title: Feedback regulations of miR-21 and MAPKs via Pdcd4 and Spry1 are involved in arsenite-induced cell malignant transformation.
abstract: OBJECTIVE
  To establish the functions of miR-21 and the roles of two feedback regulation loops, miR-21-Spry1-ERK/NF-κB and miR-21-Pdcd4-JNK/c-Jun, in arsenite-transformed human embryo lung fibroblast (HELF) cells.
  
  
  METHODS
  For arsenite-transformed HELF cells, apoptosis, clonogenicity, and capacity for migration were determined by Hoechst staining, assessment of their capacity for anchorage-independent growth, and wound-healing, respectively, after blockage, with inhibitors or with siRNAs, of signal pathways for JNK/c-Jun or ERK/NF-κB. Decreases of miR-21 levels were determined with anti-miR-21, and the up-regulation of Pdcd4 and Spry1 was assessed in transfected cells; these cells were molecularly characterized by RT-PCR, qRT-PCR, Western blots, and immunofluorescence assays.
  
  
  RESULTS
  MiR-21 was highly expressed in arsenite-transformed HELF cells and normal HELF cells acutely treated with arsenite, an effect that was concomitant with activation of JNK/c-Jun and ERK/NF-κB and down-regulation of Pdcd4 and Spry1 protein levels. However, there were no significant changes in mRNA levels for Pdcd4 and Spry1, which suggested that miR-21 regulates the expressions of Pdcd4 and Spry1 through translational repression. In arsenite-transformed HELF cells, blockages of JNK/c-Jun or ERK/NF-κB with inhibitors or with siRNAs prevented the increases of miR-21and the decreases of the protein levels but not the mRNA levels of Pdcd4 and Spry1. Down-regulation of miR-21 and up-regulations of Pdcd44 or Spry1 blocked the arsenite-induced activations of JNK/c-Jun or ERK/NF-κB, indicating that knockdown of miR-21 inhibits feedback of ERK activation and JNK activation via increases of Pdcd4 and Spry1 protein levels, respectively. Moreover, in arsenite-transformed HELF cells, inhibition of miR-21 promoted cell apoptosis, inhibited clonogenicity, and reduced migration.
  
  
  CONCLUSIONS
  The results indicate that miR-21 is both a target and a regulator of ERK/NF-κB and JNK/c-Jun and the feedback regulations of miR-21 and MAPKs via Pdcd4 and Spry1, respectively, are involved in arsenite-induced malignant transformation of HELF cells.
authors.affiliation: Institute of Toxicology, Ministry of Education, School of Public Health, Nanjing, Jiangsu, People's Republic of China.
authors.forename: Lu
authors.initials: L
authors.lastname: Shen
authors.forename: Min
authors.initials: M
authors.lastname: Ling
authors.forename: Yuan
authors.initials: Y
authors.lastname: Li
authors.forename: Yuan
authors.initials: Y
authors.lastname: Xu
authors.forename: Yun
authors.initials: Y
authors.lastname: Zhou
authors.forename: Jing
authors.initials: J
authors.lastname: Ye
authors.forename: Ying
authors.initials: Y
authors.lastname: Pang
authors.forename: Yue
authors.initials: Y
authors.lastname: Zhao
authors.forename: Rongrong
authors.initials: R
authors.lastname: Jiang
authors.forename: Jianping
authors.initials: J
authors.lastname: Zhang
authors.forename: Qizhan
authors.initials: Q
authors.lastname: Liu
mesh_terms: D051017:Apoptosis Regulatory Proteins / Q000235:genetics* / Q000378:metabolism; D018053:Arsenites; D002461:Cell Line, Transformed; D002465:Cell Movement; D049109:Cell Proliferation; D002471:Cell Transformation, Neoplastic / Q000139:chemically induced / Q000235:genetics*; D004622:Embryo, Mammalian; D025461:Feedback, Physiological; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D048670:MAP Kinase Kinase 4 / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D035683:MicroRNAs / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics* / Q000378:metabolism; D047428:Protein Kinase Inhibitors; D016755:Proto-Oncogene Proteins c-jun / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects
chemical_list: D051017:Apoptosis Regulatory Proteins; D018053:Arsenites; C524940:MIRN21 microRNA, human; D008565:Membrane Proteins; D035683:MicroRNAs; D016328:NF-kappa B; C114487:PDCD4 protein, human; D010750:Phosphoproteins; D047428:Protein Kinase Inhibitors; D016755:Proto-Oncogene Proteins c-jun; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; C486055:SPRY1 protein, human; D048670:MAP Kinase Kinase 4; C015001:arsenite