Expression of TLR5 in Different Types of Non-small Cell Lung Cancer Cell Lines and its Activation Mechanism
10.3779/j.issn.1009-3419.2015.01.02
- VernacularTitle:TLR5在不同非小细胞肺癌细胞株的表达及其活化机制的初步探讨
- Author:
ZHOU HUI
1
,
2
;
LUO MEI
;
WEN YIGE
;
MA ANDI
;
LUO YONGZHONG
;
YI QING
;
CHEN JIANHUA
;
XIAO LING
Author Information
1. 410013 长沙，湖南省肿瘤医院，中南大学湘雅医学院附属肿瘤医院，文
2. 410078 长沙，中南大学国家遗传学重点实验室
- Keywords:
Lung neoplams;
TLR5;
Flagellin;
Signaling pathway
- From:
Chinese Journal of Lung Cancer
2015;(1):8-15
- CountryChina
- Language:Chinese
-
Abstract:
Background and objective 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. Te aim of this study is to investigate the expression of TLR5 in diferent types of NSCLC cell lines, and analyze the activity of the signaling pathway afer stimulated by its specific exogenous ligand fiagellin. Methods Te TLR5 protein was detected by immunofiuorescence 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 fiagel-lin. 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. Te 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 activ-ity of TLR5 signaling pathway by Western blot at 0 min, 10 min, 30 min and 60 min, respectively. Results Te expression of TLR5 was the highest in the lung adenocarcinoma cell line SPC-A-1 by immunofiuorescence, 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 man-ner. 0.1 μg/mL fiagellin 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 fiagellin, compared with that of 0 min group, the levels of p-IKBα, p-ERK1/2, p-JNK of SPC-A-1 cells increased significantly afer 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 fiagellin (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). Conclusion Exogenous ligand fiagellin can ac-tivate TLR5 protein in NSCLC cell lines and initiate downstream signaling pathways. It may be relative to the development of NSCLC.
