N6-methyladenosine modification of hypoxia-inducible factor-1α regulates Helicobacter pylori-associated gastric cancer via the PI3K/AKT pathway

Figure 4 Methyltransferase-like protein 14 is downregulated by Helicobacter pylori and involved in hypoxia-inducible factor-1α expression. A: Detection of primary m6A modifiers under Helicobacter pylori (H. pylori) infection; B: Methyltransferase-like protein 14 (METTL14) plasmid and METTL14 small interfering RNAs (siRNAs) were successfully transfected into gastric epithelial cells (GES-1) and the mRNA expression was determined by quantitative reverse transcription polymerase chain reaction; C: Actinomycin D experiment was performed to test the stability of hypoxia-inducible factor-1α (HIF-1α) mRNA at 0 h, 6 h, 12 h, and 24 h. remaining (%) = 6/12/24 h HIF-1α mRNA level/0 h HIF-1α mRNA level; D and E: The mRNA and protein levels of HIF-1α were detected in cells that were transiently transfected with pcDNA3.1(+)-METTL14 and infected with H. pylori; F and G: Transiently transfected siRNA-METTL14 cells were infected with H. pylori, and then the mRNA and protein levels of HIF-1α were detected; H: Immunofluorescence staining and confocal microscopy images of control and Hp27-infected GES-1 cells. Nuclei (blue), METTL14 (green), and HIF-1α (Red). Scale bars = 20 μm. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001, compared with pcDNA3.1(+) or Hp27 + pcDNA3.1(+), siRNA-NC or Hp27 + siRNA-NC group, all normalized to β-actin. siRNAs: Small interfering RNA; METTL14: Methyltransferase-like protein 14; HIF-1α: Hypoxia-inducible factor-1α; NC: Negative control.