3C), indicating the specificity of the repression To

inv

3C), indicating the specificity of the repression. To

investigate whether these genes were expressed in liver cells, we performed qRT-PCR assays CT99021 cost to detect their mRNA levels in the mouse liver. As shown in Fig. 3D, mRNAs were expressed in the mouse liver throughout the development, indicating that they are potential functional targets of miR-122 during liver development. Notably, mRNA levels in the majority of genes (8 of 12) were not inversely correlated with miR-122 expression during liver development (Fig. 3D), suggesting that they might be regulated by miR-122 posttranscriptionally. To verify this hypothesis, we selected seven genes whose mRNA levels were significantly reduced from e18.5 to adulthood for further confirmation, but none of the transcripts was significantly affected by miR-122 (Supporting Fig. 4). To investigate whether the identified targets play important roles in liver cells, we selected one target for further investigation. CUTL1 was chosen for three reasons: (1) it is the most prominently repressed target of miR-122 in our reporter screening (Fig. 3B); (2) the binding site for miR-122 within its 3′-UTR is the most conserved site among the candidate targets we selected (Supporting Fig. 5); and (3) it is a known transcriptional repressor of genes specifying terminal differentiation during

development.20, 25 Western blot analysis revealed that the CUTL1 protein (p200) was clearly detected in early stage mouse embryonic livers (e12.5 selleck inhibitor and e15.5), whereas it

was barely detectable after birth (Fig. 4A). Interestingly, the amount of CUTL1 protein gradually disappeared during the progression of development, which was inversely correlated with the expression of miR-122 (Fig. 1A). Moreover, we also observed a similar correlation between the CUTL1 protein abundance (Fig. 4B) and Digestive enzyme the miR-122 level (Fig. 1D) in human HCC cell lines. CUTL1 protein was highly expressed in HepG2, 7721, and Sk-hep-1 cells, whereas it was weakly expressed in Huh7 cells. To confirm that the CUTL1 protein is suppressed by miR-122, we performed both overexpression and knockdown experiments on miR-122 in HCC cells. As shown in Fig. 4C, when HepG2, 7721, and Sk-hep-1 cells were transfected with miR-122 mimics, the CUTL1 protein was significantly reduced. Alternatively, when Huh7 cells were transfected with the miR-122 inhibitor, the CUTL1 protein expression apparently increased (Fig. 4C). These data strongly support the interpretation that CUTL1 is an in vivo target of miR-122 in hepatocytes. To assess whether miR-122 contributes to liver development, we investigated whether miR-122 suppresses cellular proliferation and promotes differentiation, because these functions are contrary to the roles of CUTL1 but are similar to the roles of the LETFs.

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