BaP对ARNT/HIF-1β介导的AhR和HIF-1信号通路的影响

ARNT/HIF-1βis a pivot protein in the AhR and HIF-1 signal pathways. AhR and HIF-1αcombined with ARNT/HIF-1βand which activated the expression of CYP1A1,CYP1B1 and VEGF,CAⅨ genes. The methods of Real-time quantitative PCR and Western blot were used to assay the expression of mRNA and protein of CYP1A1, CYP1B1 and VEGF, CAⅨ genes. The influence of BaP on the AhR and HIF-1 signal pathways were studied by estimate the level of expression of CYP1A1,CYP1B1,VEGF and CAⅨ genes in normal rat trachea-bronchial epithelial cells and human lung carcinoma cells(A549). The methods of immunoprecipitation and Western blot were used to determine the degree of HIF-1αand AhR-bound ARNT in normal rat trachea-brochial epithelial cells and A549 cells. The cross talk of AhR and HIF-1 signal pathways were studied by estimate the degree of HIF-1α and AhR-bound ARNT. To know the influence of passive smoking to gene expression of CYP1A1,CYP1B1,VEGF and CAⅨ, the expression of CYP1A1, CYP1B1, VEGF and CAⅨ genes of passive smoking mouse lung tissues were determined by immunohistochemical staining methods. The difference of expression of CYP1A1,CYP1B1,VEGF and CAⅨ genes of lung cancer lesions and paracancerous tissues of Mongolia nationality and Han nationality people in Inner Mongolia Autonomous Region were determined by immunohistochemical staining methods. These results were also used to assay if there were nationality difference in the expression of CYP1A1,CYP1B1,VEGF and CAⅨ genes.The influence of BaP on AhR and HIF-1 signal pathways which were mediated by ARNT/HIF-1βwere illuminated on the level of genes, cells,tissues and bodies. The relationship of expression of CYP1A1, CYP1B1, VEGF and CAⅨ with the initiation and progression of lung cancer was clarified also.

ARNT/HIF-1β是AhR和HIF-1信号通路的枢纽蛋白，两条通路均和ARNT/HIF-1β结合后激活下游基因的表达。采用实时定量PCR和 Western blot法检测AhR信号通路的CYP1A1、CYP1B1和HIF-1信号通路的VEGF 、CAⅨ的mRNA和蛋白水平的表达,研究BaP对AhR和HIF-1信号通路的影响。采用免疫沉淀法和Western blot法，探讨AhR和HIF-1α与ARNT结合情况，阐明两条信号通路的相互作用。采用免疫组化法检测被动吸烟对小鼠肺组织CYP1A1、CYP1B1、VEGF和CAⅨ基因的表达的影响。检测内蒙古地区蒙、汉族肺癌患者的癌组织和癌旁正常组织中CYP1A1、CYP1B1、VEGF和CAⅨ基因的表达，分析表达是否存在民族差异。从不同层次阐明BaP对ARNT/HIF-1β介导的AhR和HIF-1信号通路的影响及这些基因表达与肺癌发生发展的关系。

1.采用RT-PCR、Western blot和免疫沉淀法研究BaP激活AhR信号通路时对HIF-1信号通路的影响。结果：BaP能促进AhR信号通路下游基因CYP1B1及CYP1A1和HIF-1信号通路下游基因CAⅨ及VEGF的mRNA和蛋白表达，并呈剂量依赖性(P<0.01)；与对照组相比BaP组的AhR及HIF-1α蛋白的表达量随着BaP浓度的增加而增加(P<0.01)，表明BaP激活AhR信号时不抑制HIF-1信号通路。2.采用RT-PCR、Western blot和免疫沉淀法研究在CoCl2和一定浓度的BaP同时存在时，A549细胞中AhR和HIF-1两条信号通路的相互作用。结果：300μMCoCl2处理A549细胞后，加入不同浓度BaP作用24h，VEGF、CAⅨ、CYP1A1和CYP1B1 mRNA和蛋白的表达量与对照组相比明显增加，差异具有统计学意义(P<0.01)；AhR与HIF-1β/ARNT的结合能力与对照组相比明显降低，差异具有统计学意义(P<0.01)，进一步表明激活HIF-1信号通路时抑制AhR信号通路。3.为进一步验证以上实验结果，课题组利用荧光能量共振转移技术研究在A549细胞中AhR与HIF-1α是否竞争结合ARNT/HIF-1β，进而更明确这两条信号通路之间的相互关系，结果：不同浓度的BaP处理后FRET荧光信号强度与对照组相比明显增加，差异具有显著性(P<0.05)；CoCl2处理后较无CoCl2组的FRET荧光信号强度减弱说明HIF-1α竞争AhR结合ARNT/HIF-1β，与前期实验结果一致。4.采用免疫组化法检测被动吸烟对小鼠肺组织中CYP1A1、CYP1B1、VEGF和CAⅨ表达的影响。结果显示：被动吸烟能上调CYP1A1、CYP1B1、VEGF、CAⅨ蛋白的表达，且随着被动吸烟剂剂量的增加各基因的阳性表达率增加。5.用Cytoscape3.0软件建立肺癌-蛋白表达-影响因素的网络模型，明确癌旁正常组织和癌组织中这些基因的表达差异与肺癌发展的关系。结果：肺癌与VEGF、CYP1A1等蛋白的表达存在一定联系。采用免疫组化法检测肺癌患者肺癌组织和癌旁正常组织中CYP1A1、VEGF的表达情况。结果显示：肺癌患者肺癌组织中VEGF、CYP1A1的表达率比癌旁正常组织中高(P＜0.05)，且两种蛋白的表达呈正相关(r=0.723)