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. 2023 Apr 26:14:1078266.
doi: 10.3389/fimmu.2023.1078266. eCollection 2023.

Genome-wide analysis of RNA-binding proteins co-expression with alternative splicing events in mitral valve prolapse

Meng Zhao  1 , Jingxin Zhou  1 , Yihu Tang  1 , Mingzhu Liu  1 , Yawei Dai  1 , Hui Xie  1 , Zihao Wang  1 , Liang Chen  1 , Yanhu Wu  1
Affiliations

Genome-wide analysis of RNA-binding proteins co-expression with alternative splicing events in mitral valve prolapse

Meng Zhao et al. Front Immunol. .

Abstract

Objectives: We investigated the role and molecular mechanisms of RNA-binding proteins (RBPs) and their regulated alternative splicing events (RASEs) in the pathogenesis of mitral valve prolapse (MVP).

Methods: For RNA extraction, we obtained peripheral blood mononuclear cells (PBMCs) from five patients with MVP, with or without chordae tendineae rupture, and five healthy individuals. High-throughput sequencing was used for RNA sequencing (RNA-seq). Differentially expressed genes (DEGs) analysis, alternative splicing (AS) analysis, functional enrichment analysis, co-expression of RBPs, and alternative splicing events (ASEs) analysis were conducted.

Results: The MVP patients exhibited 306 up-regulated genes and 198 down-regulated genes. All down- and up-regulated genes were enriched in both Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Furthermore, MVP was closely associated with the top 10 enriched terms and pathways. In MVP patients, 2,288 RASEs were found to be significantly different, and four suitable RASEs (CARD11 A3ss, RBM5 ES, NCF1 A5SS, and DAXX A3ss) were tested. We identified 13 RNA-binding proteins (RBPs) from the DEGs and screened out four RBPs (ZFP36, HSPA1A, TRIM21, and P2RX7). We selected four RASEs based on the co-expression analyses of RBPs and RASEs, including exon skipping (ES) of DEDD2, alternative 3' splice site (A3SS) of ETV6, mutually exclusive 3'UTRs (3pMXE) of TNFAIP8L2, and A3SS of HLA-B. Furthermore, the selected four RBPs and four RASEs were validated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and showed high consistency with RNA sequencing (RNA-seq).

Conclusion: Dysregulated RBPs and their associated RASEs may play regulatory roles in MVP development and may therefore be used as therapeutic targets in the future.

Keywords: RNA sequencing; RNA-binding protein; alternative splicing; genome-wide analysis; mitral valve prolapse.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
DEGs in the MVP patient and healthy individual groups. (A) shows an inter-sample clustering analysis of sample correlation. The 306 up-regulated genes and 198 down-regulated genes are shown in (B) A heat map of 504 DEGs is shown in (C) DEGs, differentially expressed genes; MVP, mitral valve prolapse.
Figure 2
Figure 2
Functional enrichment analysis of DEGs in the MVP patient and healthy individual groups. The down-regulated DEGs that were enriched in GO terms are shown in panels (A–C), and up-regulated DEGs that were enriched in GO terms are shown in panels (D–F). The down- and up-regulated DEGs that were enriched in KEGG pathways are shown in (G, H), respectively. DEG, differentially expressed gene; GO, Gene Ontology; MVP, mitral valve prolapse.
Figure 3
Figure 3
The AS analysis of peripheral blood in MVP patients. (A) Principal component analysis of two sample groups of samples based on normalized NIR splicing ratio. (B) The bar chart shows the distribution of AS events in nine AS types. X-axis: the different types of AS events. Y-axis: RASE number. (C) Hierarchical clustering heat map showing expression levels of all RASEs. (D) Bubble diagram exhibiting the most enriched GO biological process results of the regulated alternative splicing genes (RASGs). (E) Bar plot showing the expression pattern and statistical difference of RASE. AS, alternative splicing; GO, Gene Ontology; MVP, mitral valve prolapse; RASE, regulated alternative splicing event. * means p<0.05, ** means p<0.01.
Figure 4
Figure 4
Analysis of differential expression of RBPs in MVP patients (A) Venn diagram showing the overlap in the number of genes of RBPs and DEGs. (B) Hierarchical clustering heat map showing the expression levels of overlapping RBPs. (C) The scatterplot shows the RBPs and the number of co-expressed RASGs. (D) Bubble diagram exhibiting the most enriched GO biological process results of the RBPs co-expressed RASGs. (E) Bar plot showing the expression pattern and statistical difference of RBPs. DEG, differentially expressed gene; GO, Gene Ontology; MVP, mitral valve prolapse; RASG, regulated alternative splicing gene; RBP, RNA-binding protein. ** means p<0.01, *** means p<0.001.
Figure 5
Figure 5
Co-expression network between DE RBPs and RASGs associated MVP patients. (A) The network diagram shows co-expression between RASGs with ZFP36, HSPA1A, TRIM21, and P2RX7. (B) Bubble diagram exhibiting the most enriched GO biological process results of the four RBPs co-expressed with RASGs. (C) Bar plot showing the expression pattern and statistical difference of RASEs. DEG, differentially expressed gene; GO, Gene Ontology; MVP, mitral valve prolapse; RASG, regulated alternative splicing gene; RBP, RNA-binding protein; DE RBPs, differentially expressed RBPs. * means p<0.05, ** means p<0.01.
Figure 6
Figure 6
Validation of important RBPs and RASGs in clinical samples. Results of the qPCR of the gene expression of four RPBs and four AS events of RASGs. AS, alternative splicing; RAS, regulated alternative splicing gene; RBP, RNA-binding protein; qPCR, quantitative polymerase chain reaction.
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