title: Downregulation of MicroRNA-644a Promotes Esophageal Squamous Cell Carcinoma Aggressiveness and Stem Cell-like Phenotype via Dysregulation of PITX2.
abstract: OBJECTIVE
  We previously reported the oncogenic role of paired-like homeodomain 2 (PITX2) in esophageal squamous cell carcinoma (ESCC). In this study, we aimed to identify the miRNA regulators of PITX2 and the mechanism underlying the pathogenesis of ESCC.
  
  
  METHODS
  Using miRNA profiling and bioinformatics analyses, we identified miR-644a as a negative mediator of PITX2 in ESCC. A series of in vivo and in vitro assays were performed to confirm the effect of miR-644a on PITX2-mediated ESCC malignancy.
  
  
  RESULTS
  ESCC cells and tissues expressed less miR-644a than normal epithelial controls. In patient samples, lower expression of miR-644a in ESCC tissues was significantly correlated with tumor recurrence and/or metastasis, such that miR-644a, PITX2, and the combination of the two were independent prognostic indicators for ESCC patient's survival (P < 0.05). Gain- and loss-of-function studies demonstrated that miR-644a inhibited ESCC cell growth, migration, and invasion in vitro and suppressed tumor growth and metastasis in vivo In addition, miR-644a dramatically suppressed self-renewal and stem cell-like traits in ESCC cells. Furthermore, the effect of upregulation of miR-644a was similar to that of PITX2 knockdown in ESCC cells. Mechanistic studies revealed that miR-644a attenuates ESCC cells' malignancy and stem cell-associated phenotype, at least partially, by inactivation of the Akt/GSK-3β/β-catenin signaling pathway through PITX2. Furthermore, promoter hypermethylation caused downregulation of miR-644a in ESCC.
  
  
  CONCLUSIONS
  Downregulation of miR-644a plays an important role in promoting both aggressiveness and stem-like traits of ESCC cells, suggesting that miR-644a may be useful as a novel prognostic biomarker or therapeutic target for the disease. Clin Cancer Res; 23(1); 298-310. ©2016 AACR.
authors.affiliation: The State Key Laboratory of Oncology in South China, Sun Yat-Sen University Cancer Center, Collaborative Innovation Center for Cancer Medicine, Guangzhou, P.R. China.|Department of Oncology, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, P.R. China.|Guangdong Esophageal Cancer Institute, Guangzhou, P.R. China.
authors.forename: Jia-Xing
authors.initials: JX
authors.lastname: Zhang
authors.affiliation: Department of Urology, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, P.R. China.
authors.forename: Zhen-Hua
authors.initials: ZH
authors.lastname: Chen
authors.affiliation: Department of Oncology, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, P.R. China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Xu
authors.affiliation: The State Key Laboratory of Oncology in South China, Sun Yat-Sen University Cancer Center, Collaborative Innovation Center for Cancer Medicine, Guangzhou, P.R. China.|Guangdong Esophageal Cancer Institute, Guangzhou, P.R. China.|Department of Pathology, Sun Yat-Sen University Cancer Center, Guangzhou, P.R. China.
authors.forename: Jie-Wei
authors.initials: JW
authors.lastname: Chen
authors.affiliation: Department of Oncology, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, P.R. China.
authors.forename: Hui-Wen
authors.initials: HW
authors.lastname: Weng
authors.affiliation: Department of Oncology, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, P.R. China.
authors.forename: Miao
authors.initials: M
authors.lastname: Yun
authors.affiliation: Department of Oncology, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, P.R. China.
authors.forename: Zou-San
authors.initials: ZS
authors.lastname: Zheng
authors.affiliation: Department of Oncology, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, P.R. China.
authors.forename: Cui
authors.initials: C
authors.lastname: Chen
authors.affiliation: Department of Biochemistry and Molecular Biology, Shantou University Medical College, Shantou, Guangdong, P.R. China.
authors.forename: Bing-Li
authors.initials: BL
authors.lastname: Wu
authors.affiliation: Department of Biochemistry and Molecular Biology, Shantou University Medical College, Shantou, Guangdong, P.R. China.
authors.forename: En-Min
authors.initials: EM
authors.lastname: Li
authors.affiliation: The State Key Laboratory of Oncology in South China, Sun Yat-Sen University Cancer Center, Collaborative Innovation Center for Cancer Medicine, Guangzhou, P.R. China.|Guangdong Esophageal Cancer Institute, Guangzhou, P.R. China.
authors.forename: Jian-Hua
authors.initials: JH
authors.lastname: Fu
authors.affiliation: Department of Oncology, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, P.R. China. xiedan@sysucc.org.cn yes20111212@163.com.
authors.forename: Sheng
authors.initials: S
authors.lastname: Ye
authors.affiliation: The State Key Laboratory of Oncology in South China, Sun Yat-Sen University Cancer Center, Collaborative Innovation Center for Cancer Medicine, Guangzhou, P.R. China. xiedan@sysucc.org.cn yes20111212@163.com.|Guangdong Esophageal Cancer Institute, Guangzhou, P.R. China.|Department of Pathology, Sun Yat-Sen University Cancer Center, Guangzhou, P.R. China.
authors.forename: Dan
authors.initials: D
authors.lastname: Xie
mesh_terms: D000818:Animals; D014408:Biomarkers, Tumor; D002294:Carcinoma, Squamous Cell / Q000235:genetics* / Q000378:metabolism / Q000401:mortality / Q000473:pathology*; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000235:genetics; D049109:Cell Proliferation; D019175:DNA Methylation; D004195:Disease Models, Animal; D018450:Disease Progression; D004938:Esophageal Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000401:mortality / Q000473:pathology*; D000077277:Esophageal Squamous Cell Carcinoma; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D038362:Glycogen Synthase Kinase 3 / Q000235:genetics; D064593:Heterografts; D018398:Homeodomain Proteins / Q000235:genetics*; D006801:Humans; D016130:Immunophenotyping; D051379:Mice; D035683:MicroRNAs / Q000235:genetics*; D009362:Neoplasm Metastasis; D014411:Neoplastic Stem Cells / Q000378:metabolism*; D010641:Phenotype; D011379:Prognosis; D011401:Promoter Regions, Genetic; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D034622:RNA Interference; D014157:Transcription Factors / Q000235:genetics*; D047368:Tumor Burden / Q000235:genetics; D060449:Wnt Signaling Pathway; D000097577:Homeobox Protein PITX2
chemical_list: D014408:Biomarkers, Tumor; D038362:Glycogen Synthase Kinase 3; D018398:Homeodomain Proteins; D035683:MicroRNAs; D051057:Proto-Oncogene Proteins c-akt; D014157:Transcription Factors; D000097577:Homeobox Protein PITX2; C579242:MIRN644 microRNA, human