title: [Effect of TRAF6 Downregulation on Malignant Biological Behavior of Lung Cancer Cell Lines].
abstract: OBJECTIVE
  It has been proven that tumor necrosis factor receptor-associated factor 6 (TRAF6) was a commonly amplified oncogene in lung cancer. However, the precise role of TRAF6 protein in lung cancer has not been extensively investigated. This study analyzed the effects of TRAF6 on the proliferation, apoptosis, cell cycle, migration, and invasion capability of lung cancer cell lines, as well as the potential molecular mechanisms involved.
  
  
  METHODS
  To address the expression of TRAF6 in lung cancer cells, four lung cancer cell lines (A549, H1650, SPC-A-1 and Calu-3) were assayed to determine the expression of TRAF6 protein by Western blot and TRAF6 mRNA via qRT-PCR. Moreover, siRNA targeting TRAF6 was introduced into SPC-A-1 and Calu-3 cells. Nuclear factor-қB (NF-қB) DNA-binding activity, apoptosis rates, cell proliferation, cell cycle, migration, and invasion were determined by electrophoretic mobility shift assay, flow cytometry, MTS assay, flow cytometry, scratch test, and transwell chamber assay, respectively. Western blot analysis was also performed to evaluate the expression of the following proteins through K63-ubiquitination: P65, CD24 and CXCR4. Whole-genome sequencing analysis was conducted using a second-generation sequencer in SPC-A-1 cells.
  
  
  RESULTS
  TRAF6 was highly up-expressed in SPC-A-1 and Calu-3 cell lines than the other two cells, which also showed K63-ubiquitinization in TRAF6. However, constitutive activation of NF-қB was observed only in SPC-A-1 lung cancer cells. Downregulation of TRAF6 suppressed the NF-κB activation, cell migration, and invasion but promoted the cell apoptosis of SPC-A-1 cells. Markedly decreased expression of CD24 and CXCR4 was observed in SPC-A-1 cells transfected by TRAF6 siRNA. Nevertheless, TRAF6 downregulation did not affect the proliferation and cell cycle of SPC-A-1 cells. Additionally, TRAF6 regulation did not affect the proliferation, apoptosis, cell cycle, migration, and invasion of Calu-3 cells. No mutations and no changes in gene copy numbers of TRAF6 were found by whole-exome sequencing of SPC-A-1 cells.
  
  
  CONCLUSIONS
  TRAF6 may be involved in cell migration, invasion, and apoptosis of SPC-A-1 cells, possibly through regulating the NF-қB-CD24/CXCR4 pathway. .
authors.affiliation: Department of Medical Oncology, Fujian Provincial Cancer Hospital, Fujian Medical University Teaching Hospital, Fuzhou 350014, China.
authors.forename: Gen
authors.initials: G
authors.lastname: Lin
authors.affiliation: Department of Medical Oncology, Fujian Provincial Cancer Hospital, Fujian Medical University Teaching Hospital, Fuzhou 350014, China.
authors.forename: Chuangzhong
authors.initials: C
authors.lastname: Huang
authors.affiliation: Department of Medical Oncology, Fujian Provincial Cancer Hospital, Fujian Medical University Teaching Hospital, Fuzhou 350014, China.
authors.forename: Guangjian
authors.initials: G
authors.lastname: Su
authors.affiliation: Department of Medical Oncology, Fujian Provincial Cancer Hospital, Fujian Medical University Teaching Hospital, Fuzhou 350014, China.
authors.forename: Huihua
authors.initials: H
authors.lastname: Hu
authors.affiliation: Department of Medical Oncology, Fujian Provincial Cancer Hospital, Fujian Medical University Teaching Hospital, Fuzhou 350014, China.
authors.forename: Haipeng
authors.initials: H
authors.lastname: Xu
authors.affiliation: Department of Medical Oncology, Fujian Provincial Cancer Hospital, Fujian Medical University Teaching Hospital, Fuzhou 350014, China.
authors.forename: Cheng
authors.initials: C
authors.lastname: Huang
mesh_terms: D017209:Apoptosis; D045744:Cell Line, Tumor; D002465:Cell Movement; D015536:Down-Regulation; D006801:Humans; D008175:Lung Neoplasms / Q000473:pathology*; D016328:NF-kappa B / Q000378:metabolism; D009361:Neoplasm Invasiveness; D048029:TNF Receptor-Associated Factor 6 / Q000235:genetics / Q000502:physiology*; D054875:Ubiquitination
chemical_list: D016328:NF-kappa B; D048029:TNF Receptor-Associated Factor 6