Fig. 4

ATAD3A is a direct target of hypoxia-induced miR-210-5p in hepatoma cells. a Gene-gene interaction network. b In situ ATAD3A and HIF-1α expression in paired HCC and normal liver tissues (n = 85). c Representative immunofluorescence images showing HIF-1α (in green) and ATAD3A (in red) expression Huh7 cells cultured under normoxic and hypoxic (1% O₂ for 24 h) conditions. Scale bar = 200 μm. d Immunoblot showing HIF-1α and ATAD3A in LM3 and Huh7 cells under 1% O₂ or CoCl₂ treatment for 24 h. e qRT-PCR analysis showing ATAD3A mRNA levels in Huh7 cells treated with CoCl₂ or DMSO for 24 h. f qRT-PCR analysis showing ATAD3A mRNA levels in Huh7 and LM3 cells under 1% or 20% O₂. g The overlap between the predicted miRNA regulators of ATAD3A and the hypoxia-responsive miRNAs from three different digestive tract cancers. h qRT-PCR analysis showing miR-210-5P levels in Huh7 and LM3 cells treated with 20% or 1% O₂. i qRT-PCR analysis showing the expression of ATAD3A in Huh7 cells treated with miR-210-5P mimic or inhibitor. J, k The binding sites of miR-210-5p in the 3’UTR region of ATAD3A based on bioinformatics prediction and the sequences of designed ATAD3A mutants (j). Luciferase reporter assay of 293 T cell transfected with miR-210-5P mimics or miR-NC and ATAD3A-3’UTR-wt or ATAD3A-3’UTR-mut (k). l Immunoblot showing ATAD3A expression in hepatoma cells transfected with miR-210-5P mimic or inhibitor. m-o Immunoblot (m) and qRT-PCR (n-o) analyses of ATAD3A expression in Huh7 and LM3 cells transfected with miR-210-5P inhibitor with/without CoCl₂ treatment. Each bar represents the mean ± SEM of three independent experiments. *P < 0.05 and ** P < 0.01