Dmd065664 1450..1457

Human cytochrome P450 2E1 (CYP2E1) catalyzes themetabolism of numerous xenobiotics, including acetaminophen and ethanol. CYP2E1 expression is known to be extensively regulated by posttranscriptional and post-translational mechanisms. A previous study had reported that a single-nucleotide polymorphism (SNP) 1561A>G in the 39-untranslated region (39-UTR) of CYP2E1 leads to a decreased CYP2E1 mRNA level in human peripheral blood mononuclear cells. In this study, we examined the possibility that microRNA(s) (miR) may be involved in the SNP-mediated modulation of CYP2E1 expression. Genotyping and sequencing analyses revealed that another SNP, 1556T>A, in the 39-UTR was in complete linkage disequilibrium with the SNP 1561A>G. We termed the alleles with 1556T and 1561A or 1556A and 1561G haplotype I or II, respectively. A luciferase assay revealed that miR-570 recognizes the CYP2E1 39-UTR of haplotype I but not haplotype II. Human embryonic kidney 293 (HEK293) cell lines stably expressing human CYP2E1 that included the 39-UTR of haplotype I or II (HEK/2E1(I) or HEK/2E1(II) cells, respectively) were established. Overexpression of miR-570 significantly decreased the CYP2E1 protein level in the HEK/2E1(I) cells but not in the HEK/2E1(II) cells. In seven human livers with diplotype I/I, the CYP2E1 protein levels were inversely correlated with the miR-570 levels, but no relationship was observed in 25 human livers with diplotypes I/II and II/II. Collectively, it was demonstrated that human CYP2E1 was regulated by miR-570 in a genotypedependent manner. This report describes the first proof that SNP in 39-UTR of human P450 affects binding of miRNA to modulate the expression in the liver.