A Novel GSK-3 beta-C/EBP alpha-miR-122-Insulin-Like Growth Factor 1 Receptor Regulatory Circuitry in Human Hepatocellular Carcinoma

miR-122 is a highly abundant, hepatocyte-specific microRNA. The biomedical significance and regulatory mechanisms of miR-122 remain obscure. We explored the role of miR-122 in tumorigenesis in the context of gene regulatory network. The miR-122 promoter and its transactivator were identified by way of luciferase reporter system, electrophoretic mobility shift, and chromatin immunoprecipitation assays. The miR-122 regulatory circuitry and its implication in hepatocarcinogenesis were identified using livers of different development stages, human hepatocellular carcinoma (HCC) tissues and cell lines, and aflatoxin B-1 (AFB(1))-transformed cells. We characterized the -5.3 to -4.8 kb region upstream of miR-122 precursor as miR-122 promoter. Further investigation revealed that deletion of predicted CCAAT/enhancer-binding protein alpha (C/EBP alpha) binding sites C/EBP alpha knockdown significantly reduced miR-122 promoter activity and endogenous miR-122 expression; and C/EBP alpha directly interacted with the miR-122 promoter in vitro and in vivo. These data suggest that C/EBP alpha is a transactivator for miR-122 transcription. We further demonstrated that miR-122 suppressed insulin-like growth factor 1 receptor (IGF-1R) translation and sustained glycogen synthase kinase-3 beta (GSK-3 beta) activity. The activated GSK-3 beta not only repressed cell proliferation, but also activated C/EBP alpha which maintained miR-122 levels and thereby enforced IGF-1R suppression. Interestingly, down-regulation of miR-122 and C/EBP alpha, and up-regulation of IGF-1R were frequently observed in HCC tissues, and decreased miR-122 levels were associated with worse survival of HCC patients. Moreover, AFB(1) exposure resulted in decreased activity in GSK-3 beta, C/EBP alpha and miR-122 and increased levels of IGF-1R, whereas restoration of miR-122 suppressed the tumorigenicity of HCC and AFB(1)-transformed cells. Conclusion: We have identified a novel GSK-3 beta-C/EBP alpha miR-122 IGF-1R regulatory circuitry whose dysfunction may contribute to the development of HCC. Our findings provide new insight into miR-122's function and the mechanisms of hepatocarcinogenesis. (HEPATOLOGY 2010;52:1702-1712)