Reciprocal interaction of Wnt and RXR-α pathways in hepatocyte development and hepatocellular carcinoma

Genomic analysis of human hepatocellular carcinoma (HCC) is potentially confounded by the differentiation state of the hepatic cell-of-origin. Here we integrated genomic analysis of mouse HCC (with defined cell-of-origin) along with normal liver development. We found a major shift in expression of Wnt and RXR-α pathway genes (up and down, respectively) coincident with the transition from hepatoblasts to hepatocytes. A combined Wnt and RXR-α gene signature categorized HCCs into two subtypes (high Wnt, low RXR-α and low Wnt, high RXR-α), which matched cell-of-origin in mouse models and the differentiation state of human HCC. Suppression of RXR-α levels in hepatocytes increased Wnt signaling and enhanced tumorigenicity, whereas ligand activation of RXR-α achieved the opposite. These results corroborate that there are two main HCC subtypes that correspond to the degree of hepatocyte differentation and that RXR-α, in part via Wnt signaling, plays a key functional role in the hepatocyte-like subtype and potentially could serve as a selective therapeutic target. Total RNA from whole livers taken at different developmental timepoints (embryonic day 14, embryonic day 18, post-natal day 5 and post-natal day 56) along with hepatoblasts isolated from E14 livers and immature hepatocytes isolated from E18 livers was extracted and purified using the Qiagen RNeasy Mini Kit. RNA purity and integrity were assayed by the Bioanalyser 2100 (Agilent Technologies). For each sample, 2 µg of total RNA was reverse transcribed and amplified by using an RNA amplification kit from Ambion. Fifteen micrograms of amplified RNA were labeled by direct chemical coupling to the Cy5 NHS ester (Amersham Biosciences). Normal adult mouse liver (Agilent) was used as control and Cy3 labeled. Labeled RNAs were purified, fragmented, and used as probes to hybridize microarrays. Gene expression profiling was done with the 4x44k mouse Agilent platform. Expression profiling of the 23 human HCC samples was previously described人类肝细胞癌（hepatocellular carcinoma, HCC）的基因组分析可能会受到起源肝细胞分化状态的干扰。本研究整合了具有明确起源细胞的小鼠肝细胞癌基因组分析数据与正常肝脏发育的组学数据。我们发现，在肝母细胞向肝细胞转化的过程中，Wnt通路与视黄醇X受体-α（retinoid X receptor alpha, RXR-α）通路基因的表达发生显著改变：Wnt通路基因表达上调，而RXR-α通路基因表达下调，二者的变化恰好与该转化进程同步。基于Wnt与RXR-α通路的联合基因特征，可将肝细胞癌划分为两个亚型——高Wnt、低RXR-α亚型与低Wnt、高RXR-α亚型，这两个亚型分别与小鼠模型中的肿瘤起源细胞以及人类肝细胞癌的分化状态相匹配。在肝细胞中抑制RXR-α的表达水平，可增强Wnt信号通路活性并提升成瘤能力；而通过配体激活RXR-α则会产生相反的效应。上述结果证实，肝细胞癌存在两种主要亚型，其分型与肝细胞的分化程度相对应；且RXR-α可部分通过调控Wnt信号通路，在肝细胞样亚型中发挥关键的功能性作用，有望成为选择性治疗靶点。本研究提取并纯化了不同发育时间点（胚胎第14天、胚胎第18天、出生后第5天及出生后第56天）的全肝脏总RNA，以及从胚胎第14天肝脏中分离的肝母细胞与胚胎第18天肝脏中分离的未成熟肝细胞总RNA，所用试剂盒为Qiagen RNeasy Mini Kit。采用Agilent Technologies公司的Bioanalyser 2100检测RNA的纯度与完整性。每份样本取2 μg总RNA，使用Ambion公司的RNA扩增试剂盒完成反转录与扩增。取15 μg扩增后的RNA，通过直接化学偶联的方式与Cy5 NHS酯（Amersham Biosciences）进行标记。以正常成年小鼠肝脏（Agilent）作为对照样本，采用Cy3进行标记。标记后的RNA经纯化、片段化后，作为探针用于基因芯片杂交实验。基因表达谱分析采用4×44k小鼠Agilent芯片平台完成。23例人类肝细胞癌样本的表达谱分析此前已有报道。