However, our expression data did not support co-regulation of SREBF2 and miR-33 expression across 10 types of chicken tissues examined. Predicting targets is an important first step to determine the function of a miRNA. Many algorithms and databases for miRNA target predictions have been established, and among them, miRanda, TargetScan, and PicTar, appear to be the most widely used miRNA target prediction methods. In this study, 378 genes were predicted as the target genes of miR-33 among the total 11,891 chicken genes within the 39UTR database using “miRanda”. The “TargetScan” principle was also applied in the prediction procedures: the target site should match to the seed region of miRNA, the 8th nucleotide of miRNA should also be a match or the target nucleotide corresponding to the first nucleotide of miRNA should be an A. One of the predicted target genes of miR-33 named FTO is a member of the non-heme dioxygenase superfamily, and has been recently implicated in regulation of lipid and energy metabolism. Dual-luciferase reporter assays and site mutation analyses validated that chicken FTO was a target gene of miR-33. Because in chickens de novo fatty acid synthesis occurs primarily in the liver, we further studied the possibility that miR-33 targets FTO in the chicken liver. One of the most powerful and straightforward ways to determine the relationship between a miRNA and a mRNA in tissues or cells is to determine the effect of knockdown of the miRNA on the expression of the mRNA of interest. Using LNAanti-miR-33, we successfully reduced the expression of endogenous miR-33 in primary chicken hepatocytes, and this reduction was associated with an up-regulated expression of FTO mRNA. This association supports that the FTO gene is targeted by miR-33 in chicken hepatocytes. We also observed that miR-33 and FTO mRNA expression were inversely correlated in chicken liver at most of the developmental ages examined. At day 35 and day 42 of age, the expressions of miR-33 and FTO mRNA were not inversely correlated. This suggests that the expression of FTO at these two stages may be regulated LDK378 predominantly by mechanisms other than miR-33. In the chicken, FTO is widely expressed. Expression of FTO in the hypothalamic nuclei involved in energy balance regulation has been shown to respond to nutritional manipulations such as feeding and fasting. Fasting has been shown to also increase FTO gene expression in the cerebrum, liver, breast muscle and subcutaneous fat. Alterations in feeding status resulted in significant changes in FTO expression in the liver, but not in other tissues of broiler chickens. In addition to this, hepatic FTO expression changes in response to metabolic states, and glucose reduces hepatic FTO mRNA expression independently of body weight. Since miR-33 inhibits the expression of FTO, it might play a role in mediating the nutritional regulation of FTO expression in chicken liver.