Wnt/beta-catenin signaling activates microRNA-181 expression in hepatocellular carcinoma

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/beta-catenin signaling with a parallel increased expression of all four microRNA-181 family members. We hypothesized that Wnt/beta-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 beta-catenin expression in HCC cell lines. MicroRNA-181 expression could be directly induced upon an activation of Wnt/beta-catenin signaling, which includes Wnt10B overexpression, inhibition of GSK3 beta signaling by LiCl, or forced expression of beta-catenin/Tcf4. Moreover, microRNA-181 expression was inhibited upon an inactivation of Wnt/beta-catenin signaling by an induction of adenomatosis polyposis coli (APC) expression or silencing beta-catenin via RNA interference. In addition, seven putative beta-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.