title: [Expression of TLR5 in different types of non-small cell lung cancer cell lines and its activation mechanism].
abstract: BACKGROUND
  It has been proven that toll-like receptor 5 (TLR5) plaied an important role in the development of tumor. In our previous study, we found that the expression of TLR5 was remarkably higher in non-small cell lung cancer (NSCLC) tissues than that in normal tissues, but the activation of TLR5 signaling pathway in NSCLC was still unknown. The aim of this study is to investigate the expression of TLR5 in different types of NSCLC cell lines, and analyze the activity of the signaling pathway after stimulated by its specific exogenous ligand flagellin.
  
  
  METHODS
  The TLR5 protein was detected by immunofluorescence and Western blot in three kinds of NSCLC cell lines, and the TLR5 mRNA was detected by RT-PCR. Select the cell line of TLR5 highest expression as the research object, and select the suitable concentration of flagellin. NF-κB luciferase activity was detected to validate the TLR5 activation pathway through inhibitory signaling pathways by 0 μg/mL, 0.01 μg/mL, 0.1 μg/mL, 1 μg/mL, 10 μg/mL TLR5 antibody. The chosen cell line was transfected by TLR5 shRNA plasmid, and p-IKBα, IKBα, p-ERK1/2, ERK1/2 and p-JNK of untrasfected and transfected cells were detected in the activity of TLR5 signaling pathway by Western blot at 0 min, 10 min, 30 min and 60 min, respectively.
  
  
  RESULTS
  The expression of TLR5 was the highest in the lung adenocarcinoma cell line SPC-A-1 by immunofluorescence, mainly expressed on the cell membrane. NF-κB luciferase activity of SPC-A-1 cells was the highest, and the activity was increased in a dose-dependent manner. 0.1 μg/mL flagellin could significantly increase the NF-κB luciferase activity (P<0.05), while its activity could be inhibited by the TLR5 antibody in a negative correlation. Treated by 0.1 μg/mL flagellin, compared with that of 0 min group, the levels of p-IKBα, p-ERK1/2, p-JNK of SPC-A-1 cells increased significantly after 10 min, reached the peak at 30 min, and declined at 60 min (P<0.05). Compared with that of 10 min and 60 min group, the levels of p-IKBα, p-ERK1/2, p-JNK significantly increased at 30 min (P<0.05). While the levels of IKBα, ERK1/2 at 0 min, 10 min, 30 min and 60 min had no significant changes (P>0.05). SPC-A-1 cells transfected TLR5-shRNA were also stimulated by flagellin (0.1 μg/mL). At 0 min, 10 min, 30 min and 60 min, p-IKBα and p-JNK proteins could not be detected, and the levels of IKBα and ERK1/2 had no significant changes (P>0.05), but the levels of p-ERK1/2 significantly increased as time went on (P<0.05).
  
  
  CONCLUSIONS
  Exogenous ligand flagellin can activate TLR5 protein in NSCLC cell lines and initiate downstream signaling pathways. It may be relative to the development of NSCLC.
authors.affiliation: Hunan Cancer Hospital, the Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha 410013, China;State Key Laboratory of Medical Genetics Central South University, Changsha 410078, China.
authors.forename: Hui
authors.initials: H
authors.lastname: Zhou
authors.affiliation: Hunan Cancer Hospital, the Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha 410013, China.
authors.forename: Mei
authors.initials: M
authors.lastname: Luo
authors.affiliation: Hunan Cancer Hospital, the Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha 410013, China.
authors.forename: Yige
authors.initials: Y
authors.lastname: Wen
authors.affiliation: Hunan Cancer Hospital, the Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha 410013, China.
authors.forename: Andi
authors.initials: A
authors.lastname: Ma
authors.affiliation: Hunan Cancer Hospital, the Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha 410013, China.
authors.forename: Yongzhong
authors.initials: Y
authors.lastname: Luo
authors.affiliation: Hunan Cancer Hospital, the Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha 410013, China.
authors.forename: Qing
authors.initials: Q
authors.lastname: Yi
authors.affiliation: Hunan Cancer Hospital, the Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha 410013, China.
authors.forename: Jianhua
authors.initials: J
authors.lastname: Chen
authors.affiliation: Department of Histology and Embryology, School of Basic Medical Sciences, Central South University, Changsha 410013, China.
authors.forename: Ling
authors.initials: L
authors.lastname: Xiao
mesh_terms: D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D005408:Flagellin / Q000235:genetics / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D016209:Interleukin-8 / Q000235:genetics / Q000378:metabolism; D008175:Lung Neoplasms / Q000235:genetics* / Q000378:metabolism; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D051198:Toll-Like Receptor 5 / Q000235:genetics* / Q000378:metabolism
chemical_list: D016209:Interleukin-8; D016328:NF-kappa B; D051198:Toll-Like Receptor 5; D005408:Flagellin