Identification of genes upregulated by recombinant interferon-alpha in HepG2 cells by suppressive subtractive hybridization analysis.
BACKGROUND
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.


METHODS
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.


RESULTS
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).


CONCLUSIONS
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.
Qu
Jian-Hui
JH
Gene Therapy Research Center, Institute of Infectious Diseases, PLA 302 Hospital, Beijing 100039, China.
Cheng
Jun
J
Zhang
Ling-Xia
LX
Zhang
Li-Ying
LY
Zhong
Yan-Wei
YW
Liu
Yan
Y
Wang
Lin
L
Dai
Jiu-Zeng
JZ
Xu
Dong-Ping
DP
D045744:Cell Line, Tumor; D018076:DNA, Complementary / Q000737:chemistry; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics; D007370:Interferon Type I / Q000494:pharmacology*; D008099:Liver / Q000378:metabolism*; D009693:Nucleic Acid Hybridization; D012333:RNA, Messenger / Q000032:analysis; D011994:Recombinant Proteins; D002785:Sterol O-Acyltransferase / Q000235:genetics; D015854:Up-Regulation; D000097587:Sterol O-Acyltransferase 2
D018076:DNA, Complementary; D036341:Intercellular Signaling Peptides and Proteins; D007370:Interferon Type I; D012333:RNA, Messenger; D011994:Recombinant Proteins; D002785:Sterol O-Acyltransferase; D000097587:Sterol O-Acyltransferase 2; C501126:DKK1 protein, human