Background: CCAAT enhancer binding protein alpha (C/EBP alpha), as an important transcription factor involved in cell proliferation, differentiation and metabolism, was up-regulated in primary hepatocellular carcinoma (HCC) and predicted poorer prognosis. In this study, we explored how histone deacetylases (HDACs) up-regulated C/EBP alpha in HCC. Methods: The protein expressions of HDAC1, HDAC2 were associated with C/EBP alpha by immunohistochemistry staining in a HCC tissue microarray. HCC cells were then treated with HDAC inhibitors or siRNAs to determine the roles of miR-124-3p and miR-25 in the regulation of C/EBP alpha mRNA expression. Results: Both HDAC1 and HDAC2 proteins were significantly associated with C/EBP alpha. Inhibition of HDAC by either pharmacological inhibitors or siRNAs decreased C/EBP alpha mRNA expression in dose-dependent manners in HCC cells. HDAC inhibitors reduced C/EBP alpha mRNA stability as shown by pmiRGLO luciferase reporter assays. HDAC inhibition consistently induced miR-124-3p and miR-25 expression. Conversely, blockage of miR-124-3p and/or miR-25 by treatment with specific synthetic inhibitors abolished C/EBP alpha reduction. More importantly, C/EBP alpha mRNA stability could be rescued by site-directed mutations of miR-124-3p or miR-25 recognition sites in the C/EBP alpha 3'UTR sequence. In summary, HDAC may up-regulate C/EBP alpha expression through miR-124-3p and miR-25 in HCC. (C) 2019 Elsevier Inc. All rights reserved.