Chronological mitochondrial proteomics of chemically induced hepatocellular carcinoma in rat

Although disruption of mitochondrial function has been associated with energetic deregulation in cancer, the chronological changes in mitochondria during cancer development remain unclear. With the aim to assess the role of mitochondria throughout cancer development, we analyzed samples chronologically obtained from induced hepatocellular carcinoma (HCC) in rats. In our analyses, we integrated mitochondrial proteomic data, mitochondrial metabolomic data and nuclear genome transcriptomic data. We used pathway over-representation and weighted gene co-expression network analysis (WGCNA) to integrate expression profiles of genes, miRNAs, proteins and metabolite levels throughout HCC development. Our results show that mitochondria are dynamic organelles presenting specific modifications in different stages of HCC development. For carcinogenesis, a modified resistant hepatocyte model was used. Rats were initiated with diethylnitrosamine (DEN) (200 mg/kg of body weight) at day 0. Then, 2-acetylaminofluorene (AAF) was administered (20 mg/kg per dose) at days 7, 8 and 9, followed by 3/5 partial hepatectomy (PH) at day 10. Three control groups of non-treated animals were sacrificed by exsanguination on the first day and at 9 and 18 months after the beginning of the experiment. Treated animals were sacrificed at 1, 7, 11 and 16 days and at 1, 9 and 18 months. Their livers were excised, washed in physiological saline solution, frozen with liquid nitrogen in 2-methyl butane and stored at -80°C. Once selected and separately collected nodular, tumoral and its adjacent tissues, total RNA was isolated using TriPureIsolation Reagent (Roche) according to the manufacturer’s protocol. The microarray analysis was performed using GeneChip Rat Exon 1.0 ST Arrays. Four replicas for each condition and controls were analyzed. This created 11 pairwise contrasts for the differential expression analysis. We grouped all evaluated conditions in three categories: i) early stages, including samples obtained since day one until one month after treatment, these were compared against rats without treatment sacrificed the first day; ii) tumor, nodular and its adjacent tissues obtained from nine months rats were compared against rats without treatment of the same age; and iii) tumor and its adjacent tissues obtained from eighteen months rats were compared against rats without treatment of the same age.

尽管线粒体功能异常已被证实与癌症的能量代谢紊乱相关，但癌症发生发展过程中线粒体的时序性变化仍尚不明确。为明确线粒体在癌症全程中的作用，本研究对大鼠诱发性肝细胞癌（hepatocellular carcinoma, HCC）的时序采集样本进行了分析。研究中，我们整合了线粒体蛋白质组学数据、线粒体代谢组学数据以及核基因组转录组学数据，并采用通路富集分析（pathway over-representation analysis）与加权基因共表达网络分析（weighted gene co-expression network analysis, WGCNA），整合了肝细胞癌发生发展全程中基因、微小RNA（microRNA, miRNA）、蛋白质及代谢物的表达谱数据。

研究结果表明，线粒体是动态变化的细胞器，在肝细胞癌发展的不同阶段呈现特异性的修饰改变。本研究采用改良的抵抗性肝细胞模型构建诱癌体系：于实验第0天，以二乙基亚硝胺（diethylnitrosamine, DEN）按200 mg/kg体重的剂量对大鼠进行造模启动；随后在第7、8、9天给予2-乙酰氨基芴（2-acetylaminofluorene, AAF），单次剂量为20 mg/kg体重，并于第10天进行3/5肝部分切除术（partial hepatectomy, PH）。

设置3组未造模的对照组大鼠，分别于实验开始当日、第9个月及第18个月通过放血法处死。造模组大鼠则分别于实验第1、7、11、16天以及第1、9、18个月处死。处死后快速摘取肝脏，用生理盐水冲洗后置于2-甲基丁烷中用液氮速冻，并于-80℃冰箱保存。选取并分别收集结节组织、肿瘤组织及其邻近正常组织后，按照试剂盒说明书，使用TriPureIsolation Reagent（罗氏）提取总RNA。采用GeneChip大鼠外显子1.0 ST芯片进行微阵列分析。每个实验条件及对照组均设置4个生物学重复，共获得11组用于差异表达分析的两两比较组合。我们将所有评估样本分为三类：① 早期阶段：采集自治疗开始当日至治疗后1个月的样本，与当日处死的未造模大鼠样本进行比较；② 中期肿瘤阶段：采集自第9个月大鼠的结节组织、肿瘤组织及其邻近正常组织，与同年龄段未造模大鼠样本进行比较；③ 晚期肿瘤阶段：采集自第18个月大鼠的肿瘤组织及其邻近正常组织，与同年龄段未造模大鼠样本进行比较。