Fig. 3

ALKBH5 downregulated PAQR4-mediated with IGF2BP1 in a m6A dependent manner. A Schematic representation exhibited totally 5 possible sites of m6A motifs in PAQR4 mRNA, and all of these positions were muted as described then constructed into psiCHECK-Vector plasmids to investigate the m6A roles on PAQR4 expression. PAQR4-WT was constructed into psiCHECK-Vector plasmids as control. B The psiCHECK PAQR4-MUT and psiCHECK PAQR4-WT plasmids were transfected into wild-type or ALKBH5-overexpression HLF cells and ALKBH5-knockdown LM3 cells for 24 h. Fluorescence intensity represented changes in PAQR4 transcriptional activity. C MeRIP-qPCR analysis indicated ALKBH5 overexpression abolish m6a modification on PAQR4 mRNA in HLF cells while ALKBH5 knockdown enriched m6a modification on PAQR4 mRNA. D Actinomycin D (ActD) assay showed overexpression of PAQR4 accelerate the degradation of mRNA whereas this process slowed down after ALKBH5 was knockdown. E PAQR4 expression was measured via RT–qPCR in two IGF2BP1 knockdown HCC cells. F The result of RIP-qPCR indicated that IGF2BP1 could bind to PAQR4 mRNA in HLF and LM3 cells. GAPDH was used as internal controls in qPCR analysis. G Representative immunohistochemical staining images of ALHBH5, IGF2BP1 and PAQR4 in human HCC tissues; scale bar: 200 μm. H Correlation analysis between ALHBH5 and PAQR4 in human HCC tissues. I Correlation analysis between IGF2BP1 and PAQR4 in human HCC tissues. *P < 0.05, **P < 0.01, ***P < 0.001