miR-148 and miR-152, which target Dnmt1 and Dnmt3b, are upregulated in the livers of the wild type mice fed the liquid alcohol diet.

<p><b>A.</b> miR-148/152 cognate site predicted by TargetScan in the 3′-UTR of Dnmt1. <b>B.</b> Northern blot analysis demonstrated increased expression of miR-148 in mice fed alcohol. Total RNA (5 µg) was separated in 15% acrylamide-8 M urea gel, transferred to nylon membrane and subjected to Northern blotting using ³²P-labeted anti-miR-148 oligo as probe, washed and subjected to autoradiography. The blot was rehybridized to 5S rRNA probe after stripping to demonstrate comparable RNA in each lane. <b>C.</b> qRT-PCR analysis confirmed upregulation of hepatic miR-148&152 in mice fed alcohol. miR-148/152 and RNU6B were measured in the liver cDNAs using Taqman probes and primers specific for each miRs and the data was normalized to RNU6B level. <b>D.</b> Dnmt1 is a validated target of miR-148&152. Hepa cells were transfected with psiCHECK2 vector harboring wild type or mutant Dnmt1 3′-UTR downstream of renilla luciferase coding region along with 50 nM miR-148b/152 mimic or scrambled RNA (NC RNA). After 48 h, renilla (RLU2) and firefly luciferase (RLU1) (expressed from the same vector) activities were measured and the data are represented as RLU2/RLU1. Each assay was performed in triplicate. <b>E.</b> Upregulation of miR-148/152 in Hepa cells transfected with the respective miRs compared to the controls (NC RNA transfected cells). Total RNAs from each sample in D was subjected to qRT-PCR as described in B. Each assay was performed in triplicate. <b>F.</b> Endogenous DNMT1 and DNMT3b protein levels were reduced in cells expressing ectopic miR-148/152. H293T cells were transfected with 50 nM miRs or NC RNA. After 24 h, cells were split and harvested at indicated time points post-transfection for western blot analysis of whole cell extracts (100 µg) with specific antibodies. Relative expression was determined after normalization of the signal to that of Gapdh. Single, double and triple asterisks represent P-values ≤0.05, ≤0.01 and ≤0.005, respectively.