Because these events occurred long after HNF4α inhibition, investigators proposed a

feedback loop that perpetuates HNF4α suppression and promotes the transformed phenotype. They then sought candidate molecules for this feedback circuit. miRs are very short RNA strands that modulate posttranscriptional buy BMS-907351 modification in eukaryotic cells. They bind to complementary sequences on target mRNA transcripts, leading to gene repression or silencing. Two miRs, miR-24 and miR-629, were identified in an miR-based genetic screen as candidates to directly regulate HNF4α expression. Both of these miRs were shown to interact directly with HNF4α: they each bind at the 3′ untranslated region of HNF4α, and their overexpression results in decreased levels of HNF4α mRNA and protein. Trichostatin A Furthermore, when HNF4α expression is transiently inhibited, expression of both miR-24 and

miR-629 are upregulated. Other experiments demonstrated that overexpression of these miRs induces transformation of hepatocytes, mimicking the effects of HNF4α inhibition in vitro. Excess miR-24 and miR-629 expression causes increased tumor volume and promotes invasiveness in tumor xenografts in immunodeficient mice. Moreover, when animals received antisense miR-24 and/or miR-629, the tumors in those mice were smaller, possessed many apoptotic cells, and showed increased HNF4α mRNA levels. These findings indicate that miR-24 and miR-629 promote hepatocellular transformation by maintaining suppression of HNF4α. Relieving this suppression seems to abrogate these effects. How are these miRs themselves regulated? The promoter regions of both medchemexpress miR-24 and miR-629 each contain a conserved binding

motif of the STAT3. Interleukin-6 (IL-6) is known to modulate STAT3 activity. Chromatin immunoprecipitation analyses showed that stimulation with IL-6 promotes STAT3 binding in the promoter regions of both miR-24 and miR-629. Other experiments demonstrated that STAT3 phosphorylation levels are affected by these miRs. Taken together, STAT3 and IL-6 seem to be components of the feedback loop that contains miR-24 and miR-629, which modulates hepatocellular carcinogenesis via HNF4α. Investigators identified another miR, miR-124, which is also involved in this feedback loop network. The promoter region of miR-124 strongly binds and interacts with HNF4α in liver-derived cells. Inhibition of HNF4α results in diminished levels of miR-124, and combined overexpression of miR-24 and miR-629 acts to markedly inhibit miR-124 expression. In addition, treatment with IL-6 reduced miR-124 activity, but this effect was abrogated when the HNF4α binding site was mutated, implicating that these actions are mediated by HNF4α.