1C,D). Notably, although the miR-122 level in Huh7 cells was also significantly down-regulated, it was still detected on northern blot analysis (Fig. 1C,D). As shown in HSP inhibitor Fig. 1E, qRT-PCR data revealed that the expression levels of C/EBPα, HNF1α, HNF3β, and HNF4α gradually increased with time from e12.5 to birth, after which they either continued to increase at a much slower rate or declined slightly. Additionally, the up-regulation of HNF4α and C/EBPα (70-fold and 40-fold, respectively) was more significant than that of HNF1α and HNF3β (<10-fold). As shown in Fig. 1F, the expression of these four LETFs
was also significantly down-regulated in HCC cell lines compared with that in the adult mouse liver. Similarly, their expression levels in Huh7 cells tended to be much higher than the levels in the other cell lines. Overall, it was evident that the expression of miR-122 was strongly correlated with the expression of HNF1α, HNF3β, HNF4α, Crizotinib in vivo and C/EBPα, especially the latter two. The data suggest that these LETFs potentially regulate the expression of miR-122. To investigate whether C/EBPα, HNF1α, HNF3β, and HNF4α are involved in the transcriptional regulation of miR-122, we analyzed the promoter region of miR-122. Previous study has shown that miR-122 is derived from the 3′ end of a 4.7-kb noncoding
RNA (hcr) in the woodchuck.11 Comparison of genomic sequences across species revealed that the 5′ end of the woodchuck hcr gene22 located in a highly conserved region (≈160 bp), which was included in the predicted promoter (predicted by the FirstEF program, available at the UCSC Genome Browser [http://genome.ucsc.edu/]) of human primary
miR-122 (Supporting Fig. 2). Rapid amplification of complementary DNA ends assay revealed that the transcription start sites of human and mouse primary miR-122 locate at the same region as that G protein-coupled receptor kinase of the woodchuck (Supporting Fig. 3), also supporting the prediction. By scanning the predicted human miR-122 promoter with TransFac (BIOBASE gene-regulation.com, Wolfenbuettel, Germany) and Genomatix (Genomatix, Munich, Germany) software, several potential LETF binding sites were identified (Fig. 2A and Table 1). We first checked if these LETFs could act on the predicted promoter using luciferase reporter assays. As shown in Fig. 2B, the miR-122 promoter activated the expression of the downstream reporter up to 20-fold, indicating that the predicted miR-122 promoter was a true promoter. Moreover, compared with red fluorescence protein, HNF1α, HNF3β, and HNF4α further elevated expression of the reporter through this promoter (Fig. 2B). These data suggest that these three HNFs might directly bind to the miR-122 promoter. C/EBPα may bind to elements outside of the promoter, including intronic regions.19 Therefore, we further analyzed the noncoding region between the promoter and the miR-122 precursor (+38 to +4811) (Fig. 2A).