miR-34a信号通路在全氟烷基化合物暴露所致小鼠肝脏毒性中的作用研究

Perfluoroalkyl substances (PFASs) are stable man-made compounds with many industrial and commercial uses. Several studies suggest many toxicological hazards in laboratory animals, and liver is one of the primary target organ for PFASs activity. Laboratory animal studies indicate that PFASs induce toxicological hazards in liver via a PPAR alpha agonistic mode, however, PPAR-agonistic mode can not explain all of the hepatotoxicity, and other factors may play important role in the processes. We found prominent changes in expression of miRNAs in mice after PFASs expsoure, specifically the increase of miR-34a. By targeting to plenty of genes, miR-34a involves in the regulation of many biological processes, including cell cycle progression, apoptosis, DNA repair and angiogenesis. In addition, miR-34a also modulates the levels of many key transcriptional factors, such as p53, SIRT, E2F3, c-Myc and c-Met, and play its physiological and pathological roles through the coordinated activation of these transcriptional targets. The alteration in miR-34a expression during PFASs exposure indicates that miR-34a network may be an important contributor to the hepatotoxicity of PFASs. To assess whether miR-34a modulated signaling pathway played a critical role in the hepatic toxicity of PFASs, now we had knocked the miR-34a gene out. Then we plan to feed WT and miR-34a knockout mice with PFASs. After exposure, the apoptosis, cell cycling and proliferation in liver of miR-34a knockout mice compared with non-knockout animals will be detected using TUNEL, DNA content assay with flow cytometry, and immunofluorescence assay with Ki-67 antibody. To assess the mechanism of miR-34a increase in PFASs exposed mice, we will study the CpG methylation level in its promoter about 30 kb upstream of the mature miR-34a using methylation-specific PCR, and the binding activity of p53 with its consensus binding sites by ChIP assay. The transcriptional and protein levels of the key regulation factors, including p53, SIRT1, c-Met, c-Myc, E2F3 will be monitored by qPCR and western blotting analyses, respectively. The regulation of potential new targets of miR-34a will be validated by 3' UTR reporter assay. To further confirm if the toxic phenomena of PFASs are highly miR-34a dependent, the primary hepatocyte cells will be isolated in miR-34a-deficient and normal animals, and a series of in vitro experiments after PFASs exposure, including cell growth assay, cell cycle analysis, caspase-3 activation, and cytotoxicity assay, will be carried out. Transfection of key factors in miR-34a pathway will also be carried out in knockout hepatocyte cells to study their rescue abilities. By these studies, we will identify the key factors from the multiple transcriptional regulators and targets in miR-34a network, and finally eliminate the role of miR-34a pathway played in the hepatic toxicity of PFASs.

全氟烷基化合物(PFASs)是一类新型、持久性有机污染物。PFASs暴露可导致多种肝脏毒性，但其分子机制尚未阐明。我们发现PFASs暴露动物引起肝脏miR-34a显著增高，且miR-34a调控的信号通路(包括p53，c-Myc，SIRT1和c-Met等重要调控因子)与PFASs所致肝脏病理间存在密切关联。由此，我们拟开展如下研究：miR-34a-/-鼠与野生型经PFASs暴露后的肝毒性效应差异；miR-34a活化的分子基础和miR-34a通路中关键调控分子及效应基因与PFASs所致肝毒性的关系；miR-34a-/-与野生型鼠原代肝细胞经PFASs暴露后增殖、应激和凋亡效应的改变，并通过细胞转染操控miR-34a，p53和c-Met等关键分子，研究对肝毒性的补救(rescue)效应，确定在PFASs肝毒性中发挥主导作用的分子。最终明确miR-34a通路在PFASs所致肝毒性中的作用。

miRNAs通过抑制靶mRNA翻译或切割靶mRNA发挥转录后基因调控功能。我们发现5 mg/kg/d PFOA灌胃BALB/c雄性小鼠28天后，miR-34a在肝脏中表达显著升高达10倍。而另外2个成员miR-34b和miR-34c并未发生显著改变。我们制作miR-34a开展了进一步研究，与miR-34a野生型经PFOA处理后在肝脏呈现10倍以上诱导形成对比，miR-34a敲除鼠miR-34a无表达迹象，表明基因敲除操作彻底成功。PFOA处理28天后，动物体重显著降低，而肝脏肿大是PFOA暴露更显著的表型，PFOA暴露导致肝脏增重近3倍。肝脏切片HE病理显示PFOA暴露导致肝细胞较未处理组增大2倍以上。血清肝功能相关生化指标ALT，AST和CHE等在PFOA处理组均明显升高，表明肝脏细胞受损明显。但是，在经PFOA处理的野生型和miR-34a敲除型小鼠之间，这些指标并无显著改变，表明miR-34a敲除并未增加或减弱PFOA对肝脏这些指标的影响。小鼠肝脏RNA-seq分析发现，各自基因型对照（未经PFOA暴露）比较，PFOA处理后，在野生型和miR-34a敲除型动物肝脏分别导致1,843和1,838基因显著改变（至少2倍差异，p < 0.05），这些DEGs经KEGG富集分析发现主要为脂代谢相关通路基因，包括PPAR信号通路等。而无论PFOA处理与否，miR-34a敲除鼠肝脏与野生鼠肝脏基因表达差异较小，主要为脂代谢相关基因，表明miR-34a不发挥细胞周期和凋亡调节作用，而是调控部分脂代谢相关基因。研究表明miR-34a的转录调控区存在保守的P53反应元件。acetyl-P53作为活性形式与P53反应元件结合发挥转录活化作用。我们发现PFOA处理后，P53总蛋白在miR-34a野生型和敲除型动物肝脏虽未改变，acetyl-p53却明显升高，表明PFOA处理下肝脏miR-34a的显著升高可能与P53活化（acetyl-P53升高）有关。另外，因FPOA和PFOS的毒性危害而面临或已被禁用，一些PFASs新型替代品不断涌现，其安全性问题更为近来关注的热点，因此，我们研究了PFASs新型替代品对肝脏的毒性，包括HFPO2，HFPO4和6:2 Cl-PFESA等，比较了它们与传统PFASs肝脏毒性的强弱。发现HFPO4和6:2 Cl-PFESA较其替代物FPOA和PFOS具