转录-代谢组学分析筛选乙型肝炎相关肝细胞癌早期诊断标志物

Transcription-metabolomics analysis in screening early diagnostic markers of hepatitis B virus-related hepatocellular carcinoma

目的采用非靶向代谢组学与转录组学技术分析乙型肝炎(hepatitis B virus,HBV)相关性肝细胞癌细胞差异表达的基因及代谢物,探讨HBV相关性肝细胞癌潜在的早期诊断标志物。方法取对数生长期Huh7细胞,分为实验组(转染HBV质粒)和对照组(转染空载质粒),转染48 h采用Western blot法检测2组乙肝表面抗原(hepatitis B surface antigen,HBsAg)、乙肝核心抗原(hepatitis B core antigen,HBcAg)蛋白相对表达量;提取2组细胞代谢物进行液相色谱-质谱分析并筛选出差异表达代谢物,进行京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)富集分析;提取2组细胞总RNA进行全转录组测序并筛选出差异表达基因,进行基因本体(gene ontology,GO)富集分析、KEGG富集分析;应用Pearson相关法和KEGG富集分析法分析差异表达基因与差异表达代谢物的相关性。结果转染48 h,实验组细胞HBsAg、HBcAg蛋白相对表达量(0.45±0.04、1.01±0.07)均高于对照组(0.10±0.05、0.25±0.06)(t=9.459,P=0.011;t=14.286,P=0.005)。实验组与对照组显著差异表达代谢物有90个,与对照组比较,实验组57个代谢物表达上调,33个代谢物表达下调;差异代谢物主要在亚油酸代谢、丙酮酸代谢、精氨酸和脯氨酸代谢、生物素代谢、视黄醇代谢等代谢通路上富集。实验组与对照组差异表达基因有431个,与对照组比较,实验组387个基因表达上调,44个基因表达下调;差异表达的基因主要在胶原蛋白结合、急性时相反应、胶原蛋白分解代谢、细胞外基质结合等条目上富集;差异表达基因高富集于紧密连接、细胞黏附分子、环磷酸腺苷信号通路等。差异表达基因和差异表达代谢物共同富集于氨基酸代谢、辅助因子和维生素、脂质代谢、碳水化合物代谢等通路。本研究绘制了明显差异表达的18个基因和15个代谢物的关联网络图。结论HBV感染使18个基因差异表达并导致肝脏代谢物异常表达;15个异常表达的代谢物有可能成为HBV相关肝细胞癌早期诊断的新标志物。

Objective To analyze the differentially expressed genes and metabolites of hepatitis B virus(HBV)-related hepatocellular carcinoma cells by untargeted metabolomics and transcriptomics,and to identify the potential new diagnostic markers of HBV-related hepatocellular carcinoma.Methods The Huh7 cells in logarithmic growth phase were divided into experimental group(transfected with HBV plasmid)and control group(transfected with empty plasmid).After transfection for 48 h,the relative expressions of hepatitis B surface antigen(HBsAg)and hepatitis B core antigen(HBcAg)were detected by Western blot.The differentially expressed metabolites were screened by liquid chromatography/mass spectrometry and were subjected to the Kyoto Encyclopedia of Genes and Genomes(KEGG)related pathway enrichment.Transcriptomics samples were collected by total RNA extraction technique,and the differentially expressed genes were screened for Gene Ontology and KEGG enrichment analysis.Pearson correlation coefficient and KEGG enrichment were applied to analyze the correlation between differentially expressed genes and metabolites.Results After 48-h transfection,the relative expressions of HBsAg and HBcAg proteins were higher in experimental group(0.45±0.04,1.01±0.07)than those in control group(0.10±0.05,0.25±0.06)(t=9.459,P=0.011;t=14.286,P=0.005).There were 90 significantly differentially expressed metabolites,in which 57 metabolites were up-regulated and 33 metabolites were down-regulated in experimental group compared with control group.The differential metabolites were mainly enriched in the metabolic pathways of linoleic acid metabolism,pyruvate metabolism,arginine and proline metabolism,biotin metabolism,and retinol metabolism.There were 431 differentially expressed genes,in which 387 genes were up-regulated and 44 genes were down-regulated in experimental group compared with control group.The differentially expressed genes were highly enriched in collagen binding,acute-phase response and collage catabolic process.The differentially expressed genes were highly concentrated in tight junction signaling pathway,cell adhesion molecules and cAMP signaling pathway.The differentially expressed genes and metabolites were co-enriched in amino acid metabolism,cofactors,vitamins,lipid metabolism,and carbohydrate metabolism.The association network of differently expressed 18genes and 15 metabolites was mapped.Conclusion HBV infection abnormally regulates the differential expression of 18genes and leads to a abnormal expression of liver metabolites,while these 15abnormally expressed metabolites identified in this study might be the new markers for the early diagnosis of HBV-related hepatocellular carcinoma.