FIG 3.

Enhanced expression of miR-373 inhibits IRF9 and ISG56 expression. (A) Introduction of the miR-373 mimic in IHH reduces IRF9 and ISG56 expression. The blot was reprobed with an antibody to actin for comparison of protein loads. (B) Fold changes of IRF9 and ISG56 expression levels after normalization to actin levels from three independent experiments, determined by densitometry scanning. (C) Hepatocytes were transfected with anti-control-miR or anti-miR-373. Cell extracts were prepared after 48 h posttransfection, and IRF9 and ISG56 expression levels were analyzed by Western blotting using specific antibodies. The blot was reprobed with an antibody to actin for normalization. (D) Fold changes of IRF9 and ISG56 expression levels after normalization to actin from three independent experiments, determined by densitometry scanning. (E) Hepatocytes were mock treated or infected with HCV genotype 2a, followed by transfection with anti-control-miR or anti-miR-373 after 24 h postinfection. Cell lysates were analyzed for IRF9 and ISG56 expression by Western blotting using specific antibodies. The blot was reprobed with an antibody to actin for normalization. (F) Fold changes in expression levels of all the proteins after normalization to actin from three independent experiments, determined by densitometry scanning. (G) In silico analysis of the IRF9 3′ UTR reveals a single putative miR-373 binding site. The miR-373 target region of IRF9 (GenBank accession number NM_006084) is indicated. (H) Hepatocytes were cotransfected with the 3′ UTR of IRF9 cloned into the pMIR-Report luciferase vector and different doses of the miR-373 mimic (10 or 50 nM). Relative luciferase activity was measured after 48 h of transfection. (I) Luciferase activity decreased with the 3′ UTR of the IRF9 reporter after cotransfection with the miR-373 mimic (50 nM) but not with the mutant 3′ UTR of IRF9. The results are shown as means and standard deviations from three independent experiments. *, P < 0.05; ***, P < 0.001.