piRNA-30473 mediates m6A methylation by regulating the expression of WTAP. (A-B) m6A levels of SU-DHL-8 cells with or without piRNA-30473 knockdown were detected using the EpiQuik m6A RNA Methylation Quantification Kit (A) and m6A dot blot assays (B). MB, methylene blue staining (as loading control). (C) SU-DHL-8 was transiently transfected with antagomir-30473 or antagomir-NC. qPCR was performed to determine METTL3, METTL14, WTAP, ALKBH5, and FTO mRNA levels. (D) Representative immunohistochemical staining of WTAP in tumor xenografts is shown (original magnification ×40). WTAP-positive particles (brown) are apparent in the nucleus (nuclei stained with hematoxylin, blue). Scale bars, 100 μm. (E) Cell lysates were subjected to coimmunoprecipitation (IP) with anti-WTAP antibody or rabbit immunoglobulin G (IgG) as a control, followed by immunoblotting with the respective antibodies, as shown. (F) A schematic representation of the interaction between piRNA-30473 and the 3′ UTR of WTAP. (G) Dual-luciferase reporter assays showed the effect of piRNA-30473 on WTAP reporters with either WT or MUT binding sites. (H) SU-DHL-8 or Farage cells were transiently transfected with antagomir-30473 or antagomir-NC. The half-life of WTAP mRNA was measured. Error bars represent the mean ± SD of 3 independent experiments. ***P < .001.