32 In another study, the authors detected HSP27 in HCC serum and validated the result by western blotting.34 Our finding that HSP27 is up-regulated in miR-17-5p overexpression in HCC cells further confirms

these results. The capacity of HSP27 to regulate actin reorganization and cell migration is modulated by the phosphorylation of serine residues.35 We measured the levels of phosphorylation and found that the levels of HSP27 phosphorylation at serines 15 and 82 in miR-17-5p-overexpressing HCC cells were increased compared with control cells (Fig. 3A). We therefore propose that miR-17-5p overexpression increases total HSP27 and also up-regulates HSP27 activity. It has also been reported that the MAPK cascade—in particular, the p38 MAPK pathway—leads to the phosphorylation of HSP27 through MAPK-activated protein kinase-2, one of the substrates of p38 MAPK.36, 37 Therefore, we investigated whether MAPKs are involved see more in HSP27 phosphorylation in HCC cells. We showed that the level of phosphorylated p38 MAPK was increased and inhibition

of p38 MAPK activation results in the suppression of HSP27 phosphorylation in miR-17-5p-overexpressing HCC cells (Figs. 3B, 4A). We also found that HSP27 knockdown reduces the phosphorylation level of HSP27 in miR-17-5p-overexpressing HCC cells (Fig. 4B). These findings indicate the likelihood that up-regulation of miR-17-5p regulates the phosphorylation of HSP27 through PD0325901 order p38 MAPK in human 上海皓元 HCC cells. It is not clear how miR-17-5p activates the p38 MAPK pathway, however. We hypothesized that miR-17-5p might indirectly modulate the p38 MAPK pathway through one of its target genes. Wip1, a serine/threonine phosphatase, is an E2F1-regulated gene that inactivates p38.24, 38 In addition, E2F1 is a proven target gene of miR-17-5p.15 Interestingly, we demonstrated that Wip1

is down-regulated in miR-17-5p-overexpressing HCC cells (Fig. 4C) and ectopic expression of Wip1 in miR-17-5p-overexpressing HCC cells can inactivate the p38 pathway (Fig. 4D). This result indicates that Wip1, an E2F1-regulated gene, is at least partly responsible for miR-17-5p-dependent activation of the p38 pathway. Many studies have shown increased phosphorylation levels of HSP27 in different metastatic cancer cells and have indicated HSP27 activation correlates with the metastatic potential of cancer cells.26, 37, 39 Our results indicate that HCC cell motility is increased by miR-17-5p (Figs. 5-7). In addition, the increased cell motility induced by miR-17-5p was accompanied by an increase in p38 MAPK and HSP27 activity, suggesting that miR-17-5p may activate p38 MAPK-dependent pathways. Interestingly, treatment with a siRNA against HSP27, an inhibitor of p38 MAPK or 2′-Ome -modified antisense oligoribonucleotides against miR-17-5p blocked the miR-17-5p-induced up-regulation of HCC cell metastasis (Figs. 5-7). Additionally, we found increased concentration of secreted MMP-2 in miR-17-5p-overexpressing HCCs.