脂肪积聚肝恶性转化模型制备及其生物信息学分析

Establishment of malignant transformation model in lipid accumulating liver and its bioinformatics analysis

目的:制备非酒精性脂肪肝(nonalcoholic fatty liver disease,NAFLD)和诱导肝细胞恶性转化模型,观察基因表达谱动态变化。方法:雄性Sprague-Dawley(SD)大鼠分别以高脂饲料喂饲制备非酒精性脂肪积聚,以含0.05%2-乙基氨基芴(2-fluorenylacetamide,2-FAA)高脂饲料喂饲诱发鼠肝细胞恶性转化,根据肝病理学苏木精-伊红(Hematoxylin&Eosin,HE)染色检查结果分为对照(normal control,NC)、NAFLD、肝细胞变性(hepatocyte degeneration,H-deg)、癌前(precanceraion,Pre-c)和癌变(liver cancer,LC)组,脂质以油红O染色行定量分析,从肝组织抽提RNA并合成cRNA、标记、杂交和扫描,利用基因芯片分析各组差异基因表达谱动态变化,对差异表达基因行基因本体论(gene ontology,GO)和京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)富集分析。结果:SD鼠摄入脂肪后,肝脂肪积聚与NC组比较,NAFLD组升高9倍(t=14.78,P<0.001)、H-deg组2.4倍(t=5.96,P<0.001)、Pre-c组2.3倍(t=8.37,P<0.001)和LC组8倍(t=11.97,P<0.001)。与NC组比较全基因表达谱变化,差异表达基因NAFLD组163个,H-deg组934个,Pre-c组1452个,LC组1738个。生物学过程主要集中在细胞对刺激反应的代谢调节。肝细胞恶化转化中信号通路主要有固醇合成、P53、细胞周期信号通路,其中固醇合成通路Cyp51,Tm7sf2显著下调,Ccnb1和周期蛋白依赖性激酶1(cyclin-dependent kinases 1,CDK1)与P53通路和细胞周期相关。癌变相关标志甲胎蛋白(alpha-fetoprotein,AFP)、磷脂酰肌醇蛋白多糖-3(glypican-3,GPC-3)、CD44和Wnt3a等表达,在Pre-c组和LC组中明显上调。结论:脂肪积聚肝细胞恶性转化过程中,众多基因和信号通路参与癌变发生过程。

Objective:To analyze the gene dynamic expressions during rat hepatocyte malignant transformation nonalcoholic fatty liver disease(NAFLD)by bioinformatics.Methods:Male Sprague-Dawley(SD)rats were fed with high fat(HF),and HF containing 0.05%2-fluorenylacetamide(2-FAA)diet.Rats according to the Hematoxylin&Eosin(HE)staining of livers were divided into normal control(NC),NAFLD,hepatocyte degeneration(H-deg),precanceraion(Pre-c),and liver cancer(LC)groups.Lipids were dyed with the Oil Red O.RNA of each group was extracted and then cRNA was synthesized,labeled,hybridized and scan.Differentially expressed genes related with hepatocyte malignant transformation were screened,and further analyzed by gene ontology(GO)&Kyoto encyclopedia of genes and genomes(KEGG)enrichment analysis.Results:After the rats with HF and/or HF+2-FAA diets,the lipid accumulation was significantly increasing with 9 folds in the NAFL group(t=14.78,P<0.001),2.4 folds in the H-deg group(t=5.96,P<0.001),2.3 folds in the Pre-c group(t=8.37,P<0.001),and 8 folds the LC group(t=11.97,P<0.001)after compared with the NC group.Differentially genes were found by the gene expression profiling chip with the NC group,there were 163 genes in the NAFLD group,934 genes in the H-deg group,1452 genes the Pre-c group and 1738 genes in the LC group,respectively.Steroid biosynthesis pathway,P53 signaling pathway,cell cycle pathway were the main signaling pathways found that related to the rat hepatocyte malignant transformation under lipid accumulation.The Cyp51 and Tm7sf2 in the sterol synthesis pathway were significantly down regulated in the whole process of malignant transformation of hepatocyte.Ccnb1 and cyclin-dependent kinases 1(CDK1)are not only in the P53 pathway but also in the cell cycle.Biological processes are mainly concentrated in metabolic regulation of cell response to stimuli.Biomarker genes of liver cancer such as alpha-fetoprotein(AFP),glypican-3(GPC-3),CD44,and Wnt3a expression were abnormally increasing in the Pre-C or LC group at transcriptional level.Conclusion:Dynamic model of adipose-accumulated hepatocytes suggested numerous genes and multiple signal pathways be involved in the development of hepatocyte malignant transformation.