Fig 2: HIF3A was targeted by miR-184. (A) RNA22-HAS database indicated miR-184 bound to the predicted target sequence in the 3ʹ UTR of the HIF3A and dual luciferase reporter assay verified the target relationship between HIF3A and miR-184. *P < 0.05. (B) The miR-184 expressions in 90 paired GA and adjacent tissues were detected using Western blot and qRT-PCR, *P < 0.05, compared with the group of adjacent tissues. (C) The miR-184 expressions in human gastric adenocarcinoma cells (AGS and NCI-N87) and human gastric mucosa cell (GES 1) were detected using Western blot and qRT-PCR. *P < 0.05, compared with the group of GES 1. (D) qRT-PCR was applied to detect the successful upregulation of miR-184 in the AGS cell. *P < 0.05, compared with the NC group. (E) Western blot and qRT-PCR were applied to detect the HIF3A expression in the AGS cell after overexpressing miR-184. *P < 0.05, compared with the NC group.

Fig 3: HIF3A inhibited AGS cell property both in normoxic and hypoxic conditions. (A) The HIF1A expressions in AGS cells with normoxic or hypoxic induction were detected by qRT-PCR. *P < 0.05, compared with normoxic group. (B) Phosphoenolpyruvate (PEP) and lactic acid were detected using Phosphoenolpyruvate Fluorometric Assay Kit and Lactic Acid Detection kit, respectively, in the AGS cells with normoxic or hypoxic induction. *P < 0.05, compared with normoxic group. (C) Cell proliferation of AGS cells with or without overexpressing HIF3A in normoxic and hypoxic conditions was determined using colony formation assay. *P < 0.05, compared with corresponding NC group. #P < 0.05, compared with corresponding normoxic group. &P < 0.05, compared with corresponding normoxic group. (D, E) Cell migration and invasion of AGS cells with or without overexpressing HIF3A in normoxic and hypoxic conditions were determined using transwell migration and invasion assays. *P < 0.05, compared with corresponding NC group. #P < 0.05, compared with corresponding normoxic group. &P < 0.05, compared with corresponding normoxic group.

Fig 4: HAND2-AS1 regulated glycolysis in AGS cells induced by hypoxia. (A, B) The HAND2-AS1 and miR-184 expressions were detected by qRT-PCR in AGS cells with or without HAND2-AS1 overexpression in normoxic or hypoxic induction. (C, D) The HIF3A and HIF1A expressions were detected by Western blot and qRT-PCR in AGS cells with or without HAND2-AS1 overexpression in normoxic or hypoxic induction. (E, F) PEP and lactic acid were detected using Phosphoenolpyruvate Fluorometric Assay Kit and Lactic Acid Detection kit, respectively, in AGS cells with or without HAND2-AS1 overexpression in normoxic or hypoxic induction. *P < 0.05, compared with normoxic group. #P < 0.05, compared with hypoxic group.

Fig 5: Histone acetylation (H3K9/14ac; H3K27ac) and DNA methylation (5-methylcytosine, 5mC) changes reveal relaxed chromatin architecture at the ATAC-seq peak regions near the transcription start site (TSS) of Hif3a and Slc10a6 genes.A Schematic showing the location of ATAC-seq peaks and location of primers for qPCR validation for Hif3a and Slc10a6. B The “open peak” regions at the transcriptional control regions of Hif3a and Slc10a6 were evaluated by chromatin immunoprecipitation (ChIP) assay representing fold changes of H3K27ac and H3K9/14ac occupancy at selected regions of Hif3a and Slc10a6 in ethanol-treated and control rats. C Changes in DNA methylation were also evaluated at the same loci of Hif3a and Slc10a6 in amygdala of ethanol-treated and control rats and represented as fold changes in DNA methylation (5mC levels). Values are represented as the mean ± SEM and individual values are shown on bar diagrams with circle dots for control and ethanol groups (n = 7–8; two-tailed Student’s t test *p < 0.05; **p < 0.01).