Fig. 3

HBV downregulated NFAT5 via inhibiting miR-30e-5p and activating MAPK signaling pathway. a MiR-30e-5p overexpression induced NFAT5 mRNA and protein expression in Hep3B, as verified by RT-qPCR and western blot analysis. b MiR-30e-5p relative expression was lower in HepG2.2.15 than HepG2 (P = 0.0008). (C) Top panel: MiR-30e-5p was downregulated in HCC tissues (P < 0.0001), as determined by RT-qPCR in 55 pairs of HCC tissues and para-tumor tissues. The relative expression of miR-30e-5p was calculated by log2 (2^-ΔΔCT) and normalized to U6 expression. Botton panel: MiR-30e-5p was downregulated in HBV-associated HCC (n = 39), compared to non-viral HCC (n = 19). P = 0.0056. d MiR-30e-5p expression was downregulated in the HCC cell lines HepG2 (P = 0.0217), Huh7 (P = 0.0015) and Hep3B (P < 0.0001) compared with that in the normal liver cell line L02. e Western blot analyses revealed that MAP4K4, p-ERK1/2, and c-MYC expression decreased when cells were transfected with miR-30e-5p mimics. f A luciferase reporter assay showed that co-transfection with miR-30e-5p mimics and the wild-type MAP4K4 3’UTR decreased relative luciferase activity (P = 0.0015). WT = wild-type, MUT = mutant, mimics = miR-30e-5p mimics, vector = empty vector. g ChIP-PCR revealed that the c-MYC protein directly interacted with DNA sequence fragments containing the NFAT5 promoter. The picture displays the AGE result after ChIP-PCR. IP = immunoprecipitation group (with the c-MYC antibody), input = group without any antibody as a positive control, IgG = group with IgG as a negative control. Quantitative analysis of AGE showed that IP group was significantly higher than the IgG group (P < 0.0001). h RT-qPCR and western blot showed that HCC cells treated with the c-MYC inhibitor 10,058-F4 expressed higher levels of NFAT5 mRNA (P < 0.0001) and protein. The efficiency of 10,058-F4 was verified by a western blot analysis of c-MYC