Wang's group reported1 that they performed a genome-wide screening to identify microRNAs associated with epithelial cell-adhesion molecule (EpCAM)-positive hepatic cancer stem cells. They demonstrated that all four microRNA-181 (miR-181) molecules play important roles in the maintenance of hepatic cancer stem cells by posttranslational down-regulation of two hepatic transcriptional regulators of differentiation and an inhibitor of Wnt/β-catenin signaling (nemo-like kinase [NLK]). Their study defined a novel regulatory link between miR-181s and EpCAM-positive hepatic cancer stem cells and implied that molecular targeting of miR-181 may eradicate hepatic cancer cells.1 However, this study raises several confusing questions.
First, to establish the relationship between miR-181s and EpCAM, the authors showed the expression of miR-181s and EpCAM in figure 2B,C.1 The expression pattern of EpCAM was heterogeneous in Huh7 and Huh1 cells. In another study from the same group, they demonstrated that the EpCAM expression was transcriptionally up-regulated by β-catenin and the expression of EpCAM and β-catenin was detected by immunofluorescence.2, 3 But there was no obvious heterogeneous expression of these two molecules. So, how to explain this phenomenon? We think that a TOP/FOP reporter assay could be designed as an experiment to observe the activation of β-catenin in hepatic cancer cells with the different expression patterns of miR-181s and EpCAM.
Second, are miR-181s transcriptionally regulated by β-catenin transactivation? To date, the transcriptional regulation of microRNA itself is a hotspot, but little evidence has been made to elucidate the detailed mechanism. In this study, the authors informed the existence of the putative β-catenin/transcription factor 4 (TCF4) binding sites in the promoter regions of all the miR-181s.1 However, the explanation cannot avoid the possibility that miR-181s are regulated by β-catenin in an indirect manner. Although Wang's group performed genetic and pharmacological methods to identify β-catenin transactivation in the transcriptional regulation of downstream molecules,3 we still think that the promoter reporter assay should be an optimally functional experiment to confirm the authors' conclusion.
Third, is the mechanism of microRNA biogenesis also involved in the regulation of miR-181s? We found a very interesting phenomenon in that, although all the mature miR-181s have similar expression trends in the process of the differentiation of the liver (see figure 2D,F in Ji et al.1), the different magnitudes between miR-181d and miR-181c are almost 10-fold. Using bioinformatics methods, we found a difference of only 50 nucleotides between miR-181c and miR-181d. So, it is hard to explain why β-catenin is the only key factor in the regulation of miR-181 expression. We hypothesize that the mechanism of microRNA biogenesis might also be involved in the process of miR-181 regulation in liver tissue.
Finally, from this study, can we delineate a positive feedback loop that Wnt signaling activation is essential for the expression of EpCAM and miR-181s, miR-181s can down-regulate a Wnt/β-catenin signaling inhibitor (NLK), and this can positively activate Wnt signaling to further increase the percentage of EpCAM-positive hepatic cancer stem cells? In our own opinion, we think this study gives momentumto the theory of hepatic cancer stem cells and explores a novel strategy for the therapeutics of hepatic cancer cells by disruption of the Wnt signaling pathway.