Liu Xiao-hong, Chen Ying, Wang Li, Zhao Hai-hua, Zhu Ming-hua
Department of Pathology, Changhai Hospital, Shanghai 200433, China.
Zhonghua Yi Xue Za Zhi. 2005 Dec 21;85(48):3425-9.
To investigate the mechanism of the different biological impacts of HBx3'-40, an engineered deletion mutant lacking the last 40 C-terminal amino acids.
Human hepatocellular cells of the line Huh7 were transfected with HBx3'-40 or wtHBx (wild type HBx) constructs. An oligo cDNA microarray containing 21074 human genes and Ests was utilized to examine the different gene expression between HBx3'-40 and paired control wtHBx cells. A series of methods, including immobilized pH gradient two-dimensional polyacrylamide gel electrophoresis (2-DE), silver staining, and PDQuest 2-D analysis software were used to analyze the differential protein-spots between HBx3'-40 and wtHBx cells. Selected differential protein-spots were identified with peptide mass fingerprinting based on matrix-assisted laser desorption/ionization time-of-fight mass spectrometry (MALDI-TOF-MS) and database searching.
165 (0.82%) candidate genes showing aberrant expression under HBx3'-40 induction were identified, of which 144 were up-regulated while 21 were down-regulated. Compared with wtHBx group, there were 135 +/- 13 differently protein spots in HBx3'-40 group by 2-DE. Of them, 7 significantly different protein spots were identified using mass spectrometry and computer matching with protein database. Most of the differently expressed genes and proteins were involved in transcription, oncogenes and tumor suppressor genes or protein, cell adhesion, signal transduction pathways, metabolisms, etc.
The mutant HBx affects cell genes and proteins involved in various processes, especially in regulation of liver metabolism.
研究HBx3'-40（一种缺失C末端最后40个氨基酸的工程缺失突变体）产生不同生物学影响的机制。
用HBx3'-40或野生型HBx（wtHBx）构建体转染人肝癌细胞系Huh7。利用包含21074个人类基因和EST的寡核苷酸cDNA微阵列检测HBx3'-40与配对的对照wtHBx细胞之间的基因表达差异。采用一系列方法，包括固定化pH梯度二维聚丙烯酰胺凝胶电泳（2-DE）、银染和PDQuest二维分析软件，分析HBx3'-40与wtHBx细胞之间的差异蛋白质斑点。基于基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF-MS）和数据库搜索，通过肽质量指纹图谱鉴定选定的差异蛋白质斑点。
鉴定出165个（0.82%）在HBx3'-40诱导下表现出异常表达的候选基因，其中144个上调，21个下调。与wtHBx组相比，HBx3'-40组通过2-DE有135±13个不同的蛋白质斑点。其中，通过质谱分析和与蛋白质数据库的计算机匹配，鉴定出7个显著不同的蛋白质斑点。大多数差异表达的基因和蛋白质涉及转录、癌基因和肿瘤抑制基因或蛋白质、细胞粘附、信号转导通路、代谢等。
突变型HBx影响参与各种过程的细胞基因和蛋白质，尤其是肝脏代谢的调节。
