BACKGROUND: Interferon-alpha (IFN-α) is an important cytokine with multiple functions, but the target genes transactivated by IFN-α remain largely unknown. A study of such genes will help to understand the mechanism of function of IFN-α. To isolate the gene transcripts specifically upregulated by IFN-α in HepG2 cells, we conducted suppressive subtractive hybridization (SSH) analysis.
METHODS: SSH was used to analyze the target genes transactivated by recombinant IFN-α protein, and a subtractive cDNA library was constructed from HepG2 cells treated with recombinant IFN-α (rIFN-α, 2000 IU/ml) for 16 hours as tester, and cells not treated with rIFN-α 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.
RESULTS: The subtractive cDNA library of genes upregulated by IFN-α was constructed successfully. rIFN-α 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).
CONCLUSIONS: These results suggest that rIFN-α can upregulate the expression of important genes to exert its functions, and provide new clues for discovering the molecular mechanisms of action of IFN-α.