Induction and mechanism of growth arrest DNA damage-inducible gene 45 β in human hepatoma HepG2 cells by S-adenusylmethionine

Abstract

Objective To identify the active region of proximal promoter growth arrest DNA damage-inducible gene 45 β (GADD45β) and to evaluate the influence of S-adenosylmethionine on HepG2. Methods The proximal promoter fragments (-618/-520) were synthesized in vitro by ev-ery 30-50 nucleotides, cloned into pGL3 basic luciferase expression plasmid respectively, then trans-fected into HepO2 cells by electroporation. The promoter regions were identified with reference to TRANSFAC database. Following S-adenosylmethionine administration, quantitative real-time PCR was employed to validate the expression of GADD45β in HepG2. The effect of S-adenosylmethionine to promoter activity was further focused on. Results Using the luciferase assay, several transcription factor binding sites were identified in the proximal promoter of GADD45β, which included three NF-kBs (- 602/- 593, - 581/- 572, and - 537/- 528) according to the consensus sequences in TRANSFAC database. After S-adenosylmethionine administration, the increasing expression of GADD45β was marked in a dose-dependent manner. The luciferase assay also demonstrated the pro-moter activity of fragment constructed the first NF-kB binding site could be induced by S-adenosylme-thionine. Conclusion S-adenosylmethionine can induce the expression of GADD45β, which is specific-ally down-regulated in HCC. The possible mechanism may be involved in the regulation of transcrip-tion factor NF-kB in GADD45β proximal promoter. Key words: Carcinoma,hepatocellular; S-adenosylmethionine; GADD45β; Promoter