henan Cancer Hospital, #127 Dongming Road, Zhengzhou, Henan, 450008, People's Republic of China. minyan@fdyg.edu.cn.
authors.forename: Min
authors.initials: M
authors.lastname: Yan
authors.affiliation: Department of Breast surgery, Breast Cancer Center, Affiliated Cancer Hospital of Zhengzhou University, Henan Cancer Hospital, #127 Dongming Road, Zhengzhou, Henan, 450008, People's Republic of China. cuishude73@gmail.com.
authors.forename: Shude
authors.initials: S
authors.lastname: Cui
authors.affiliation: Department of Breast surgery, Breast Cancer Center, Affiliated Cancer Hospital of Zhengzhou University, Henan Cancer Hospital, #127 Dongming Road, Zhengzhou, Henan, 450008, People's Republic of China. zhenzhenliu73@163.com.
authors.forename: Zhenzhen
authors.initials: Z
authors.lastname: Liu
mesh_terms: D000818:Animals; D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology*; D045744:Cell Line, Tumor; D002465:Cell Movement; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015536:Down-Regulation / Q000235:genetics; D058750:Epithelial-Mesenchymal Transition; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D018398:Homeodomain Proteins / Q000378:metabolism; D006801:Humans; D008175:Lung Neoplasms / Q000556:secondary*; D051379:Mice; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D009361:Neoplasm Invasiveness; D009363:Neoplasm Proteins / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism*; D012097:Repressor Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D000072160:Tumor Protein p73; D025521:Tumor Suppressor Proteins / Q000378:metabolism*; D015854:Up-Regulation / Q000235:genetics; D000074165:Zinc Finger E-box Binding Homeobox 2; D000071799:Zinc Finger E-box-Binding Homeobox 1
chemical_list: D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; C533896:MIRN200 microRNA, human; D035683:MicroRNAs; D008869:Microtubule-Associated Proteins; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D012097:Repressor Proteins; C000606460:TP73 protein, human; D014157:Transcription Factors; C000606368:Trp73 protein, mouse; D000072160:Tumor Protein p73; D025521:Tumor Suppressor Proteins; C486307:ZEB1 protein, human; C492363:ZEB2 protein, human; D000074165:Zinc Finger E-box Binding Homeobox 2; D000071799:Zinc Finger E-box-Binding Homeobox 1; C076744:cytoplasmic linker protein 170