Among the validated targets, we chose the transcriptional repressor CUTL1 (also known as CDP [CCAAT displacement protein], Cut, or Cux-1)20 for PD98059 price further investigation. Finally, we employed a lentiviral-mediated stable expression system to confirm the role of miR-122 in regulating hepatocyte proliferation and differentiation. In combination,

we placed miR-122 both upstream and downstream of the known gene regulatory network in liver development, which provides an exciting basis for understanding the regulation of liver development. In addition, our results also shed light on the cause and contribution of the down-regulation of miR-122 in HCCs. C/EBP, CCAAT/enhancer-binding protein; CTCF, CCCTC-binding factor; e, embryonic day; HCC, hepatocellular carcinoma; HNF, hepatocyte nuclear factor; LETF, liver-enriched transcription factor; MAP3K, mitogen-activated protein kinase kinase kinase; miR-122, microRNA-122; miRNA, microRNA; mRNA, messenger RNA; qRT-PCR, quantitative reverse-transcription polymerase chain reaction; SRF, serum response factor; UTR, untranslated region. Detailed materials and methods are described in the Supporting Information. Fetal, neonatal, and adult livers were isolated from C57BL/6J mice. Cell lines used were HepG2, Huh7, Sk-hep-1, SMMC-7721, and 293FT. miR-122 and control mimics were

synthesized by GenePharma (Shanghai, China). Anti-miR inhibitors for miR-122 and negative control were obtained from Ambion. All primary antibodies used for chromatin immunoprecipitation assays and western blot analyses of target genes were obtained from Santa Cruz Biotechnology. Quantitative MCE公司 reverse-transcription Pembrolizumab ic50 polymerase chain reaction (qRT-PCR) data are expressed as the mean ± standard deviation (SD) and luciferase data are presented as the mean + SD.

The differences between groups were analyzed using one-way analysis of variance, with P < 0.05 considered statistically significant (two-tailed). To search for regulators that might control in vivo transcription of miR-122, we focused primarily on the transcription factors that play important roles in regulating hepatocyte differentiation during liver development. Among the six families of liver-enriched transcription factors (LETFs) that have been characterized,18 several LETFs (including C/EBPα, HNF1α, HNF3α, HNF3β, and HNF3γ, and HNF4α,) are essential for expression of the complete repertoire of proteins that define hepatocyte function.21 C/EBPα, HNF1α HNF3β, and HNF4α, were further selected because they are highly abundant in both human and mouse liver (Supporting Fig. 1). We first investigated whether LETF expression correlated with miR-122 levels in both mouse embryonic livers and human HCC cell lines. The expression of miR-122 was detected by way of northern blot analysis and qRT-PCR. As shown in Fig. 1A,B, consistent with the previous report,11 miR-122 was gradually up-regulated in the fetal liver from e12.5 to birth.