title: Wnt/beta-catenin signaling activates microRNA-181 expression in hepatocellular carcinoma.
abstract: BACKGROUND
  Hepatocellular carcinoma (HCC) is a malignant cancer with an observable heterogeneity and microRNAs are functionally associated with the tumorigenesis of HCC. We recently identified that EpCAM (CD326)-positive cells isolated from alpha-fetoprotein (AFP)-positive HCC samples are hepatic cancer stem cells (HepCSCs). EpCAM+AFP+ HepCSCs have an activated Wnt/β-catenin signaling with a parallel increased expression of all four microRNA-181 family members. We hypothesized that Wnt/β-catenin signaling transcriptionally activates microRNA-181s in HCC.
  
  
  RESULTS
  Using both western blot and quantitative reverse transcriptase-PCR analyses, we found that the expression of all four microRNA-181 family members was positively correlated with β-catenin expression in HCC cell lines. MicroRNA-181 expression could be directly induced upon an activation of Wnt/β-catenin signaling, which includes Wnt10B overexpression, inhibition of GSK3β signaling by LiCl, or forced expression of β-catenin/Tcf4. Moreover, microRNA-181 expression was inhibited upon an inactivation of Wnt/β-catenin signaling by an induction of adenomatosis polyposis coli (APC) expression or silencing β-catenin via RNA interference. In addition, seven putative β-catenin/Tcf4 binding sites were identified in the promoter region of the microRNA-181a-2 and microRNA-181b-2 transcripts. Consistently, we found that Tcf4 interacted with these regions in vivo using chromatin immunoprecipitation assay.
  
  
  CONCLUSIONS
  Taken together, our results demonstrate that microRNA-181s are transcriptionally activated by the Wnt/beta-catenin signaling pathway in HCC.
authors.affiliation: Liver Carcinogenesis Section, Laboratory of Human Carcinogenesis, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, USA. xw3u@nih.gov.
authors.forename: Junfang
authors.initials: J
authors.lastname: Ji
authors.forename: Taro
authors.initials: T
authors.lastname: Yamashita
authors.forename: Xin W
authors.initials: XW
authors.lastname: Wang