表皮生长因子受体对苯并芘诱导的人支气管上皮细胞SIRT1活化的影响

doi:10.13481/j.1671-587x.20160419

摘要/Abstract

摘要：

目的：探讨表皮生长因子受体（EGFR）在调控苯并芘（B[a]P）诱导人支气管上皮BEAS-2B细胞沉默信息调节因子1（SIRT1）活化中的作用，阐明EGFR/SIRT1信号转导通路与肺癌发生发展的关系。方法：MOTIF Search^TM分析软件预测SIRT1启动子序列上转录DNA结合位点。培养BEAS-2B细胞，设不加苯并芘的细胞为对照组，苯并芘组细胞暴露于苯并茈（8 μmol·L^-1）不同时间（6、12、24和48h），RT-PCR和蛋白免疫印迹法检测各组细胞中EGFR mRNA和蛋白表达水平。将SIRT1启动子荧光素酶报告载体转染入BEAS-2B细胞中，采用人表皮细胞生长因子（hEGF）和酪氨酸蛋白激酶抑制剂Genistein处理细胞24h，倒置显微镜观察各组BEAS-2B细胞形态表现，荧光素酶报告基因技术检测SIRT1荧光素酶活性。收集相关肺组织临床标本，免疫组织化学法检测肺组织中EGFR蛋白表达水平。结果：SIRT1启动子序列上含有表皮细胞生长因子（EGF）、铁氧还蛋白（2Fe-2S）和血管性血友病因子（vWF）3个转录因子的DNA结合位点。与对照组比较，苯并芘组细胞中EGFR mRNA表达水平升高（P < 0.05），且在12h达到峰值，同时EGFR蛋白表达水平均不同程度升高（P < 0.05）。与对照组比较，hEGF组和苯并芘组BEAS-2B中细胞SIRT1荧光素酶活性明显增加（P < 0.05），hEGF联合苯并芘组BEAS-2B细胞中SIRT1荧光素酶活性增加更为明显（P < 0.001）；Genistein大于30 μmol·L^-1时，细胞排列逐渐稀疏，形态改变明显；Genistein（30 μmol·L^-1）能够抑制苯并芘诱导的SIRT1荧光素酶活性的增加，与苯并芘组比较差异有统计学意义（P < 0.05）。免疫组织化学法，肺癌组织中EGFR蛋白表达水平明显高于正常肺组织（P < 0.001）。结论：苯并芘暴露下，EGFR能够促进SIRT1的表达，从而诱导肺慢性炎症反应，EGFR/SIRT1信号转导通路在肺癌的发生发展过程中发挥一定的作用。

关键词: 苯并芘, 表皮生长因子受体, 沉默信息调节因子1, 信号转导

Abstract:

Objective: To investigate the role of epidermal growth factor receptor (EGFR) in the regulation of B[a]P-induced silent information regulator 1 (SIRT1) activation in the human bronchial epithelial cells (BEAS-2B),and to clarify the relationship between EGFR/SIRT1 signal transduction pathway and the occurrence and development lung cancer. Methods: The prediction analysis of transcriptional factor binding sites of SIRT1 was performed by MOTIF Search^TM software. The primary cultivated BEAS-2B cells were divided into control group (without B[a]P exposure) and B[a]P groups (the cells were exposed to B[a]P for 6,12,24,48 h). RT-PCR and Western blotting method were carried out to detect the EGFR mRNA and protein expression levels. The BEAS-2B cells transfected with SIRT1 promotor luciferase reporter gene plasmid were treated with human epidermal growth factor (hEGF) and Genistein (tyrosine protein kinase inhibitor) for 24 h,the morphology of BEAS-2B cells was observed by inverted microscope,and luciferase reporter assay was used to test the SIRT1 transcriptional activity. The human lung tissue biopsies were acquired and the immunohistochemical analysis was used to determine the EGFR protein expression level. Results: The transcriptional factor binding sites of SIRT1 contained EGF, 2Fe-2S and vWF. Compared with control group,the expression levels of EGFR mRNA in B[a]P groups were increased in a time-dependent manner,and reached the peak at 12 h (P < 0.05);the EGFR protein expression levels were also increased (P < 0.05).The SIRT1 luciferase activity in hEGF group was increased compared with control group (P < 0.05);when hEGF and B[a]P worked together,the SIRT1 luciferase activity was increased even further (P < 0.001). The cells showed arrangement and morphologic changes gradually when the B[a]P concentration was above 30 μmol·L^-1. Genistein (30 μmol·L^-1) inhibited the increase of SIRT1 luciferase activity induced by B[a]P ,and there was significant difference compared with control group(P < 0.05).The immunohistochemistry results showed that EGFR expression level in lung cancer tissue was higher than that in normal lung tissue (P < 0.001). Conclusion: EGFR can regulate the B[a]P-induced SIRT1 expression in BEAS-2B cells,and to cause lung chronic inflammation; EGFR/SIRT1 signal transduction pathway may play a role in the occurrence and development of lung cancer.

Key words: benzo(a)pyrene, epidermal growth factor receptor, silent information regulator 1, signaling transduction

中图分类号:

R734.2

张敏, 崔运芹, 宋焕芳, 吕建祎, 徐笑红, 高基民. 表皮生长因子受体对苯并芘诱导的人支气管上皮细胞SIRT1活化的影响[J]. 吉林大学学报(医学版), 2016, 42(04): 731-736.

ZHANG Min, CUI Yunqin, SONG Huanfang, LYU Jianyi, XU Xiaohong, GAO Jimin. Influence of EGFR in B[a]P-induced SIRT1 activation in human bronchial epithelial cells[J]. Journal of Jilin University Medicine Edition, 2016, 42(04): 731-736.

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