Fig. 3 | Signal Transduction and Targeted Therapy

Fig. 3

From: m6A RNA methylation-mediated HNF3γ reduction renders hepatocellular carcinoma dedifferentiation and sorafenib resistance

Fig. 3

M6A analysis of HNF3γ mRNA in HCC. a The correlation between HNF3γ levels and METTL3, METTL14, WTAP, or FTO expression in HCC tissues was evaluated in 57 HCC specimens by Pearson’s correlation analysis. b The correlation between HNF3γ levels and METTL14 expression in HCC tissues from Cohort 1 was evaluated using a chi-square test. Representative IHC staining images are shown. Scale bar, 100 μm. c HCCLM3 and Huh7 cells were transfected with si-NC or siMETTL14 and then subjected to immunoprecipitation of m6A-modified RNA followed by real-time PCR analysis. d To analyze the effect of METTL14 on HNF3γ mRNA degradation, HCCLM3 or Huh7 cells transfected with si-NC or si-METTL14 were incubated with actinomycin D (10 μM) for 0, 15, 30, 60, 120, 180, 240, or 300 min. The levels of HNF3γ mRNA were determined by real-time PCR and the values were normalized to time zero. e Relative luciferase activity of pMIR-REPORT-HNF3γ-CDS (left panel) and pMIR-REPORT-HNF3γ-3′ UTR (right panel) with either wild-type or mutant (A-to-T mutation) m6A sites in HCCLM3 cells co-transfected with si-NC or si-METTL14, respectively. Firefly luciferase activity was measured and normalized to Renilla luciferase activity. Results were expressed as means ± SEM. * Indicates the significant difference between WT + si-NC group and WT + si-METTL14 group. # Indicates the significant difference between WT + si-NC group and MUT + si-NC group. See Supplementary materials for the details. f The expression of HNF3γ in HCCLM3 or Huh7 cells transfected with si-NC or si-IGF2BP1/2/3 was determined by real-time PCR. g RIP assay was carried out using anti-IGF2BP1/2/3 antibodies. The RNA was extracted from protein G-agarose with anti-IGF2BP1/2/3 antibody, or with control IgG. The specific anti-IGF2BP1/2/3 binding mRNA regions of HNF3γ were amplified by PCR

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