Demethylase ALKBH5 suppresses invasion of gastric cancer via PKMYT1 m6A modification.

ABSTRACT

BACKGROUND: Gastric cancer (GC) is one of the most pernicious tumors that seriously harm human healthcare. GC metastasis is one of the prime cause of failed cancer treatment, but correlation between N6-methyladenosine (m6A) and GC metastasis was less reported. METHODS: Methylated RNA immunoprecipitation sequencing (MeRIP-seq) of GC tissues was conducted. Quantitative real-time PCR (qRT-PCR), western blotting and immunohistochemistry (IHC) were taken to determine the expression of ALKBH5 in GC tissues and cell lines. RNA-seq together with MeRIP-qRT-PCR was used to screen the target gene of ALKBH5. RNA pulldown, mass spectrometry and RNA immunoprecipitation (RIP) were used to search the "reader" protein of target gene. The mechanism was also validated via a tail vein injection method for lung metastasis model. RESULTS: Decreased expression of ALKBH5 was detected in GC samples, and it was correlated with clinical tumor distal metastasis and lymph node metastasis. ALKBH5 interference promoted metastasis of GC cells and this effect was closely related to the demethylase activity of ALKBH5. PKMYT1, as a downstream target of ALKBH5, promoted invasion and migration in GC. Caused by ALKBH5 knockdown or its demethylase activity mutation, upregulated expression of PKMYT1 indicated that ALKBH5 modulates expression of PKMYT1 in an m6A-dependent manner. IGF2BP3 helped stabilize the mRNA stability of PKMYT1 via its m6A modification site. CONCLUSIONS: This study established an ALKBH5-PKMYT1-IGF2BP3 regulation system in metastasis, representing a new therapeutic target for GC metastasis.