Qu Jian-Hui, Cheng Jun, Zhang Ling-Xia, Zhang Li-Ying, Zhong Yan-Wei, Liu Yan, Wang Lin, Dai Jiu-Zeng, Xu Dong-Ping
Gene Therapy Research Center, Institute of Infectious Diseases, PLA 302 Hospital, Beijing 100039, China.
Hepatobiliary Pancreat Dis Int. 2007 Jun;6(3):290-3.
Interferon-alpha (IFN-alpha) is an important cytokine with multiple functions, but the target genes transactivated by IFN-alpha remain largely unknown. A study of such genes will help to understand the mechanism of function of IFN-alpha. To isolate the gene transcripts specifically upregulated by IFN-alpha in HepG2 cells, we conducted suppressive subtractive hybridization (SSH) analysis.
SSH was used to analyze the target genes transactivated by recombinant IFN-alpha protein, and a subtractive cDNA library was constructed from HepG2 cells treated with recombinant IFN-alpha (rIFN-alpha, 2000 IU/ml) for 16 hours as tester, and cells not treated with rIFN-alpha as driver. The SSH PCR products from the library were cloned into pGEM-T easy vector and with BLASTX, the positive clones were randomly selected, sequenced and compared to the database in GenBank of the 35 differentially expressed gene fragments from the library, 6 clones showed significant homology to other known proteins.
The subtractive cDNA library of genes upregulated by IFN-alpha was constructed successfully. rIFN-alpha upregulated the expression of the RAN binding protein 5 (RANBP5), NADH dehydrogenase, exosome component 3 (EXOSC3), zinc finger RNA binding protein, Dickkopf homolog 1 (DKK1) and acetyl-coenzyme A acetyltransferase 2 (ACAT2).
These results suggest that rIFN-alpha can upregulate the expression of important genes to exert its functions, and provide new clues for discovering the molecular mechanisms of action of IFN-alpha.
α-干扰素（IFN-α）是一种具有多种功能的重要细胞因子，但其激活的靶基因仍大多未知。对这些基因的研究将有助于了解IFN-α的功能机制。为了分离出在HepG2细胞中被IFN-α特异性上调的基因转录本，我们进行了抑制性消减杂交（SSH）分析。
采用SSH分析重组IFN-α蛋白激活的靶基因，以经重组IFN-α（rIFN-α，2000 IU/ml）处理16小时的HepG2细胞作为测试样本，未经rIFN-α处理的细胞作为驱动样本，构建消减cDNA文库。将文库中的SSH PCR产物克隆到pGEM-T easy载体中，通过BLASTX随机选择阳性克隆进行测序，并与GenBank数据库进行比较。从文库中35个差异表达的基因片段中，有6个克隆与其他已知蛋白具有显著同源性。
成功构建了IFN-α上调基因的消减cDNA文库。rIFN-α上调了RAN结合蛋白5（RANBP5）、NADH脱氢酶、外泌体成分3（EXOSC3）、锌指RNA结合蛋白、Dickkopf同源物1（DKK1）和乙酰辅酶A乙酰转移酶2（ACAT2）的表达。
这些结果表明，rIFN-α可上调重要基因的表达以发挥其功能，为揭示IFN-α的分子作用机制提供了新线索。
