【作者】 刘海峰；

【导师】 刘秉慈；

【作者基本信息】 中国疾病预防控制中心 ， 劳动卫生与环境卫生学， 2010， 博士

【摘要】 石英粉尘(简称石英)是我国最严重的职业危害因素之一,既可致矽肺,也可致癌。石英可诱导细胞周期改变,也可引起DNA双链断裂损伤(DNA double strand breaks, DSBs)。DNA损伤后,在信号分子介导下细胞周期发生阻滞,DNA损伤修复蛋白被激活进行DNA损伤修复。DNA依赖性蛋白激酶(DNA dependent protein kinase, DNA-PK)是DSBs的传感器之一,识别受损伤的DNA,磷酸化组蛋白H2AX,参与DSBs的非同源末端连接。DNA-PK由调节亚单位Ku和催化亚单位(DNA-PK catalytic subunit, DNA-PKcs)组成,Ku是Ku70与Ku80蛋白通过非共价键紧密结合形成的异二聚体结构。以往研究表明,DNA-PK作为蛋白激酶可将多种重要的功能蛋白底物,如Akt、JNK、AP-1、p53等磷酸化,激活DNA损伤信号转导通路,促进修复相关因子的转录、引起细胞周期阻滞或诱导细胞凋亡。本课题组前期研究发现,Akt/JNK/AP-1信号通路可调节CyclinD1和CDK4表达,参与石英诱导的细胞周期S期阻滞；DNA-PKcs具有DSBs传感器功能,通过DNA-PKcs/JNK信号通路,促进石英致DSBs的修复。然而,Ku在石英致DSBs修复的DNA-PK/JNK信号通路中的作用仍不清楚。其次,DNA-PK与磷脂酰肌醇-3激酶(Phosphatidylinositol 3 kinase, PI3K)有相同的结构域,均与MAPKs及细胞周期有关,两者在石英致DSBs的修复通路中作用的异同仍有待研究。针对以上问题,本研究以人胚肺成纤维细胞(human embryo lung fibroblasts, HELF)为实验模型,采用RNAi、显性失活突变体以及化学抑制剂等研究手段,将我室以往的MAPKs通路研究向上溯源至传感器DNA-PK的调节亚单位Ku,向下追踪到DSBs损伤及修复,重点研究Ku80在石英致DSBs修复及细胞周期改变中的作用,以及Ku80与DNA-PK/JNK通路中信号分子的上下游关系。其次,探讨PI3K在石英致DSBs修复中的作用以及对DNA-PK的影响。采用Ku80的siRNA及阴性对照质粒稳定转染HELF;中性彗星实验和／或yH2AX识别抗体技术检测石英致DSBs损伤程度；免疫印迹法测定蛋白的表达及其磷酸化水平；免疫荧光技术检测yH2AX焦点在细胞内的定位及其水平；流式细胞术检测细胞周期。结果如下：1成功建立了Ku80 siRNA质粒及阴性对照质粒稳定转染HELF的细胞系。2抑制Ku80表达,石英诱导的γH2AX水平的升高被抑制,即H2AX磷酸化是Ku80依赖性的；DSBs修复能力降低；石英诱导的Akt、JNK、AP-1 (c-Jun)磷酸化水平增加受抑制。3抑制Ku80表达,石英诱导的G1期细胞百分比进一步减少,S期细胞百分比进一步增加；E2F1蛋白表达及pRb-Ser780水平进一步增加；Cyclin D1、CDK4、CyclinE、CDK2、p53、p53(serl5)、p21表达及磷酸化水平增加受抑制。4抑制PI3K活性,石英诱导的Ku70、Ku80和γH2AX表达及磷酸化水平升高受抑制,对DNA-PKcs表达无影响；DSBs修复能力降低。根据以上结果,本研究结论如下：1 Ku80是DNA-PK发挥DSBs感受器功能的必要组成成分,通过调节DNA-PK/JNK信号通路促进石英致DSBs的修复。2 Ku80通过DNA-PK/JNK信号通路正性调控CyclinD1、CDK4、CyclinE、CDK2表达水平；通过DNA-PK/p53信号通路正性调控p21的表达,负性调控E2F1表达及pRb-Ser780磷酸化水平。Ku80抑制石英诱导的G1期细胞比例减少及S期细胞比例增加。3 PI3K通过调节Ku70、Ku80的表达,影响DNA-PK全酶的活性,促进石英致DSBs的修复。综上所述,石英诱导的细胞周期改变是各信号分子综合调控的结果。Ku80参与石英诱导的细胞周期改变,引起G1／S期阻滞,为DSBs修复赢得时间,并通过调节DNA-PK/Akt/JNK/AP-1信号通路,促进石英致DSBs的修复。本研究探讨了Ku80在石英致DSBs修复的DNA-PK/JNK信号转导通路中的作用,所得结果加深了对石英致病分子机制的理解,为深入研究石英的致病机理提供了线索。更多还原

【Abstract】 Background and objective:Silica is one of the most serious occupational hazards capable of inducing lung fibrosis and lung cancer after chronic exposure. Previous studies showed that silica exposure can induce cell cycle alternations and DNA double-strand breaks (DSBs).Our previous studies showed that silica exposure can induce cell cycle alternations, accompanied with the increased percentages of cells in S phase, and the marked activation of serine/threonine kinase protein kinase B (PKB/Akt), activator protein-1 (AP-1) as well as mitogen activated protein kinase (MAPK). Moreover, Akt/ERK, JNK pathway medicates silica-induced activator protein 1 (AP-1) transactivation, the expression of cyclin Dl and cyclin-dependent kinase 4 (CDK4) as well as cell cycle alternations.DNA-dependent protein kinase (DNA-PK), composing of a large catalytic subunit, DNA-PKcs, and a regulatory component, the Ku70-Ku80 heterodimer, is a molecular sensor for DNA damage, and is involved in the repair of DSBs by nonhomologous end-joining (NHEJ) pathway, in which Ku70-Ku80 plays a key role.It has been reported that DNA-PK phosphorylates a number of proteins, including Akt, JNK, p53, and many transcription factor such as c-Jun, c-Fos, which eventually contribute to damage-induced cell cycle arrest or cell apoptosis. Our previous studies showed that DNA-PKcs involves silica-induced cell cycle arrest and DSBs repair by DNA-PK/Akt/JNK/AP-1 signaling pathway.However, Roles of Ku80 in DNA-PK/JNK signaling pathway mediates silica-induced DSBs repair are not clear. Another, DNA-PK comprises same catalytic domain with Phosphatidylinositol 3 kinase (PI3K), which also involves silica-induced cell cycle alternations and activation of MAPK. Whether PI3K involves in repair of DSBs is neither known.Based on above studies, our current studies, which take DSBs and the repair as the breakthrough point, further focused on tracing the upstream sensor of our previous MAPKs pathway, the regulatory component (Ku80 mainly) of DNA-PK, and the biological endpoints of DSBs damage repair effect. In this study, RNAi, dominant negative mutants as well as chemical inhibitors, were used to investigate the roles of Ku80 in DNA-PK/JNK pathway mediates silica-induced DSBs repair as well as the potential effect of this pathway on silica-induced cell cycle and cell cycle regulatory proteins alternation in human embryonic lung fibroblast (HELF), and to detect the upstream or downstream relationship of signaling pathway.MethodsKu80 siRNA expression vectors was transfected into HELF by lipofectamine. Neutral comet assay and/or yH2AX recognition technology were applied to detect silica-induced DNA double strand breaks. According to the neutral comet experimental result, the DNA repair ability (DNA repair compentence, DRC) was calculated. According to the DRC value, the roles of Ku80 in the silica-induced DSBs repair were analyzed. The expression levels and activity of protein in HELF, such as DNA-PKcs, Akt, JNK, c-Jun, p53, p21, Cyclin D1, CDK4, Cyclin E, CDK2, E2F1, pRb were determined by WB. Cell cycle changes were identified by flow cytometry in HELF. The formation of yH2AX foci in HELF were analyzed by immunofluorescence microscopy.Result1. Stable transfectants, H-NC and H-Ku80 were established successfully.2. Both western blot and immunofluorescence assay analysis indicated that siRNA-mediated silencing of Ku80 strikingly downregulated the silica-induced expression of yH2AX in HELF. It indicates that H2AX phosphorylation is through Ku80 dependent pathway, and Ku80 effects the function of DNA-PK which acts as silica-induced DNA double strand breaks damage sensor.Silencing of Ku80 in HELF cells resulted in a decreased of silica-induced DNA damage repair competence compared with the negative control cell.After the expression of Ku80 was inhibited, the increase of phosphorylation of Akt at Ser473, JNK at Thr183/Tyr185, and c-Jun at ser63, which induced by silica exposure were potently blocked in HELF cells.3. When Ku80 expression was inhibited, the number of S phase cells was marked increased, and the overexpression of p21, p53, CyclinD1, CDK4, Cyclin E, CDK2, and the phosphorylation level of p53 at ser15 were potently blocked. however, the overexpression of E2F1 and the phosphorylation level of pRb-Ser780 were further increased.4. Blocking activation of PI3K by dominant negative mutant in DN-p85 cell, silica-induced DNA damage repair competence was decreased compared with the negative control cell induced by silica. And expression levels of Ku70 and Ku80, not DNA-PKcs, were also decreased.Conclusion1. Ku80 regulates the function of DNA-PK as silica-induced DSBs damage sensor and promotes DSBs repair by DNA-PK/Akt/JNK/AP-1 signaling pathway.2. Ku80 mediates silica-induced cell cycle change by DNA-PK/JNK and DNA-PK/p53 signaling pathway.3. PI3K promotes silica-induced DSBs damage repair by regulated expression levels of Ku70 and Ku80.In brief, this research had initially proven the roles of Ku80 in DNA-PK/JNK pathway in silica-induced DSBs repair. These findings will help us to understand the signal transduction mechanisms involved in the pathogenesis effects of silica at DNA damage reponse level.更多还原