Because SREBP2 and miR-33a are coexpressed by the SREBP2 gene, we hypothesized that miR-33a might be also induced in Cyp7a1-tg mice to participate in the regulation of cholesterol metabolism. Indeed, hepatic miR-33a expression was coinduced with SREBP2 in Cyp7a1-tg mice under both chow- and WD-feeding conditions (Fig. 2A,B).[9] These results suggest that increasing bile acid synthesis in Cyp7a1-tg mice may induce miR-33a expression by inducing cholesterol-regulated SREBP2 expression. To further test whether miR-33a regulates

bile acid metabolism, we used adenovirus-mediated gene delivery to overexpress miR-33a specifically in WT mouse liver (Supporting Fig. 3). mRNA analysis by real-time PCR showed MAPK Inhibitor Library order that overexpression of miR-33a reduced the mRNA expression of CYP7A1 and CYP8B1 and Na+-dependent taurocholate cotransport peptide (NTCP), the basolateral bile acid uptake transporter (Fig. 3A). HM781-36B research buy mRNA levels of BSEP, ABCG5, and ABCG8 were also reduced by miR-33a (Fig. 3A). As a positive control, miR-33a inhibited ABCA1 and carnitine palmitoyl-CoA transferase 1 mRNA (Fig. 3A).[9-11] Consistent with down-regulation of CYP7A1 mRNA, miR-33a overexpression reduced microsomal CYP7A1 enzyme activity by ∼40% (Fig. 3B) and total bile acid pool size by ∼25% (Fig. 3C). In addition, miR-33a reduced total serum cholesterol levels by ∼50%

(Fig 3D), but increased hepatic cholesterol content by ∼20% (Fig 3E). Such changes in serum and hepatic cholesterol levels are likely resultant from inhibition of both ABCA1

and CYP7A1. To investigate whether miR-33a regulation of CYP7A1 is conserved in the human CYP7A1 gene, we used adenovirus-mediated gene delivery to overexpress miR-33a in “humanized” mice, which express the human CYP7A1 gene. miR-33a overexpression resulted in ∼40% reduction of human CYP7A1 mRNA and ∼25% reduction in total bile acid pool size (Supporting Fig. 4A,C). As a positive control, miR-33a repressed ABCA1 mRNA in the “humanized” CYP7A1 mouse liver (Supporting Fig. 4B). We next transfected miR-33a mimic or miR-33a hairpin inhibitor into HepG2 cells to confirm our in vivo observations. miR-33a mimic dose dependently decreased mRNA levels MCE公司 of CYP7A1, CYP8B1, and ABCA1 (positive control) (Fig. 4A,B). In addition, antagonism of miR-33a by a miR-33a hairpin inhibitor dose dependently increased mRNA levels of CYP7A1, CYP8B1, and ABCA1 (Fig. 4D-F). In summary, these data suggest that miR-33a regulates CYP7A1 and bile acid synthesis and may coordinately regulate hepatic cholesterol and bile acid homeostasis. miR-mediated target gene repression commonly occurs through binding to the 3′-untranslated region (3′-UTR) in target mRNAs, which usually results in degradation of the mRNA and inhibition of protein translation.