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. 2024 Dec 28;14(1):31349.
doi: 10.1038/s41598-024-82846-1.

Genome-wide analysis of alternative splicing differences in hepatic ischemia reperfusion injury

Yongliang Hua  1   2 , Xinglong Li  1 , Bing Yin  1 , Shounan Lu  1 , Baolin Qian  1 , Yongzhi Zhou  1 , Zhongyu Li  1 , Zhanzhi Meng  1 , Yong Ma  3
Affiliations

Genome-wide analysis of alternative splicing differences in hepatic ischemia reperfusion injury

Yongliang Hua et al. Sci Rep. .

Abstract

Alternative splicing (AS) contributes to transcript and protein diversity, affecting their structure and function. However, the specific transcriptional regulatory mechanisms underlying AS in the context of hepatic ischemia reperfusion (IR) injury in mice have not been extensively characterized. In this study, we investigated differentially alternatively spliced (DAS) genes and differentially expressed transcripts (DETs) in a mouse model of hepatic IR injury using the high throughput RNA sequencing (RNA-seq) analysis and replicate multivariate analysis of transcript splicing (rMATS) analysis. We further conducted Gene ontology (GO) term enrichment, the Kyoto Encyclopedia of Genes and Genomes (KEGG) database and the protein-protein interaction (PPI) network. A total of 898 DAS genes (p ≤ 0.05) were screened out in the hepatic IR group compared to the sham group, while functional enrichment analysis revealed that DETs and DAS genes were significantly associated with the ATP-dependent chromain, splicesome and metabolic pathways. The expression level of the DAS genes: Gabpb2, Smg1, Tnrc6c, Mettl17, Smpd4, Kcnt2, D16Ertd472e, Rab3gap2, Echdc2 and Ssx2ip were verified by RT-PCR and qRT-PCR. Our findings provide a comprehensive genome-wide view of AS events in hepatic IR injury in mice, enhancing our understanding of AS dynamics and the molecular mechanisms governing alternative pre-mRNA splicing.

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Conflict of interest statement

Declarations. Ethical approval: All procedures in the present study were approved by the Ethics Committee of The First Affiliated Hospital of Harbin Medical University (approval number: No. 2021087), and were also conducted in accordance with the Guide for the Care and Use of Laboratory Animals and with the ARRIVE guidelines. Competing interests: The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Analysis of RNA-seq data of hepatic IR injury in mice. (A) Scatter plot of the PCA analysis for the IR and sham groups. (B) Heatmap of hierarchical clustering analysis the ranscript expression patterns in the hepatic IR and sham groups. (C) Volcano plots showing differential expression genes in hepatic IR groups compared with sham groups (log2FC ≥ 0.585/log2FC ≤ -0.585 and p ≤ 0.05). (D) The scatter diagram showing the levels of mRNA genes with AS events in the hepatic IR and sham groups.
Fig. 2
Fig. 2
The diagram presents the AS modes in hepatic IR injury.
Fig. 3
Fig. 3
Analysis of DAS genes and distribution of the five main AS events in the hepatic IR and sham groups. (A) Number of alternative exons between IR and sham groups in mice (p ≤ 0.05). (B) The venn diagram shows the distribution of AS events in the hepatic IR and sham groups.
Fig. 4
Fig. 4
Validation of the expression of DAS genes in the hepatic IR and sham groups. (A) RNA sequencing read density plot. (B) Validation of AS genes by qRT-PCR. (C) RNA-seq of AS genes. Data are shown as the mean ± SD of three independent experiments performed in triplicate.
Fig. 5
Fig. 5
GO and KEGG analyses of DAS genes in the hepatic IR vs. sham groups. (A) the top ten significant GO enrichment terms for the up-regulated genes. (B) the top ten significant GO enrichment terms for the down-regulated genes. (C) the top ten enrichment scores of the enrichment pathway for the up-regulated genes. (D) the top ten enrichment scores of the significant enrichment pathway for the down-regulated genes.
Fig. 6
Fig. 6
AS events interaction network and the distribution of the DAS genes in the PPI network. (A) The distribution of the DAS genes in the PPI network (p ≤ 0.05). (B) The distribution of differential mRNAs with DAS events (fold change ≥ 1.5, p value ≤ 0.05).
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