Cai Zhipeng, Zheng Fuli, Ding Yan, Zhan Yanting, Gong Ruijie, Li Jing, Aschner Michael, Zhang Qunwei, Wu Siying, Li Huangyuan
Fujian Provincial Key Laboratory of Environmental Factors and Cancer.
The Key Laboratory of Environment and Health, School of Public Health, Fujian Medical University, Fuzhou 350122, China.
Toxicol Sci. 2019 Oct 1;171(2):515-529. doi: 10.1093/toxsci/kfz162.
Laboratorial and epidemiological research has established a relationship between paraquat (PQ) exposure and a risk for Parkinson's disease. Previously, we have investigated the effects of nuclear factor erythroid 2 related factor 2 (Nrf2) and microRNAs in PQ-induced neurotoxicity, addressing the function of miR-380-3p, a microRNA dysregulated by PQ, as well as Nrf2 deficiency. Nrf2 is known to mediate the expression of a variety of genes, including noncoding genes. By chromatin immunoprecipitation, we identified the relationship between Nrf2 and miR-380-3p in transcriptional regulation. qRT-PCR, Western blots, and dual-luciferase reporter gene assay showed that miR-380-3p blocked the translation of the transcription factor specificity protein-3 (Sp3) in the absence of degradation of Sp3 mRNA. Results based on cell counting analysis, annexin v-fluorescein isothiocyanate/propidium iodide double-staining assay, and propidium iodide staining showed that overexpression of miR-380-3p inhibited cell proliferation, increased the apoptotic rate, induced cell cycle arrest, and intensified the toxicity of PQ in mouse neuroblastoma (N2a [Neuro2a]) cells. Knockdown of Sp3 inhibited cell proliferation and eclipsed the alterations induced by miR-380-3p in cell proliferation. Two mediators of apoptosis and cell cycle identified in previous studies as Sp3-regulated, namely cyclin-dependent kinase inhibitor 1 (p21) and calmodulin (CaM), were dysregulated by PQ, but not Sp3 deficiency. In conclusion, Nrf2-regulated miR-380-3p inhibited cell proliferation and enhanced the PQ-induced toxicity in N2a cells potentially by blocking the translation Sp3 mRNA. We conclude that CaM and p21 were involved in PQ-induced toxicity.
实验室和流行病学研究已证实百草枯（PQ）暴露与帕金森病风险之间存在关联。此前，我们研究了核因子红细胞2相关因子2（Nrf2）和微小RNA在PQ诱导的神经毒性中的作用，探讨了受PQ失调的微小RNA miR-380-3p的功能以及Nrf2缺乏的影响。已知Nrf2可介导多种基因的表达，包括非编码基因。通过染色质免疫沉淀，我们确定了Nrf2与miR-380-3p在转录调控中的关系。定量逆转录聚合酶链反应（qRT-PCR）、蛋白质免疫印迹法和双荧光素酶报告基因检测表明，miR-380-3p在不降解Sp3 mRNA的情况下阻断了转录因子特异性蛋白3（Sp3）的翻译。基于细胞计数分析、膜联蛋白V-异硫氰酸荧光素/碘化丙啶双染检测和碘化丙啶染色的结果表明，miR-380-3p的过表达抑制了小鼠神经母细胞瘤（N2a [Neuro2a]）细胞的增殖，增加了凋亡率，诱导细胞周期停滞，并增强了PQ的毒性。Sp3的敲低抑制了细胞增殖，并消除了miR-380-3p诱导的细胞增殖变化。先前研究中确定为受Sp3调控的两种凋亡和细胞周期介质，即细胞周期蛋白依赖性激酶抑制剂1（p21）和钙调蛋白（CaM），受PQ失调，但不受Sp3缺乏的影响。总之，Nrf2调控的miR-380-3p可能通过阻断Sp3 mRNA的翻译来抑制N2a细胞的增殖并增强PQ诱导的毒性。我们得出结论，CaM和p21参与了PQ诱导的毒性作用。
