METTL14-mediated N6-methyladenosine modification of ITGB4 mRNA inhibits metastasis of clear cell renal cell carcinoma

Abstract

Abstract Background Integrin β4 (ITGB4) participates in tumorigenesis and progression of several malignancies, but its role and related mechanisms in clear cell renal cell carcinoma (ccRCC) remain unclear. Methods Quantitative real-time PCR (qRT-PCR), western blot and immunohistochemistry were used to detect mRNA and protein levels of relevant genes. Biological functions of ITGB4 and methyltransferase-like 14 (METTL14) were determined by in vitro and in vivo experiments. The levels of N6-methyladenosine (m6A) in ccRCC tissues and adjacent normal tissues were calculated via total RNA m6A quantification assay. The m6A modification of ITGB4 was demonstrated via m6A RNA immunoprecipitation (MeRIP), RIP and luciferase reporter assays. Results ITGB4 was significantly overexpressed in ccRCC tissues and high level of ITGB4 predicted poor prognosis as well as metastasis. Functionally, ITGB4 stimulated ccRCC cell migration and invasion in vitro and metastasis in vivo with epithelial–mesenchymal transition (EMT) strengthened. Mechanically, the total levels of m6A were reduced in ccRCC tissues. METTL14, a favorable factor for ccRCC patients’ prognosis, facilitated m6A modification on ITGB4 3′UTR and subsequently accelerated ITGB4 mRNA degradation, leading to its declined expression. Furthermore, the METTL14-mediated inhibition of ITGB4 expression was dependent on the YTH domain family protein 2 (YTHDF2), which acted as an m6A reader to bind to ITGB4 mRNA and to promote its decay. In addition, we demonstrated that knockdown of METTL14 promoted ccRCC cell migration, invasiveness and metastasis as well as stimulating the EMT process and the PI3K/AKT signal by overexpressing ITGB4. Conclusion Our study reveals that METTL14 inhibits ITGB4 expression via m6A modification to attenuate metastasis and EMT of ccRCC cells, suggesting the METTL14/ITGB4 axis as a potential prognostic biomarker and therapeutic target for ccRCC.