环境镉和多氯联苯联合暴露慢性损伤环状RNA circHIPK3分子调控机制及分析方法研究

Typical pollutants cadmium (Cd) and polychlorinated biphenyl (PCB) often cause environmental pollution by incineration of e-waste. Chronic health hazards attributed to their long-term, continuous and joint exposure have been paid much attentions in environmental medicine and toxicology. However, the chronic damage caused by co-exposure and control mechanism of the network signaling are not fully clear. circHIPK3 as one kind of circRNA, is considered as a newly active regulatory molecule for miRNA, which is closely correlated to environment-related diseases. To clarify the molecular regulation mechanism of circHIPK3 during the Cd-PCB co-exposure resulted in chronic damage, we will treat the target cell lines in groups according to their different doses. Firstly, the damage effect will be quantitatively analyzed, and the differences in gene expression profiling and expression level can be determined through circRNA-Seq and bioinformatics technology. Then, we will screen the binding sites of circHIPK3 and obtain the sponge microRNAs. By exploring the role of ceRNA in the regulation of corresponding target gene and the expression of downstream regulation protein, the regulation network mechanism of downstream miRNA-mRNA signaling will be analyzed. Afterwards, the key parameters will be analyzed in real-time by total internal reflection imaging ellipsometry biosensor and biomarkers will be screened combining with the high-throughput circRNA chip and suspension array. It will provide scientific evidences for ecological security assessment of co-exposure of multiple pollutants.

典型污染物镉(Cd)和多氯联苯(PCB)常通过电子垃圾焚烧污染环境，二者长期持续联合暴露所致的慢性健康危害，备受环境医学和毒理学界关注，但联合暴露所致慢性损伤及网络信号调控机制尚不完全清楚。环状RNA circHIPK3作为新型的miRNA活性调控分子，与环境相关疾病密切关联。为阐明circHIPK3在以Cd和PCB联合暴露致慢性损伤的分子调控机制，本项目拟以二者不同分组作用于靶细胞系，从定量分析损伤效应入手，通过CircRNA-Seq和生物信息学技术，确定表达谱和表达水平差异，筛选circHIPK3结合位点，获取海绵作用miRNA；通过分析ceRNA调控相应靶基因及下游结合蛋白表达的作用，探寻下游miRNA-mRNA信号调控网络机制；采用电偏置相敏成像生物传感实时在线分析关键参数变化，结合高通量circRNA芯片和悬浮芯片筛选生物标志物，为多种污染物联合暴露的生态安全性评估提供科学依据。

典型污染物镉(Cd)和多氯联苯(PCB)常通过电子垃圾焚烧污染环境，二者长期持续联合暴露所致的慢性健康危害，备受环境医学和毒理学界关注，但联合暴露所致慢性损伤及网络信号调控机制尚不完全清楚。环状RNA circHIPK3作为新型的miRNA活性调控分子，与环境相关疾病密切关联。为阐明circRNA在以环境Cd和PCB联合暴露致损伤的分子调控机制，本项目选取了人类肝脏L-02细胞和秀丽隐杆线虫作为模式生物，分别对上述两种细胞系和模式生物进行分组染毒暴露研究，确定表达谱和表达水平差异；通过分析ceRNA调控相应靶基因及下游结合蛋白表达的作用，探寻了下游miRNA-mRNA信号调控网络机制；通过转录组测序和RT-qPCR筛选得到差异表达的cul-6，mom-2，dsh-2和vang-1，通过使用相关突变株，发现Wnt通路的关键调控因子，mom-2，dsh-2和vang-1，可以负调控诱导的生殖细胞凋亡。通过转录组测序和生物信息学分析筛选基因并验证环境Cd和PCB联合暴露潜在的生殖毒性机制，为环境污染物暴露的精准防控和靶点治疗提供新的理论依据。环境污染物重金属Cd和有机污染物PCB153联合暴露会严重损伤线虫的生长发育过程和生殖功能，长期暴露时会造成神经损伤；还可以减少线虫有丝分裂区和减数分裂区细胞数目，使线虫粗线期凋亡水平升高，并影响终变期卵母细胞的减数分裂成熟，但并未影响细线期和偶线期染色体的浓缩和联会；还可以减弱Wnt通路的信号传导，使生殖凋亡水平升高并影响减数分裂成熟。本研究建立了一套可以检测单分子在传感表面吸附时的最高占据能级（HUMO）以及最低未占据能级（LUMO）的单分子成像检测方案，部分实现细胞形态、circRNA 浓度、 生物标志物蛋白或核酸、电化学等多参数即时监测和分析。