As shown in Fig. 4C, E2F1 and Wip1 proteins were clearly down-regulated in miR-17-5p-overexpressing cells. To investigate whether Wip1 is responsible for the activation of the p38 MAPK-HSP27 signaling pathway, we overexpressed Wip1 in clone 1 cells. Figure 4D shows that the phosphorylation of HSP27and p38 MAPK is reduced when Wip1 is up-regulated. Together, these results

suggest that E2F1-dependent down-regulation of Wip1 is necessary for p38 MAPK-HSP27 pathway activation by miR-17-5p. To verify whether the p38 MAPK-HSP27 pathway contributes to miR-17-5p-facilitated cell growth, we performed CCK-8 assays to detect cell proliferation in vitro. In accordance with the results of the xenograft models in nude mice, clone 1 cells proliferated much faster than control cells, and

SB203580 was able to reduce this effect TGF-beta inhibitor in clone 1 cells. However, siRNAs against HSP27 had no effect (Fig. 5A). These results indicate that miR-17-5p facilitates cell growth, but this process does not involve the p38 MAPK-HSP27 pathway. To verify whether the p38 MAPK-HSP27 pathway contributes to the miR-17-5p-induced increase in HCC cell motility, we performed a series of assays to detect cell migration in vitro. Migration of Huh-7 cells, as assayed by a scrape assay, was significantly increased in clone 1 and 2 cells compared with pEZX-MR01-Huh-7 cells. Treatment Crizotinib ic50 with SB203580 or the siRNA against HSP27 abolished this effect (Fig. 5B). Transwell experiments were then performed with clone 1, clone 2, and pEZX-MR01-Huh-7 cells with or without SB203580 or siRNA against HSP27. As shown in Fig. 6A,B, miR-17-5p induced Huh-7 cell migration in the transwell experiments, and this migration was inhibited by SB203580 or siRNA against HSP27. Together, these results indicate that p38 MAPK and HSP27 play a crucial role in the control of Huh-7 cell migration. Similar phenomena were also observed in HepG2 cells (Supporting Fig. 1B). We investigated

the effect of miR-17-5p on the organization of the actin cytoskeleton in Huh-7 cells. The actin cytoskeleton was stained with phalloidin (Fig. 6C). miR-17-5p increased the cortical localization of actin, which was inhibited by the siRNA against HSP27 or treatment with SB203580, demonstrating the involvement of miR-17-5p-p38 MAPK-HSP27 in actin cytoskeleton remodeling. To address in greater detail the function of miR-17-5p in HCC cells medchemexpress and to avoid potential overexpression artifacts, we transfected clone 1 cells with 2′-Ome-modified antisense oligoribonucleotides against miR-17-5p. As shown in Fig. 7A, when introduced into clone 1 cells, phosphorylated HSP27, total HSP27, and phosphorylated p38 MAPK levels were reduced by 50%-60% compared with negative controls, indicating efficient up-regulation by miR-17-5p. Transfection with 2′-Ome-modified antisense oligoribonucleotides, but not the scrambled oligoribonucleotides, significantly reduced the migration of clone 1 cells (Fig.