m6A-related bioinformatics analysis and functional characterization reveals that METTL3-mediated NPC1L1 mRNA hypermethylation facilitates progression of atherosclerosis via inactivation of the MAPK pathway.

objectiveAccumulating evidence has demonstrated that N6-methyladenosine (m6A) plays important roles in many major diseases, including atherosclerosis (AS). In the present study, we aimed to explore the transcriptomic m6A landscape of endothelial function-associated genes and identify potential regulators in AS progression.

methodsThe GEO data (GSE142386) from MeRIP-seq in human umbilical vein endothelial cells (HUVECs) with METTL3 knocked down or not were analyzed. RNA-seq was performed to identify differences in gene expression. Gene ontology (GO) functional and Kyoto encyclopedia of genes and genomes (KEGG) pathway analyses were conducted to evaluate the potential functions of the differentially expressed genes. MeRIP-qPCR was used to measure the m6A and mRNA levels of the top 8 downregulated genes, and NPC1L1 was selected as the candidate gene. Oxidized low-density lipoprotein (ox-LDL) was used to stimulate HUVECs, and METTL3 or NPC1L1 was silenced in ox-LDL-treated cells. And Transwell, ELISA, and cell apoptosis assays were performed to assess cell functional injury. ApoE-/- mice were fed with high-fat diet for 8 weeks to establish an AS model, and adenovirus-mediated NPC1L1 shRNA or NC shRNA was injected into the mice through the tail vein. Mouse aortic tissue damage and plaque deposition were evaluated by H&E, Oil Red O, and TUNEL staining.

resultsOne hundred and ninety-four hypermethylated m6A peaks and 222 hypomethylated peaks were detected in response to knockdown of METTL3. Genes with altered m6A peaks were significantly involved in the histone modification, enzyme activity, and formation of multiple complexes and were predominantly enriched in the MAPK pathway. NPC1L1 was a most significantly downregulated transcript in response to knockdown of METTL3. Moreover, knockdown of NPC1L1 or de-m6A (METTL3 knockdown)-mediated downregulation of NPC1L1 could improve ox-LDL-induced dysfunction of HUVECs in vitro and high-fat diet-induced atherosclerotic plaque in vivo, which was associated with the inactivation of the MAPK pathway.

conclusionMETTL3-mediated NPC1L1 mRNA hypermethylation facilitates AS progression by regulating the MAPK pathway, and NPC1L1 may be a novel target for the treatment of AS.