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.
背景与目的 已有的研究表明：Toll样受体5（toll-like receptor 5, TLR5）在肿瘤起始和发展中发挥重要作用。我们前期研究发现， TLR5在非小细胞肺癌（non-small cell lung cancer, NSCLC）组织中高表达，但其在NSCLC高表达后的信号通路活化情况的研究并不多见。本研究旨在探讨TLR5在不同NSCLC细胞株上的表达，及其在NSCLC细胞中活化的机制。方法 用免疫荧光、RT-PCR和Western blot方法检测TLR5在三种不同NSCLC细胞株中的表达。分别用0 μg/mL、0.01 μg/mL、0.1 μg/mL、1 μg/mL、5 μg/mL、10 μg/mL的鞭毛蛋白刺激，用NF-κB荧光素酶报告基因质粒瞬时转染后，检测细胞内NF-κB荧光素酶的活性。选择TLR5表达最高的SPC-A-1细胞株为实验对象，选择0.1 μg/mL的鞭毛蛋白，分别用0 μg/mL、0.01 μg/mL、0.1 μg/mL、1 μg/mL、10 μg/mL的TLR5抗体抑制通路活化，检测细胞内NF-κB荧光素酶的活性，验证TLR5活化通路。构建TLR5-shRNA，转染SPC-A-1细胞48 h后，以0.1 μg/mL浓度鞭毛蛋白分别刺激SPC-A-1细胞及转染的SPC-A-1细胞，在刺激0 min、10 min、30 min、60 min，用Western blot方法比较TLR5信号通路因子p-IKBα、p-ERK1/2、p-JNK、IKBα、ERK1/2的变化。结果 TLR5在肺腺癌细胞株SPC-A-1中呈高表达，且主要表达在细胞膜上。三种细胞株中SPC-A-1细胞NF-κB荧光素酶的活性最高，呈浓度依赖性，0.1 μg/mL鞭毛蛋白即可明显增强NF-κB荧光素酶的活性（P<0.05）；而SPC-A-1细胞内NF-κB荧光素酶的活性可被TLR5抗体抑制，与TLR5抗体浓度负相关（P<0.05）。与0 min相比较，SPC-A-1细胞内p-IKBα、p-ERK1/2、p-JNK水平在鞭毛蛋白刺激10 min即明显增高，30 min达到高峰，60 min开始下降（P<0.05），且与10 min和60 min组相比，p-IKBα、p-ERK1/2、p-JNK水平在30 min增高（P<0.05）；而IKBα、ERK1/2的水平无明显变化（P>0.05）。以适合浓度鞭毛蛋白刺激转染的SPC-A-1细胞，p-IKBα、p-JNK蛋白均未检出，IKBα、ERK1/2蛋白的水平无明显变化（P>0.05），p-ERK1/2蛋白水平随着时间延长明显增高（P<0.05）。结论 外源性配体鞭毛蛋白可激活NSCLC细胞株TLR5蛋白，启动下游信号通路，可能与NSCLC的发生发展有关。