Fig. 5.

ALDOA was regulated by HIF‐1α and the m⁶A modification under hypoxia. (A) Correlation analysis of the RNA levels of HIF‐1α and ALDOA in HCC tumor samples. (B) Silencing of HIF‐1α decreased ALDOA expression at the mRNA level in HCC cells under hypoxia. (C) Correlation and colocalization analysis of ALDOA and HIF‐1α in a subcutaneous xenograft HCC mouse model. (D) Correlation and colocalization analysis of ALDOA and HIF‐1α in tumor tissues of patients with HCC by immunofluorescence histochemistry. (E) Wild‐type and mutants of HIF‐1α binding elements of ALDOA promoter were fused into a firefly luciferase reporter. Three potential HIF‐1α binding elements were found in the ALDOA promoter region, and mutant forms of ALDOA promoter were deleted as indicated. (F) Relative luciferase activity of the wild‐type and three mutant forms of ALDOA promoter in SMMC‐7721 cells under hypoxia. (G) Half‐life of ALDOA mRNA in SMMC‐7721 cells under normoxia or under hypoxia treated with actinomycin D. (H) Global mRNA m⁶A levels in SMMC‐7721 cells under hypoxia and in subcutaneous xenograft HCC tumors were determined by RNA m⁶A dot‐blotting assay. (I) Knockdown of key regulators of m⁶A modification affected the expression of ALDOA mRNA under hypoxia. (J) Knockdown of FTO significantly reduced the expression of ALDOA mRNA under hypoxia in HCC cells. (K) Knockdown of FTO significantly reduced the expression of ALDOA protein under hypoxia in HCC cells. (L) Half‐life of ALDOA mRNA in FTO‐knockdown SMMC‐7721 cells under hypoxia treated with actinomycin D. (M) Knockdown of FTO promoted m⁶A methylation in ALDOA mRNA by the m⁶A methylated RIP analysis in SMMC‐7721 cells. (N) Knockdown of YTHDF2, but not YTHDF1, rescued expression of ALDOA in FTO‐depleted SMMC‐7721 cells. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Abbreviations: ACTD, actinomycin D; CDS, coding sequence; FC, fold change; WT, wild type.