Hepatocyte nuclear factor 4a (HNF4a) is a liver enriched transcription factor and is indispensable for liver development. However, the role of HNF4a in hepatocellular carcinoma (HCC) progression remains to be elucidated. We report that reduced HNF4a expression correlated well with the aggressive clinicopathological characteristics of HCC and predicted poor prognosis of patients. HNF4a levels were even lower in metastatic HCCs, and ectopic HNF4a expression suppressed the metastasis of hepatoma cells both in vitro and in vivo. Forced HNF4a expression attenuated the expression and nuclear translocation of RelA (p65) and impaired NF-jB activation through an IKKindependent mechanism. Blockage of RelA robustly attenuated the suppressive effect of HNF4a on hepatoma cell metastasis. MicroRNA (miR)-7 and miR-124 were transcriptionally up-regulated by HNF4a, which repressed RelA expression by way of interaction with RelA-3 0 untranslated region (UTR). In addition, nuclear factor kappa B (NF-jB) up-regulated the expression of miR-21 in hepatoma cells, resulting in decreased HNF4a levels through down-regulating HNF4a-3 0 UTR activity. Conclusions: Collectively, an HNF4a-NF-jB feedback circuit including miR-124, miR-7, and miR-21 was identified in HCC, and the combination of HNF4a and NF-jB exhibited more powerful predictive efficiency of patient prognosis. These findings broaden the knowledge of hepatic inflammation and cancer initiation/progression, and also provide novel prognostic biomarkers and therapeutic targets for HCC.

Hepatocyte nuclear factor-4a (HNF4a) is a dominant transcriptional regulator of hepatocyte differentiation and hepatocellular carcinogenesis. There is striking suppression of hepatocellular carcinoma (HCC) by HNF4a, although the mechanisms by which HNF4a reverses HCC malignancy are largely unknown. Herein, we demonstrate that HNF4a administration to HCC cells resulted in elevated levels of 28 mature microRNAs (miRNAs) from the miR-379-656 cluster, which is located in the delta-like 1 homolog (DLK1) -iodothyronine deiodinase 3 (DIO3) locus on human chromosome 14q32. Consistent with the reduction of HNF4a, these miRNAs were down-regulated in human HCC tissue. HNF4a regulated the transcription of the miR-379-656 cluster by directly binding to its response element in the DLK1-DIO3 region. Interestingly, several miRNAs in this cluster inhibited proliferation and metastasis of HCC cells in vitro. As a representative miRNA in this cluster, miR-134 exerted a dramatically suppressive effect on HCC malignancy by down-regulating the oncoprotein, KRAS. Moreover, miR-134 markedly diminished HCC tumorigenicity and displayed a significant antitumor effect in vivo. In addition, inhibition of endogenous miR-134 partially reversed the suppressive effects of HNF4a on KRAS expression and HCC malignancy. Furthermore, a positive correlation between HNF4a and miR-134 levels was observed during hepatocarcinogenesis in rats, and decreases in miR-134 levels were significantly associated with the aggressive behavior of human HCCs. Conclusion: Our data highlight the importance of the miR-379-656 cluster in the inhibitory effect of HNF4a on HCC, and suggest that regulation of the HNF4a-miRNA cascade may have beneficial effects in the treatment of HCC. (HEPATOLOGY 2013;58:1964-1976 H epatocyte nuclear factor-4a (HNF4a), a member of the nuclear hormone receptor superfamily, is essential for the differentiation of the hepatic lineage and for maintaining the function of mature hepatocytes.1-3 Loss of HNF4a expression is a critical event in the progression of hepatocellular carcinoma (HCC) and is inversely associated with HCC differentiation status.4,5 A previous study from this laboratory demonstrated that up-regulating HNF4a could reverse the malignant phenotypes of HCC by inducing redifferentiation of HCC cells to hepatocytes. 6 We also demonstrated that HNF4a administration could attenuate liver fibrosis and block hepatocarcinogenesis in rats. 7,8 Interestingly, a recent study by others reported that transient inhibition of HNF4a could initiate hepatocellular oncogenesis

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