Molecular characterisation of a mutant kras-driven zebrafish model of hepatocellular carcinoma

We used doxycycline (dox) treatment to generate a larval model of hepatocellular carcinoma (HCC)22. In this model, we induced the expression of a single EGFP-krasG12V transgene, denoted TO(krasG12V)T/+ in developing livers by treating with doxycycline between 2-7 days post-fertilisation (dpf). This led to the accumulation of a constitutively active, EGFP-tagged, potently oncogenic form of Kras specifically in hepatocytes, causing hepatocyte hyperplasia and a substantial increase in total liver volume. We used RNA sequencing to analyse the gene expression patterns of hepatocytes expressing the krasG12V transgene compared to wildtype (WT) livers expressing no transgene. We detected more than 6000 significantly upregulated genes in dox-treated TO(krasG12V)T/+ livers compared WT livers, and a further 6000+ genes were significantly downregulated. Of the upregulated genes, many were significantly enriched in KEGG pathways associated with highly proliferative cancers, including DNA replication, cell cycle and DNA damage repair. Geneset enrichment analysis identified a positive correlation between the differential gene expression data from the dox-treated TO(krasG12V)T/+ versus WT livers and the differential gene expression data for the HCC (LIHC) and healthy liver subsets available from The Cancer Genome Atlas. We also found a positive correlation between the DEGs from the dox-treated TO(krasG12V)T/+ versus WT livers and a small HCC expression signature based on four patient samples carrying KRAS G12 or KRAS G13 mutations. These observations build on previous reports that dox-treated TO(krasG12V)T/+ zebrafish provide an authentic model of human HCC. Overall design: Paired-end RNA-sequencing data was generated for 8 samples of zebrafish liver. Of these samples, 3 are wild type (WT) and 5 are kras mutants. Differential analyses were performed, identifying differentially expressed genes between the kras mutant and wild type groups. Please note, all WT and 2 of the mutant samples are sequenced over 2 runs.

本研究采用多西环素（doxycycline, dox）处理，构建了肝细胞癌（hepatocellular carcinoma, HCC）的斑马鱼幼鱼模型[22]。
在该模型中，我们通过在受精后2~7天（days post-fertilisation, dpf）施加多西环素处理，在发育中的肝脏中诱导单个EGFP-krasG12V转基因（transgene）的表达，该转基因被命名为TO(krasG12V)T/+。
这使得组成型激活、带有增强绿色荧光蛋白（enhanced green fluorescent protein, EGFP）标签且具有强致癌活性的Kras突变体特异性富集于肝细胞中，进而引发肝细胞增生，并使肝脏总体积显著增大。
本研究采用RNA测序（RNA sequencing, RNA-seq），对比分析了表达krasG12V转基因的肝细胞与未携带转基因的野生型（wildtype, WT）肝脏的基因表达谱。
相较于野生型肝脏，多西环素处理的TO(krasG12V)T/+肝脏中检出超过6000个显著上调基因，另有6000余个基因显著下调。
在上述上调基因中，大量基因显著富集于与高增殖性癌症相关的京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）通路，包括DNA复制、细胞周期及DNA损伤修复通路。
基因集富集分析（Gene Set Enrichment Analysis, GSEA）结果显示，多西环素处理的TO(krasG12V)T/+肝脏与野生型肝脏的差异基因表达数据，与癌症基因组图谱（The Cancer Genome Atlas, TCGA）中肝细胞癌（hepatocellular carcinoma, HCC，对应队列代号LIHC）及健康肝脏亚组的差异基因表达数据呈显著正相关。
本研究同时发现，上述两组肝脏的差异表达基因（differentially expressed genes, DEGs），与一项基于4例携带KRAS G12或KRAS G13突变患者样本的小型肝细胞癌表达特征集呈显著正相关。
上述结果佐证了既往研究结论，即多西环素处理的TO(krasG12V)T/+斑马鱼可作为模拟人类肝细胞癌的可靠模型。
实验整体设计：本研究共获取8份斑马鱼肝脏样本的双端RNA测序数据，其中3份为野生型（WT）样本，5份为kras突变型样本；通过差异表达分析，鉴定得到kras突变型与野生型肝脏样本间的差异表达基因。
需说明的是，所有野生型样本及2份突变型样本均通过2轮测序完成。