Background
N6-Methyladenosine (m6A) methylation is the most prevalent post-transcriptional modification in mRNA, and plays significant roles in various diseases. Nevertheless, the precise functions of m6A modification in the formation of ALI remain unclear. In this study we explore the transcriptome distribution of m6A methylation and its probable roles of in ALI.
Conclusions
m6A modification was paramount in the pathogenesis of ALI, and provided a foundation for the further investigation in the prevention and treatment of ALI.
Methods
Lipopolysaccharide (LPS) was utilized to establish an ALI mouse model. Real-time qPCR, Western blotting and m6A dot blot were utilized to assess m6A methylation level and the expression of m6A methylation enzymes. MeRIP-Seq and RNA-seq were utilized to explore differential m6A modifications and differentially expressed genes in ALI mice. The hub genes and enriched pathways were assessed by Real-time qPCR and Western blotting.
Results
Our findings showed that overall m6A methylation level was increased in ALI mice lung tissues, accompanied by lower levels of METTL3 and FTO. Notably, the protein expression of these methylases were different in various cells. There were 772 differently expressed m6A peaks in ALI as compared to the control group, with 316 being hypermethylated and 456 being hypomethylated. GO and KEGG analyses demonstrated these differentially methylated genes were associated with the calcium signaling pathway and cAMP signaling pathway. Furthermore, we identified 50 genes with distinct m6A peaks and mRNA expressions by combined analysis of MeRIP-Seq and RNA-Seq. KEGG analysis also demonstrated that these overlapped genes were closely associated with the calcium signaling pathway, cGMP-PKG signaling pathway, etc. Besides, Western blotting results demonstrated that the protein expression of Fibronectin leucine-rich transmembrane protein 3 (Flrt3) as well as the calcium signaling pathway and cGMP-PKG signaling pathway, increased significantly after ALI. Conclusions: m6A modification was paramount in the pathogenesis of ALI, and provided a foundation for the further investigation in the prevention and treatment of ALI.