Fig. 4

MiR-743a-3p downregulation directly contributes to alcohol-upregulated GSTM1. A Venn plot for the nine overlapped miRNAs. B Relative expression of miRNAs in the Lieber-DeCarli diet plus single binge model. C, D GSTM1 mRNA and protein expression in mouse AML-12 hepatocytes. The cells were cultured with miR-743a-3p mimic or inhibitor for 24 h. E Alignments of miR-743a-3p binding to the 3′-UTRs of GSTM1 mRNAs. F Relative luciferase activity in HEK293T cells. The cells were cotransfected with miR-743a-3p mimic or inhibitor and GSTM1-3′-UTR reporter. G, H GSTM1 mRNA and protein expression, and I GST activity in the hepatocyte-specific miR-743a-3p knockdown mice. Hepatocyte-specific miR-743a-3p knockdown mice were generated by injection into the caudal vein with recombinant AAV8 gene transfer vectors bearing a liver-specific promoter combination (TBG) with mouse miR-743a-3p full length sequence. Mice injected with null vector are served as control. Protein bands intensity was quantified by ImageJ. Data are presented as means ± SD. Statistical comparisons were made using one-way ANOVA with Tukey’s post hoc test or one Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001 versus corresponding control. PF pair-fed, AF alcohol-fed. B, D (n = 4); C (n = 3); F–I (n = 6)