Comprehensive Analysis of N6-Methyladenosine (m6A) Methylation in human Calcific Aortic Valve Tissues by High-Throughput m6A Sequencing
Background: Recent research shows cardiovascular diseases have been strongly linked to N6-methyladenosine (m6A) methylation. However, there is still much to learn about the specific changes in m6A RNA methylation in patients with calcified aortic valve dBackground: Recent research shows cardiovascular diseases have been strongly linked to N6-methyladenosine (m6A) methylation. However, there is still much to learn about the specific changes in m6A RNA methylation in patients with calcified aortic valve disease (CAVD).  Methods: In this study, high-throughput methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA sequencing (RNA-seq) were performed to identify the differentially expressed m6A methylation pattern in human calcified valves tissue for the first time.  Results: A total of 7,412 significantly changed m6A methylation peaks and 2,380 differentially expressed genes were identified. MeRIP-seq and RNA-seq analyses screened out 131 genes with significant m6A methylation and expression changes. Gene Ontology (GO) analysis also revealed a relationship between m6A methylated genes and inflammatory responses, calcium ion binding, extracellular matrix organization, and cell adhesion, among other. Furthermore, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that these genes had an increased role in focal adhesion. Using real-time qPCR, we also confirmed the presence of 4 genes involving the focal adhesion signaling pathway (SPP1, COL1A1, ITGA8, and MYL9).  Conclusions: Taken together, our study firstly revealed the distinct m6A methylation profiles in calcified valve tissue and associated biological roles and signaling pathways. The information in this study will be useful for future mechanism research and for developing new treatment targets for patients with CAVD. Overall design: The m6A methylation profiles of calcified valve tissues and non-calcified valve tissues