METTL16/IGF2BP2 axis enhances malignant progression and DDP resistance through up-regulating COL4A1 by mediating the m6A methylation modification of LAMA4 in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is the third most common malignant tumor after gastric cancer and esophageal cancer, which is a serious threat to human health. Methyltransferase-like protein 16 (METTL16) regulates the occurrence and development of various cancers, but its molecular mechanism in HCC has not been fully investigated. METHODS: A series of databases were used to predict gene expression, methylation sites, correlation analysis, and protein interaction analysis. Gene expression levels were detected by quantitative real-time polymerase chain reaction (qRT-PCR), western blot, and immunohistochemistry (IHC). What's more, drug-resistant cell lines were established for drug resistance analysis. Cell proliferation was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and 5-ethynyl-2'-deoxyuridine (EdU) staining. Flow cytometry, transwell and wound healing assays were used for apoptosis, invasion and migration, respectively. In addition, the regulatory mechanism of METTL16 in HCC was investigated by methylated RNA immunoprecipitation (MeRIP), RNA immunoprecipitation (RIP) and co-immunoprecipitation (Co-IP). Finally, constructing subcutaneous transplanted tumor in nude mice confirmed the effect of METTL16 in vivo. RESULTS: METTL16 was up-regulated in HCC drug-resistant tissues and cells. Knockdown of METTL16 inhibited Cisplatin (DDP) resistance, proliferation, invasion and migration of HCC cells, but promoted apoptosis. Besides, laminin subunit alpha 4 (LAMA4), which was overexpressed in HCC drug-resistant tissues and cells, was selected as the target of METTL16. Mechanistically, METTL16 and m6A reader insulin like growth factor 2 mRNA binding protein 2 (IGF2BP2) co-regulated the m6A modification and mRNA stability of LAMA4, and LAMA4 weakened the effects of METTL16 knockdown on HCC drug-resistance. Meanwhile, LAMA4 bound to collagen type IV alpha 1 chain (COL4A1) and facilitated DDP resistance and HCC progression via COL4A1. Similarly, in vivo, METTL16 induced tumor growth, as well as LAMA4 and COL4A1 expression, and increased DDP resistance. CONCLUSION: METTL16 and IGF2BP2 jointly mediated the m6A methylation modification of LAMA4, thereby promoting DDP resistance and malignant progression of HCC through regulation of COL4A1.