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Caveolin-1介导张氏肝细胞X射线诱导的DNA损伤修复的信号转导

李洪艳¹, 屈超¹, 张叶军¹, 孙佳¹, 韩朝², 刘晶², 邹伟^1,*

¹辽宁师范大学生命科学学院，大连 116081；²大连医科大学附属第一医院中英再生医学中心，大连 116011

摘要

已知Caveolin-1 ( Cav-1)是胞膜窖的重要结构蛋白，通过介导多条信号转导途径广泛参与细胞的生理和病理过程。引人关注的是，Cav-1的表达通常具有组织特异性和功能多样性，在乳腺癌、肝癌等肿瘤的发生和发展中起到重要的调节作用。近年研究表明，Cav-1参与胰腺癌细胞放射引起的DNA损伤修复过程，从而对放疗产生抵抗。但其是否对其它种类细胞的DNA损伤也存在类似作用并不清楚。本文采用RNA干扰技术建立稳定敲低Cav-1表达的张氏肝细胞系(Chang liver cell line, CHL)，即CHL-CAV7细胞，旨在探讨Cav-1在CHL细胞DNA损伤修复中的作用。Western blot结果显示，与转染非靶向质粒的对照细胞(CHL-C细胞)相比，CHL-CAV7细胞Cav-1蛋白表达水平被抑制了60%。采用X射线对细胞进行辐射，并对细胞的存活能力及DNA损伤修复相关蛋白的表达水平进行检测。克隆形成实验结果显示，在8和10 Gy X射线照射下，CHL-CAV7细胞的存活能力相对CHL-C细胞明显降低。Western blot结果显示，与CHL-C细胞相比，CHL-CAV7细胞照射后γH2AX蛋白表达量增加，p-ATM、DNA依赖蛋白激酶催化亚单位(DNA-dependent protein kinase, catalytic subunit, DNA-PKcs)和p53蛋白表达量减少。免疫荧光分析结果显示，CHL-CAV7细胞Mdm2和Cav-1的共定位相对CHL-C细胞显著减少。上述结果提示，Cav-1表达下降后，CHL细胞DNA的损伤加重，这可能与Cav-1与Mdm2相互作用减少，进而导致p53的降解增加有关。本文为了解肝细胞的放疗抵抗作用机制提供了一定的实验依据。

关键词： 小窝蛋白-1; 张氏肝细胞 ; DNA损伤 ; DNA修复

分类号：Q291; R34

Caveolin-1 is involved in DNA damage and repair signaling in X-irradiated Chang liver cells

LI Hong-Yan¹, QU Chao¹, ZHANG Ye-Jun¹, SUN Jia¹, HAN Chao², LIU Jing², ZOU Wei^1,*

¹College of Life Science, Liaoning Normal University, Dalian 116081, China；²Regenerative Medicine Centre, the First Affiliated Hospital of Dalian Medical University, Dalian 116011, China

Abstract

Caveolin-1 (Cav-1), as an important structural protein of caveolae, has been proven to be correlated with several signal transduction pathways. Recent studies have shown that Cav-1 may play a critical role in response to DNA damage in irradiated pancreatic cancer cells. However, it is not known whether down-regulation of Cav-1 is required to enhance the damage of other kinds of human cells exposed to X-radiation. In this study, the role of Cav-1 in Chang liver cell line (CHL) exposed to X-radiation was investigated. Cav-1 knockdown cell line (CHL-CAV7) was stably established by the siRNA plasmids transfection, and Cav-1 expression was suppressed by 60%, compared with that of control group (CHL-C) which was transfected with non-targeting plasmids. Cellular survival ability and the expressions of proteins related to DNA damage and repair were examined by colony formation assay and Western blot, respectively. Down-regulation of Cav-1 expression induced a significant decrease of the survival rate in CHL-CAV7 cells exposed to 8 and 10 Gy X-radiation. Compared with CHL-C cells, CHL-CAV7 cells showed increased γH2AX expression, as well as decreased p-ATM, DNA-dependent protein kinase, catalytic subunit (DNA-PKcs) and p53 protein expressions when treated with X-radiation. Meanwhile, the colocalization of Mdm2 and Cav-1 was decreased in CHL-CAV7 cells compared with that in CHL-C cells. These results suggest that the down-regulation of Cav-1 may aggravate DNA damage of CHL cells through reducing the interaction of Cav-1 and Mdm2, which results in the promotion of p53 degradation.

Key words: caveolin-1; Chang liver cells ; DNA damage and repair ;

收稿日期：2017-03-05　　录用日期：2017-11-13

通讯作者：邹伟　　E-mail: weizou60@126.com

DOI: 10.13294/j.aps.2017.0085

引用本文：

李洪艳, 屈超, 张叶军, 孙佳, 韩朝, 刘晶, 邹伟. Caveolin-1介导张氏肝细胞X射线诱导的DNA损伤修复的信号转导[J]. 生理学报 2017; 69 (6): 759-766.

LI Hong-Yan, QU Chao, ZHANG Ye-Jun, SUN Jia, HAN Chao, LIU Jing, ZOU Wei. Caveolin-1 is involved in DNA damage and repair signaling in X-irradiated Chang liver cells. Acta Physiol Sin 2017; 69 (6): 759-766