Integrative analyses of N6-methyladenosine-associated single-nucleotide polymorphisms (m6A-SNPs) identify tumor suppressor gene AK9 in lung cancer

N⁶ -methyladenosine (m⁶ A) modification has been identified as one of the most important epigenetic regulation mechanisms in the development of human cancers. However, the association between m⁶ A-associated single-nucleotide polymorphisms (m⁶ A-SNPs) and lung cancer risk remains largely unknown. Here, we identified m⁶ A-SNPs and examined the association of these m⁶ A-SNPs with lung cancer risk in 13,793 lung cancer cases and 14,027 controls. In silico functional annotation was used to identify causal m⁶ A-SNPs and target genes. Furthermore, methylated RNA immunoprecipitation and quantitative real-time polymerase chain reaction (MeRIP-qPCR) assay was performed to assess the m⁶ A modification level of different genotypes of the causal SNP. In vitro assays were performed to validate the potential role of the target gene in lung cancer. A total of 8794 m⁶ A-SNPs were detected, among which 397 SNPs in nine susceptibility loci were associated with lung cancer risk, including six novel loci. Bioinformatics analyses indicated that rs1321328 in 6q21 was located around the m⁶ A modification site of AK9 and significantly reduced AK9 expression (β = -0.15, p = 2.78 × 10^-8 ). Moreover, AK9 was significantly downregulated in lung cancer tissues than that in adjacent normal tissues of samples from the Cancer Genome Atlas and Nanjing Lung Cancer Cohort. MeRIP-qPCR assay suggested that C allele of rs1321328 could significantly decrease the m⁶ A modification level of AK9 compared with G allele. In vitro assays verified the tumor-suppressing role of AK9 in lung cancer. These findings shed light on the pathogenic mechanism of lung cancer susceptibility loci linked with m⁶ A modification.