Cancer stem cells (CSCs) are thought to be responsible for tumor invasion, 
metastasis, and recurrence. We previously showed that the pluripotency factor 
Nanog not only serves as a novel biomarker of CSCs but also potentially plays a 
crucial role in maintaining the self-renewal ability of liver CSCs. However, how 
CSCs maintain Nanog gene expression has not been elucidated. Here, we 
demonstrated that microRNA-449a (miR-449a) is overexpressed in poorly 
differentiated hepatocellular carcinoma tissues, drug-resistant liver cancer 
cells, cultured liver tumorspheres, and Nanog-positive liver cancer cells. The 
upregulation of miR-449a in non-CSCs increased stemness, whereas the 
downregulation of miR-449a in Nanog-positive CSCs reduced stemness. Furthermore, 
transcription factor 3 (TCF3), a target of miR-449a, could downregulate Nanog 
expression, and restoring TCF3 expression in miR-449a-expressing Nanog-negative 
cells abrogated cellular stemness. These data establish that the 
miR449a-TCF3-Nanog axis maintains stemness in liver CSCs.