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Inducing Acute Lung Injury in Mice by Direct Intratracheal Lipopolysaccharide Instillation
Published on: July 6, 2019
Comprehensive analysis of m6A modification in lipopolysaccharide-induced acute lung injury in mice
Chenzhen Xu1, Congkuan Song1, Wenjie Wang1
1Department of Thoracic Surgery, Renmin Hospital of Wuhan University, Wuhan, 430060, China.
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.
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.
Insights
N6-Methyladenosine (m6A) methylation plays a key role in acute lung injury (ALI). This study reveals altered m6A patterns and associated signaling pathways, offering insights into ALI pathogenesis and potential treatments.
Area of Science:
- Molecular Biology
- Epigenetics
- Pulmonology
Background:
- N6-Methyladenosine (m6A) is the most abundant mRNA modification, crucial in various diseases.
- The specific role of m6A in acute lung injury (ALI) pathogenesis remains largely unknown.
- This study investigates the transcriptome-wide distribution and function of m6A in ALI.
Purpose of the Study:
- To explore the transcriptome distribution of m6A methylation in ALI.
- To identify the functional roles of m6A modification in ALI development.
- To uncover potential therapeutic targets for ALI based on m6A regulation.
Main Methods:
- Established an ALI mouse model using lipopolysaccharide (LPS).
- Assessed m6A levels and enzyme expression using qPCR, Western blotting, and m6A dot blot.
- Employed MeRIP-Seq and RNA-seq to identify differential m6A peaks and gene expression.
Main Results:
- Increased overall m6A methylation in ALI lung tissues with altered METTL3 and FTO levels.
- Identified 772 differentially methylated m6A peaks (316 hypermethylated, 456 hypomethylated).
- Associated differentially methylated genes with calcium and cAMP signaling pathways; identified FLRT3 as upregulated in ALI.
Conclusions:
- m6A modification is critically involved in ALI pathogenesis.
- Findings provide a basis for further research into ALI prevention and treatment strategies.
- Altered m6A patterns and associated signaling pathways are key features of ALI.
