DNA methylation of the promoter of soluble epoxide hydrolase silences its expression by an SP-1-dependent mechanism

Epoxyeicosatrienoic acids, derived from arachidonic acid, function as antihypertensive and antihypertrophic mediators in the cardiovascular system. They are hydrolyzed by soluble epoxide hydrolase (sEH). Pharmacological inhibition of sEH increases the level of epoxyeicosatrienoic acids, which may have a cardiovascular protective effect. However, the regulation and function of sEH in cancer are largely unknown. The present study investigated whether DNA methylation regulates the expression of sEH in carcinoma HepG2 cells. The mRNA and protein expressions of sEH in HepG2 cells were lower than those in transformed human embryonic kidney cells and in primary cultured human endothelial cells. Bioinformatic analysis revealed a putative CpG island and 5 SP-1 binding sites located in the promoter region of the sEH gene. Furthermore, the sEH expression was significantly enhanced by demethylation treatment with 5-Aza-CdR, a DNA methyltransferase inhibitor, and the sEH promoter was transformed from hypermethylation to hypomethylation as detected by methylation-specific PCR and bisulfite sequencing. Transient transfection assays showed that the activity of the human sEH promoter was increased in HepG2 cells in response to 5-Aza-CdR. Five SP-1 binding sites in the promoter region responding to treatment with 5-Aza-CdR were identified by construct deletion and mutation analysis and chromatin immunoprecipitation assay. Interestingly, adenoviral overexpression of sEH in HepG2 cells decreased cell proliferation. Thus, SP-1 is involved in the decrease in the transcription of sEH as a result of DNA methylation in HepG2 cells, which might contribute to epigenetic mechanism-induced carcinogenesis in hepatocytes.

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► A putative CpG island and 5 SP-1 binding sites were located in the promoter region of the sEH gene.
► sEH expression and promoter activity were increased by demethylation treatment.
► Five SP-1 binding sites in the promoter region were identified.
► Overexpression of sEH decreased cell proliferation.