m^(6)A-mediated regulation of PBX1-GCH1 axis promotes gastric cancer proliferation and metastasis by elevating tetrahydrobiopterin levels

Background:Methyltransferase 3(METTL3)-mediated N6-methyladenosine(m^(6)A)RNA modification has been demonstrated to be a potential factor in promoting gastric cancer(GC).METTL3 regulates a series of signaling pathways by modifying various mRNAs.This study aimed to identify novel METTL3-mediated signaling pathways and explored possible targets for use in the clinical setting of gastric cancer.Methods:To investigate the proliferation and metastatic capacity ofGCcell lines with METTL3 knockdown,a xenograft,lung metastasis,and popliteal lymph node metastasis model was used.Them^(6)A-modified RNA immunoprecipitation(Me-RIP)sequence was utilized to explore the target mRNAs of METTL3.Cell counting kit 8 and transwell assays were performed to investigate the promoting function of pre-B cell leukemia homeobox 1(PBX1)and GTP cyclohydrolase 1(GCH1).Western blotting and chromatin immunoprecipitation were employed to confirm the involvement of the METTL3-PBX1-GCH1 axis.ELISA and liquid chromatography-mass spectrometry were used to explore the biological function of tetrahydrobiopterin(BH_(4)).Results:Knockdown of METTL3 suppressed xenograft tumor growth and lung/lymph node metastasis in vivo.Mechanistically,we found that METTL3 combined with and stabilized PBX1 mRNAs.Chromatin immunoprecipitation(ChIP)and further experiments suggested that PBX1 acted as a transcription factor inducing GCH1 expression.Moreover,the METTL3-PBX1-GCH1 axis increased BH_(4)levels in GC cells,thereby promoting tumor progression.Conclusions:This study suggested that METTL3 enzymes promote tumor growth and lung/lymph node metastasis via METTL3-PBX1-GCH1 axis increasing BH_(4)levels in GC.