title: Intelectin 1 suppresses the growth, invasion and metastasis of neuroblastoma cells through up-regulation of N-myc downstream regulated gene 2.
abstract: BACKGROUND
  Recent studies have revealed the potential roles of intelectin 1 (ITLN1) in tumorigenesis. However, its functions and underlying mechanisms in neuroblastoma (NB), the most common extracranial solid tumor in childhood, still remain largely unknown.
  
  
  METHODS
  Human neuroblastoma cell lines were treated with recombinant ITLN1 protein or stably transfected with ITLN1 expression and short hairpin RNA vectors. Gene expression and signaling pathway were detected by western blot and real-time quantitative RT-PCR. Gene promoter activity and transcription factor binding were detected by luciferase reporter and chromatin immunoprecipitation assays. Growth and aggressiveness of tumor cells were measured by MTT colorimetry, colony formation, scratch assay, matrigel invasion assay, and nude mice model.
  
  
  RESULTS
  Mining of public microarray databases revealed that N-myc downstream regulated gene 2 (NDRG2) was significantly correlated with ITLN1 in NB. Gain- and loss-of-function studies indicated that secretory ITLN1 facilitated the NDRG2 expression, resulting in down-regulation of vascular endothelial growth factor (VEGF) and matrix metalloproteinase 9 (MMP-9), in NB cell lines SH-SY5Y, SK-N-BE(2), and SK-N-SH. Krüppel-like factor 4 (KLF4), a transcription factor crucial for NDRG2 expression, was up-regulated by ITLN1 in NB cells via inactivation of phosphoinositide 3-kinase (PI3K)/AKT signaling. Ectopic expression of ITLN1 suppressed the growth, invasion and metastasis of NB cells in vitro and in vivo. Conversely, knockdown of ITLN1 promoted the growth, invasion, and metastasis of NB cells. In addition, rescue experiments in ITLN1 over-expressed or silenced NB cells showed that restoration of NDRG2 expression prevented the tumor cells from ITLN1-mediated changes in these biological features. In clinical NB tissues, ITLN1 was down-regulated and positively correlated with NDRG2 expression. Patients with high ITLN1 or NDRG2 expression had greater survival probability.
  
  
  CONCLUSIONS
  These findings indicate that ITLN1 functions as a tumor suppressor that affects the growth, invasion and metastasis of NB through up-regulation of NDRG2.
authors.affiliation: Department of Pediatric Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, 430022, P. R. China. d.li26@hotmail.com.
authors.forename: Dan
authors.initials: D
authors.lastname: Li
authors.affiliation: Department of Pediatric Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, 430022, P. R. China. drmeihong@gmail.com.
authors.forename: Hong
authors.initials: H
authors.lastname: Mei
authors.affiliation: Department of Pediatric Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, 430022, P. R. China. jiaruipu82@gmail.com.
authors.forename: Jiarui
authors.initials: J
authors.lastname: Pu
authors.affiliation: Department of Pediatric Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, 430022, P. R. China. xuan_xiang86@hotmail.com.
authors.forename: Xuan
authors.initials: X
authors.lastname: Xiang
authors.affiliation: Department of Pediatric Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, 430022, P. R. China. xiangzhaotongji@hotmail.com.
authors.forename: Xiang
authors.initials: X
authors.lastname: Zhao
authors.affiliation: Department of Pediatric Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, 430022, P. R. China. quhongxia02@gmail.com.
authors.forename: Hongxia
authors.initials: H
authors.lastname: Qu
authors.affiliation: Clinical Center of Human Genomic Research, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, 430022, P. R. China. kaihuangunion@gmail.com.
authors.forename: Kai
authors.initials: K
authors.lastname: Huang
authors.affiliation: Clinical Center of Human Genomic Research, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, 430022, P. R. China. ld_zheng@hotmail.com.|Department of Pathology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, 430022, P. R. China. ld_zheng@hotmail.com.
authors.forename: Liduan
authors.initials: L
authors.lastname: Zheng
authors.affiliation: Department of Pediatric Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, 430022, P. R. China. qs_tong@hotmail.com.|Clinical Center of Human Genomic Research, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, 430022, P. R. China. qs_tong@hotmail.com.
authors.forename: Qiangsong
authors.initials: Q
authors.lastname: Tong
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000235:genetics*; D016207:Cytokines / Q000235:genetics*; D015536:Down-Regulation / Q000235:genetics; D058851:GPI-Linked Proteins / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics; D006801:Humans; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000235:genetics; D037102:Lectins / Q000235:genetics*; D008297:Male; D020780:Matrix Metalloproteinase 9 / Q000235:genetics; D051379:Mice; D008819:Mice, Nude; D009361:Neoplasm Invasiveness / Q000235:genetics*; D009447:Neuroblastoma / Q000235:genetics*; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics; D015398:Signal Transduction / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics; D025521:Tumor Suppressor Proteins / Q000235:genetics*; D015854:Up-Regulation / Q000235:genetics*; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics
chemical_list: D016207:Cytokines; D058851:GPI-Linked Proteins; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; D037102:Lectins; D020780:Matrix Metalloproteinase 9; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; D042461:Vascular Endothelial Growth Factor A; C432397:ITLN1 protein, human; C000715467:KLF4 protein, human; C000715468:Klf4 protein, mouse; C466228:NDRG2 protein, human