BACKGROUND & AIMS: Most hepatocellular carcinoma (HCC) patients die from metastasis and recurrence.Emerging evidence suggests that epithelial-mesenchymal transition (EMT) contributes to the promotion of tumor metastasis and recurrence.Important associations between EMT and cancer stem cell (CSC) generation have been implicated in breast cancer.Several advances have been made in understanding the EMT regulation of cancer cells including liver cancer cells.Understanding the molecular mechanisms that regulate the EMT is crucial for improving treatment of HCC.microRNAs (miRNAs) play important roles in cancer cells;however, the mechanisms by which miRNAs target EMT and their associated therapeutic potential remains largely unknown.We sought to identify EMT-associated miRNAs and revealed the therapeutic potential for HCC.METHODS: To better identify miRNAs in the EMT process, we established an EMT model in HCC cells by TGF-β1 treatment.As an initial step toward identifying miRNAs that may inhibit EMT, 17 tumor suppressors miRNAs reported in liver cancer was screened.We confirmed the role of miR-125b in EMT and cancer stem cell (CSC) generation through HCC cell lines, the mouse model and patients samples.Direct target genes of miR-125b were predicted by TargetScan and confirmed by dual luciferase reporter system and the functional studies.To investigate the therapeutic potential of miR-125b for HCC, an HCC metastasis mouse model by subcutaneous injection of highly metastatic HCCLM3 cells were used.RESULTS: Four miRNAs (let-7a, miR-99a, miR-100 and miR-125b) were downregulated in the model, of which miR-125b was clustered with the others and most strongly downregulated.Among these miRNAs,miR-125b was demonstrated to negatively correlate with EMT phenotypes and EMT-associated traits,including chemoresistance, migration and CSC generation, in both HCC cells and clinical tumor tissues.miR-125b overexpression reduced CD13+ and/or EpCAM+ populations and the side population, which have been used to enrich liver CSCs.miR-125b overexpression suppressed EMT and EMT-associated traits of HCC cells in vitro and in vivo by targeting SMAD2 and SMAD4.Importantly, the therapeutic delivery of a synthetic miR-125b mimic decreased the target molecule in cancer stem cells and inhibited metastasis in a mouse model.CONCLUSIONS: Our study revealed the inhibitory role of miR-125b on EMT and CSC generation in HCC.Our work sheds light on therapeutics targeting EMT and CSCs through an miRNA paradigm in HCC therapy.