C-Myc-Regulated-microRNA-Protein Functional Pathways Affect Hepatocarcinogenesis

c-Myc (Myc) plays an important role in normal individual development and tumorigenesis, and is frequently deregulated in many kinds of human cancers including hepatocellular carcinoma(HCC). MicroRNA (miRNA) as a key regulator of genes expression whose deregulation contributes to HCC progression. As a transcription factor Myc can regulate transcription of many protein genes and microRNAs. To investigate the mechanisms between Myc and its regulated miRNAs and how miRNA act as the mediator of Myc's function can help us to understand more about the pathogenesis of HCC.In recent years, many studies have shown that Myc can promote various tumors progression though regulating the expression of some miRNAs, but it is still known little in hepatocarcinogenesis. In order to identify miRNAs which are regulated by Myc, we confirm that Myc is pathologically activated in and essential for promoting human HCC, then analysis the loci of all miRNAs in human to find the candidate miRNAs by using bioinformation methodology. We find Myc induces HCC through a novel, microRNA (miRNA)-mediated feedback loop comprised of miR-148a-5p, miR-363-3p, and ubiquitin-specific protease 28 (USP28). Myc directly binds to conserved regions in the promoters of the two miRNAs and represses their expression. miR-148a-5p directly targets and inhibits Myc, whereas miR-363-3p destabilizes Myc by directly targeting and inhibiting USP28. Activation of miR-148a-5p or miR-363-3p inhibits hepatocellular tumorigenesis by blocking Gl to S phase progression, whereas inhibition of them has the opposite effects. The Myc-miRNA feedback loop is deregulated in human HCC.In order to further study the miRNA deregulation contributes to hepatocellular carcinoma progression, we created a human miRNA expression library by cloning nearly all annotated human miRNAs [1254 miRNA loci,1746 mature miRNAs, miRBase release 18.0 (2012)] into the miRNA expression vector. We subsequently performed a high-content, function-based miRNA screen and identified miR-129-5p as a miRNA that inhibits HCC cell growth. miR-129-5p targets the mitochondrial matrix protein PDK4, which leads to decreased phosphorylation of the El alpha subunit of PDH complex, inhibition of the glycolysis, decreased tumor growth and impaired metastases. Enforced expression of PDK4 refractory to inhibition by miR-129-5p rescued each of these phenotypes. Targeting PDK4 by shRNA or small molecule inhibitor of PDK4, DCA, recapitulated the effects caused by miR-129-5p. Myc directly represses miR-129-5p expression by epigenetic silence through interacting with HDAC3 and EZH2. Levels of miR-129-5p negatively correlated with clinical stage and tumor differentiation in human HCC, and levels of Myc or PDK4 stand on the opposite side. To restore Mir-129-5p expression or inhibit Pdk4 activity by DCA suppressed the DEN-induced hepatocarcinogenesis in mice.Taken together, we clarify the important roles of the functional miRNA-Protein pathways which are regulated by Myc in hepatocarcinogenesis. On one side, Myc transcriptionally represses miR-148a-5p and miR-363-3p, which are also negatively regulating Myc through a direct or indirect manner, thus keeping a high level expression of itself to promote cell cycles progression. On the other hand, Myc also represses miR-129-5p and enforces the glycolysis to contribute to hepatocellular tumorigenesis. The mechanism of this novel network of Myc in the progression of HCC suggests us potential points of therapeutic intervention for this disease.