Several target genes of miR-21 were

previously reported.20 However, to potentially identify new targets of miR-21 involved in liver regeneration, we chose an unbiased Doxorubicin order approach. We first used the TargetScan algorithm to identify genes targeted by miR-21 in both mice and humans.23 Focusing on conserved miR-21 targets not only increased the probability of target gene prediction but also assured that our results could be extended to human liver regeneration. We then used the PicTar algorithm to scan the 3′UTR of the conserved miR-21 target genes and eliminate genes with a lower score or free energy (Supporting Information Table 2).24 Our findings of impaired G1 to S phase progression in miRNA-deficient hepatocytes and induction of miR-21 at a time when entry into S phase is negotiated suggested that miR-21 acts to promote liver regeneration. Therefore, among the 63 genes meeting the selection criteria, we focused on 17

genes PD-0332991 manufacturer with established negative effects on proliferation (Supporting Information Table 2). Among these genes were the previously reported miR-21 targets Timp3, Reck, and Pdcd4.20 Potential new miR-21 targets included Tgfbi and Smad7, components of the transforming growth factor β (TGFβ) signaling pathway, which is known to restrict liver regeneration.25 Most interestingly, however, our search retrieved Btg2, a gene restraining G1 to S phase transition that, paradoxically, is induced by 2/3 PH.18 Because Btg2 also had the highest score and free energy of the predicted conserved miR-21 target genes with established proliferation-inhibiting function, we investigated whether it is directly targeted by miR-21 (Supporting Information Fig. 4A, Supporting Information MCE公司 Table 2). BTG2 inhibits proliferation

by interfering with activating phosphorylation of FoxM1.26 FoxM1 is activated after 2/3 PH and its deficiency impairs DNA synthesis and Ccnb1 gene expression in regenerating mouse hepatocytes.26, 27Btg2 was previously reported to be immediately induced and peak at 4 hours after 2/3 PH.18 When we investigated the expression of Btg2 at later stages, we found that it returns to baseline levels between 6 and 18 hours after 2/3 PH. Thus, the expression pattern of Btg2 is the mirror opposite of that of miR-21 (Fig. 3A). Analysis of Dgcr8del/fl, Alb-Cre+/− mice lacking oval cells showed that miR-21 is mainly expressed in hepatocytes in the liver (Fig. 3B). Taken together with the similar nature of the cell cycle defect in hepatocytes with FoxM1 or global miRNA deficiency (Fig. 1A,B), our findings suggested that miR-21 antagonizes Btg2 in regenerating hepatocytes to facilitate efficient cell cycle progression. Indeed, Btg2 messenger RNA (mRNA) levels and activity of a reporter gene linked to its 3′UTR readily responded to miR-21 mimic or inhibitor transfection into well-differentiated mouse hepatoma cells (Fig. 3C). These manipulations also caused induction or suppression of the FoxM1 target gene Ccnb1, respectively (Fig. 3D).