Differentially expressed genes in the livers of mice exposed to microcystin-LR (MC-LR). Differentially expressed genes in the livers of mice exposed to microcystin-LR (MC-LR)

To explore the molecular mechanisms involved in the toxicity in the livers exposed to MC-LR at the environmental level, the hepatic transcriptome was performed. A total of 210 genes were differentially expressed (P<0.05, |fold change|≥2) in response to MC-LR exposure; among them, 143 genes were significantly upregulated, and 67 genes were downregulated. Pathway enrichment analysis identified the top biological functions associated with the genes differentially expressed in response to MC-LR exposure, which were circadian regulation of gene expression, negative regulation of glucocorticoid receptor signaling pathway, the epoxygenase P450 pathway, regulation of insulin secretion, lipid metabolic process, and cell cycle pathway. Overall design: Six-week-old male BALB/c mice were assigned randomly to two groups (one control and one treatment). Mice in treatment were exposed to 100 μg/L microcystin-LR through drinking water for 12 months. The control mice were given normal sterile water. At the end of exposure time, three liver samples from independent mice in each group were obtained to analyze the gene expression by microarray assay. Total RNA was extracted, amplified and labeled using a One-Color LowInput Quick Amp Labeling Kit (Agilent, Santa Clara, CA, USA). Labeled cRNA was purified with an RNeasy Mini Kit (Qiagen, Hilden, Germany). The slides were then hybridized with Cy3-labeled cRNA using Gene Expression Hybridization Kit in a hybridization oven (Agilent, Santa Clara, CA, USA), followed by washing in staining dishes (Thermo, Waltham, MA, USA) with Gene Expression Wash Buffer Kit (Agilent, Santa Clara, CA, USA). An Agilent Microarray Scanner was used to scan the slides. Data were extracted by the Feature Extraction software and normalized by the Quantile algorithm of Gene Spring software.

为探究环境浓度下微囊藻毒素-LR（MC-LR）暴露致肝脏毒性的分子机制，本研究开展了肝脏转录组学分析。结果显示，MC-LR暴露后共有210个基因呈现差异表达（P<0.05，|折叠变化|≥2），其中143个基因显著上调，67个基因显著下调。通路富集分析鉴定出与MC-LR暴露响应的差异表达基因相关的核心生物学功能，包括基因表达的昼夜节律调控、糖皮质激素受体信号通路负调控、细胞色素P450环氧酶通路、胰岛素分泌调控、脂质代谢过程以及细胞周期通路。
实验设计：将6周龄雄性BALB/c小鼠随机分为两组（对照组与处理组）。处理组小鼠通过饮用水暴露于100 μg/L的微囊藻毒素-LR，持续12个月；对照组小鼠则给予正常无菌饮用水。暴露结束后，从每组各3只独立小鼠中获取肝脏样本，通过微阵列实验分析基因表达水平。
总RNA提取、扩增与标记采用One-Color LowInput Quick Amp Labeling Kit（安捷伦，美国加利福尼亚州圣克拉拉）完成。标记后的cRNA通过RNeasy Mini Kit（凯杰，德国希尔德）进行纯化。随后使用Gene Expression Hybridization Kit（安捷伦，美国加利福尼亚州圣克拉拉）在杂交炉中与Cy3标记的cRNA进行玻片杂交，再使用Gene Expression Wash Buffer Kit（安捷伦，美国加利福尼亚州圣克拉拉）在染色皿（赛默飞，美国马萨诸塞州沃尔瑟姆）中完成洗涤。采用安捷伦微阵列扫描仪对玻片进行扫描。数据通过Feature Extraction软件提取，并通过Gene Spring软件的Quantile算法完成标准化处理。