Among a collection of 1,049 miRNA loci (miRBase-Release 1631), we identified one or more E-boxes in the promoters, first exons, or first introns in 834 cases (Supporting Fig. 4A and Supporting Table 3). We then determined whether these E-boxes are located in CpG islands, because Myc-binding E-boxes are usually located within such contexts. As shown in Supporting Fig. 4A and Supporting Table 3, 94 of the 834 loci resided within CpG islands. Further analyses showed 21 of these loci

to be conserved between humans and mice (Supporting Fig. 4A and Supporting Table 3). Interestingly, miRNAs from 10 of these loci have been reported to be regulated by Myc and to mediate a variety of Myc functions32, 33(Table 1 and Supporting Fig. 4B,C). Therefore, miRNAs from the other 11 loci may also be regulated by Myc GS-1101 solubility dmso and have important roles in mediating Myc http://www.selleckchem.com/products/Erlotinib-Hydrochloride.html function. To address whether Myc regulates expression of miRNAs

from the remaining 11 loci, we tested the effects of Myc inhibition or Myc induction on the expression of these miRNAs. As shown in Fig. 1A and Supporting Table 2, induction of MycER activation in HL7702 cells resulted in the down-regulation of several of the miRNAs, including miR-148a-5p and miR-363-3p, whereas inhibition of Myc in HepG2 and BEL-7402 cells resulted in up-regulation of other miRNAs, including miR-148a-5p and miR-363-3p. Taken together, Myc differentially regulates these miRNAs in a cell context–dependent manner. Stem-loop quantitative RT-PCR results in all three cell lines indicated miR-363-3p to be the most significantly regulated miRNA in response to Myc activation or inhibition (Supporting Table 2). In addition, prediction of miRNA targets showed the 3′-UTR of Myc to contain one highly conserved miR-148a-5p-binding site this website from human to dog. Based on these findings, we selected miR-363-3p and miR-148a-5p for additional analyses. To address whether Myc directly binds the promoter regions of miR-148a and miR-363,

we conducted a chromatin IP assay in HepG2 and BEL-7402 cells. This revealed that Myc binds both miR-148a and miR-363 promoter regions containing the highly conserved E-box regions in CpG islands (Fig. 1B-D and Supporting Fig. 5). These results provided strong evidence that both miRNAs are directly regulated by Myc. In addition, our unpublished data show that inhibition of both miRNAs by Myc is accompanied by a prominent decrease in active histone marks around the transcription start sites of both miRNAs. To examine the consequences of relieving the suppression of miR-148a-5p and miR-363-3p in hepatocarcinoma, we tested whether ectopic expression of these miRNAs affected the biology of liver cells. As shown in Fig. 2A-C and Supporting Fig.