The nuclear and cell boundaries could not be seen clearly. Moreover, many dead cells or cell debris with bright green fluorescence were floating Selleckchem Decitabine above the living cell layer. These phenomena suggest that G2-122×8 cells might have been undergoing apoptosis or/and differentiation. To determine whether miR-122 promoted hepatocyte differentiation, we quantified the mRNA expression of three cytochrome P450 family genes (CYP1A2, CYP2C9, and

CYP7A1) that are hepatic functional proteins specifically expressed in mature hepatocytes.18, 27, 28 Notably, CYP7A1 is a known target of CUTL1 in HepG2 cells.27 As shown in Fig. 5F, in G2-122×4 cells, only the expression of CYP7A1 increased, Saracatinib whereas in G2-122×8 (B7) cells, all three cytochrome P450 genes were significantly up-regulated. This result indicates that the continuous high level of miR-122 eventually induces the differentiation of hepatoblastoma cells. In addition,

it suggests that CUTL1 is an important functional target of miR-122. In combination, our studies suggest that the activation of miR-122 plays an important role in guiding hepatocyte differentiation during development. This study demonstrates that four LETFs (C/EBPα, HNF1α, HNF3β, and HNF4α) are involved in the transcriptional regulation of miR-122, which could directly regulate a group of target genes involved in proliferation and differentiation regulation. In line with this, restoration of miR-122 in hepatoblastoma cells suppresses cellular proliferation and activates the expression of hepatocyte functional genes. We show that CUTL1 is a biological target of miR-122 during liver development. Our findings support a role of miR-122 in liver development, as shown in Fig. 6. According to this model, miR-122 acts as an important bridge connecting the two different types of regulators that control the balance between the proliferation and differentiation of hepatocytes selleck during liver development. The transcriptional regulation of the majority of miRNAs is currently unknown.

Because miR-122 is the most abundant and specific miRNA in the liver, clarification of its regulatory mechanism is necessary to reveal the transcriptional regulation of liver miRNAs. Here, we provide the first direct evidence that the transcription of miR-122 is regulated by several LETFs. The involvement of several transcription factors in the transcriptional regulation of a single miRNA has not been reported previously. However, this mechanism is a common principle for liver gene regulation.17, 18 While our study was underway, others identified LETFs as central regulatory molecules in gene networks associated with the loss of miR-122 in human HCCs, and their knockdown experiments suggest that miR-122 is under the transcriptional control of HNF1α, HNF3α, and HNF3β.