Liu J, Lian Z, Pan J
Institute of Digestive Diseases, Xijing Hospital, Xi'an 710032, China.
Zhonghua Yi Xue Za Zhi. 2000 Jun;80(6):456-60.
To clone full length differentially expressed genes which are related with HBxAg.
HepG2-cells were infected with prepared recombinant retroviruses encoding the X antigen. The differences in gene expression between HepG2 x and HepG2Cat cells were evaluated by suppression subtractive hybridization and PCR. In situ hybridization (ISH) and Northern blot analysis were carried out to screen the differentially expressed genes. The full length cDNA clone of the gene was obtained by 5' and 3' rapid amplification of cDNA ends(race) PCR. HepG2 cells transiently transfected with the new full length gene were subjected to fluorescence activated cell sorting (FACS) analysis for DNA content. HepG2 cells stably transfected with the new full length gene were tested for anchorage independent growth in soft agar and for tumorigenicity in nude mice.
The expression of multiple genes were turned on (8) or off (2) in HepG2X compared to HepG2CAT cells. One differentially expressed gene C2, the human homology of Sui1, encoded a translation initiation factor whose expression was suppressed by X antigen in HepG(2) cells. The full length of this gene was 1.35 kb, which encoded a small protein of 113 amino acids. Introduction of C2 into HepG2 cells could inhibit cell growth in culture, in soft agar, and partially inhibit tumor formation in nude mice. Cells transfected with pcDNA3-HBx showed little or no detectable C2, which was consistent with the suppression of this protein in the presence of HBxAg. C2 was also expressed in nontumor liver, but not in tumor cells from patients with hepatocellular carcinoma.
HBX can regulate the expression of genes whose products may be positive or negative regulators of cell growth. Our work for the first time demonstrates that the mechanism of DNA virus associated carcinogenesis involves altered patterns of gene expression regulated at the level of translation initiation.
克隆与乙肝病毒X抗原（HBxAg）相关的全长差异表达基因。
用制备的编码X抗原的重组逆转录病毒感染HepG2细胞。通过抑制性消减杂交和聚合酶链反应（PCR）评估HepG2 x细胞和HepG2Cat细胞之间的基因表达差异。进行原位杂交（ISH）和Northern印迹分析以筛选差异表达基因。通过cDNA末端的5'和3'快速扩增（race）PCR获得该基因的全长cDNA克隆。用新的全长基因瞬时转染的HepG2细胞进行DNA含量的荧光激活细胞分选（FACS）分析。用新的全长基因稳定转染的HepG2细胞在软琼脂中进行非锚定依赖性生长测试，并在裸鼠中进行致瘤性测试。
与HepG2CAT细胞相比，HepG2X细胞中有多个基因的表达开启（8个）或关闭（2个）。一个差异表达基因C2，即Sui1的人类同源物，编码一种翻译起始因子，其表达在HepG(2)细胞中被X抗原抑制。该基因的全长为1.35 kb，编码一个由113个氨基酸组成的小蛋白。将C2导入HepG2细胞可抑制培养物、软琼脂中的细胞生长，并部分抑制裸鼠中的肿瘤形成。用pcDNA3-HBx转染的细胞几乎检测不到C2，这与在存在HBxAg的情况下该蛋白的抑制作用一致。C2也在非肿瘤肝脏中表达，但在肝细胞癌患者的肿瘤细胞中不表达。
HBX可以调节其产物可能是细胞生长的正或负调节因子的基因的表达。我们的工作首次证明DNA病毒相关致癌作用的机制涉及在翻译起始水平调节的基因表达模式的改变。
