circRNA_0114605调控miRNA-29b在水通道蛋白4介导的1,2-二氯乙烷中毒性脑水肿中的作用机制研究

Brain edema caused by 1,2-dichloroethane (1,2-DCE) exposure induces adverse health effects on occupational workers in China. The key to preventing this disease is to elucidate the poisoning mechanisms and to find early biomarkers. Aquaporin 4 (AQP4) plays a central role in brain edema. There are currently few investigative publications on the role of AQP4 and its microRNAs (miRNAs) and circleRNAs (circRNAs) regulation mechanisms in the occurrence and progression of 1,2-DCE-induced brain edema. Our preliminary results have established cell and animal models for brain edema treated with 1,2-DCE and/or its metabolites. Based on these results, we are going to study the role of circRNA_0114605-regulated miRNA-29b on the AQP4 and its function in the 1,2-DCE-induced brain edema on three levels of cell, animal, exposure population and poisoning cases, respectively. First, an in vitro cell model was applied to analyze the pattern of miRNA-29b and circRNA_0114605 regulations on AQP4 and its function in 1,2-DCE-induced brain edema in the presence or absence of high expression plasmid of AQP4 or CRISPR-Cas9 application, the miRNA-29b mimic or inhibitor, high expression plasmid or siRNA of circRNA_0114605, respectively. Then, miRNA-29b and AQP4 wild type and knockout mice were used to confirm the pattern of AQP4 expression and its regulation by miRNA-29b, as well as to explore the key molecular pathways between the miRNA-29b alteration and pathological change in brain edema induced by 1,2-DCE by using proteomic analysis. Finally, an occupational epidemiology study with healthy exposed subjects and poisoning cases was carried out to confirm the results of the in vitro and in vivo studies. This study will shed light on the role of AQP4 and its miRNA and circRNA regulations in 1,2-DCE-induced brain edema. It will also provide scientific evidence on targets for the development of biomarkers and treatment of this occupational hazard.

1,2-二氯乙烷（1,2-DCE）中毒性脑水肿严重危害我国职业工人健康，防治的关键在于阐明中毒机制和寻找早期生物学标志。水通道蛋白4（AQP4）在脑水肿中发挥核心作用，微小RNA（miRNA）和环状RNA（circRNA）调控AQP4影响1,2-DCE中毒性脑水肿发生发展鲜见报道。本项目在构建1,2-DCE中毒性脑水肿细胞和动物模型的基础上，应用AQP4高表达质粒或CRISPR-Cas9抑制、miR-29b mimic或抑制剂、circRNA_0114605高表达质粒或siRNA、野生型和基因工程小鼠以及不同暴露水平的人群和病例，从正向和反向在细胞、动物和人群三个水平探讨1,2-DCE通过下调circRNA_0114605的表达促进miR-29b对AQP4的负性调控，AQP4的功能改变介导中毒性脑水肿的发生发展，揭示1,2-DCE中毒性脑水肿发生发展的分子机制和发现生物学标志及治疗靶标。

1,2-二氯乙烷（1,2-DCE）是一种普遍存在的环境污染物，过量的高浓度接触1,2-DCE可致动物和人类的中毒性脑水肿。表观遗传调控是1,2-DCE致脑水肿发生的重要机制之一。CircRNA作为转录后剪切生成的一类重要的非编码环状RNA分子，可通过miRNA调控下游靶基因的表达进而影响蛋白功能。但目前circRNA是否可以调控miR-29b影响AQP4的表达在1,2-DCE致中毒性脑水肿发挥作用尚不明确。通过对人源性星形胶质细胞（SVG p12）进行1,2-DCE（0、12.5、25.0、50.0 μM）处理。应用circRNA微阵列芯片、电镜形态学检查、同位素3H标记结合3- O-甲基葡萄糖（3-OMG）摄取法测量细胞的含水量，以及分子间相互调控作用的功能研究，探讨1,2-DCE引起星形胶质细胞水肿的circRNA-miR-AQP4表观遗传调控机制。1,2-DCE可引起星形胶质细胞水肿，表现为含水量增加，空泡增加和线粒体肿胀，并伴随miR-29b-3p的下调和AQP4的上调。分别对miR-29b-3p和AQP4的功能以及两者的相互调控关系进行验证，并结合双荧光素酶标记基因报告实验结果，证明1,2-DCE通过下调miR-29b-3p，靶向上调AQP4引起的星形胶质细胞水肿。结合circRNA芯片结果，筛选出2个circRNA（circPRSS12和circBCL11B）与1,2-DCE的处理以及对miR-29b-3p调控密切相关。通过在细胞水平上的调控关系和功能验证，我们发现只有circBCL11B可通过miR-29b-3p对AQP4进行调控。即过表达circBCL11B可发挥其内源性竞争作用，通过miR-29b-3p引起AQP4的表达上调，并导致细胞水肿；但敲减circBCL11B可逆转由1,2-DCE诱导的miR-29b-3p的下调和AQP4的上调，缓解细胞水肿。最后，我们通过双荧光素酶标记基因法以及荧光原位杂交技术，证明了circBCL11B与miR-29b-3p的靶向结合。本研究发现1,2-DCE通过circBCL11B、miR-29b-3p和AQP4之间的靶向内源性竞争调控关系，引起星形胶质细胞水肿，诱发脑水肿的发生。