6C). Strikingly, EZH2-regulated miRNAs can potentially modulate cell motility-associated pathways and key signaling pathways. It was interesting to note that the first- and second-rated pathways were focal adhesion (Pathway ID hsa04510) and adherens junction (Pathway ID hsa04520), two crucial pathways in cancer cell invasion and metastasis. Consistent findings were obtained by PicTar, another miRNA target prediction algorithm (Supporting Fig. 5, Supporting Table 7). We also noticed that the RhoGTPase-associated cytoskeleton reorganization

axis was recurrently engaged RXDX-106 datasheet in six of the top-rated KEGG pathways, including those for focal adhesion (Pathway ID hsa04510), adherens junction (Pathway ID hsa04520), transforming growth factor

beta (TGF-β) signaling (Pathway ID hsa04350), noncanonical Wnt singling (Pathway ID hsa04310), axon guidance (Pathway ID hsa04360), and the actin cytoskeleton regulation (Pathway ID hsa04810) (Supporting Fig. 6). We previously reported that the RhoGTPase signaling pathway is frequently altered in human HCCs and is tightly associated with HCC metastasis.21, 35, 36 Our present findings further suggest that the EZH2-tumor suppressor miRNA axis may act upstream of the pathway to mediate its perturbation. Consistent with this notion, we found that knockdown of EZH2 resulted in down-regulation of RhoA and ROCK2 protein and inhibited stress fiber formation in HCC cells (Supporting Fig. 7). Taken together, the in silico analysis reinforces the tumor suppressive functions of EZH2-regulated miRNAs, and suggests their combinational effects in modulating key cell movement STI571 concentration and metastasis-related pathways in driving however HCC metastasis. Epigenetic regulation machinery involves multiple proteins with distinct functions. In our study, we first revealed that deregulation of epigenetic modifiers is common in HCCs. These epigenetic modifiers, including DNA methyltransferases, histone deacetylases, SET domain-containing histone methyltransferases, and

PcG proteins are direct mediators of epigenetic mechanisms. Their concordant deregulation reflects HCC epigenome is likely to be affected in multiple aspects. In line with our observation, not only are some of these proteins reported to be up-regulated in HCC,5, 37 but genome-wide DNA hypomethylation and promoter DNA hypermethylation of tumor-suppressors,38 as well as changes in global histone modification such as an increase of H3K27me3 level,39 are also noted in HCC, suggesting functional implication of these epigenetic regulators in HCC development. We further identified EZH2 and its associated PRC2 as one of the critical epigenetic regulators in HCC and demonstrated its tumor and metastasis promoting role in HCC development. Our findings are consistent with other previous reports on EZH2 up-regulation40 and tumorigenesis in HCC.41 Beyond this, our present findings provide new knowledge to understand how EZH2 contributes to HCC metastasis.