Transcriptome profiling of intact bowel wall reveals that PDE1A and SEMA3D are possible markers with roles in enteric smooth muscle apoptosis, proliferative disorders, and dysautonomia in Crohn's disease

Abstract

Background: Inflammatory bowel disease (IBD) is a complex and multifactorial inflammatory condition, comprising Crohn's disease (CD) and ulcerative colitis (UC). While numerous studies have explored the immune response in IBD through transcriptional profiling of the enteric mucosa, the subtle distinctions in the pathogenesis of Crohn's disease and ulcerative colitis remain insufficiently understood. Methods: The intact bowel wall specimens from IBD surgical patients were divided based on their inflammatory status into inflamed Crohn's disease (iCD), inflamed ulcerative colitis (iUC) and non-inflamed (niBD) groups for RNA sequencing. Differential mRNA GO (Gene Ontology), and KEGG (Kyoto Encyclopedia of Genes and Genomes), and GSEA (Gene Set Enrichment Analysis) bioinformatic analyses were performed with a focus on the enteric autonomic nervous system (ANS) and smooth muscle cell (SMC). The transcriptome results were validated by quantitative polymerase chain reaction (qPCR) and immunohistochemistry (IHC). Results: A total of 2099 differentially expressed genes were identified from the comparison between iCD and iUC. Regulation of SMC apoptosis and proliferation were significantly enriched in iCD, but not in iUC. The involved gene PDE1A in iCD was 4-fold and 1.5-fold upregulated at qPCR and IHC compared to that in iUC. Moreover, only iCD was significantly associated with the gene sets of ANS abnormality. The involved gene SEMA3D in iCD was upregulated 8- and 5-fold at qPCR and IHC levels compared to iUC. Conclusion: These findings suggest that PDE1A and SEMA3D may serve as potential markers implicated in enteric smooth muscle apoptosis, proliferative disorders, and dysautonomia specifically in Crohn's disease.

Keywords: Crohn’s disease; PDE1A; RNA seq; Sema3D; apoptosis; autonomic nervous system; proliferation; smooth muscle cell.

FIGURE 1

PCA and differential expression analysis of transcriptomic profiling. (A,B) Principal Component Analysis (PCA) plot displaying mRNA transcriptomic data. Each point represents a specific specimen, while each color represents a different group. Numbers along the perimeter indicate principal components (PC1-PC2), and numbers in parentheses represent the percentage variance accounted for by each PC. (C) Venn diagram illustrating the overlaps between differentially expressed mRNAs identified in iCD vs. niBD, iUC vs. niBD, and iCD vs. iUC comparisons. The criteria for differential expression were log2 fold change (log2FC) greater than absolute value of 0.58 and an adjusted p-value less than 0.05. (D) Bar graph showing the number of dysregulated mRNAs. Upregulated mRNAs are depicted in red, while downregulated mRNAs are shown in blue. The criteria for dysregulation were log2FC greater than absolute value of 0.58 and an adjusted p-value less than 0.05. (E–G) Heatmap representing differentially expressed mRNAs identified through whole transcriptomic analysis of IBD specimens. Each row corresponds to one mRNA, and each column represents a specimen. Upregulated and downregulated mRNAs are highlighted in red and blue, respectively. The dendrograms on top of the heatmap display hierarchical clustering correlation between the specimens.

FIGURE 2

Pathway analysis of dysregulated mRNAs reveals dysregulated enteric smooth muscle cell apoptosis and proliferation in CD. (A) Selected Gene Ontology (GO) biological processes that exhibit significant enrichment among dysregulated mRNAs(log2FC >|0.58| and adjusted p-value <0.05). The enrichment score for each dysregulated gene annotated to the corresponding GO term is indicated. p-values greater than 0.05 are labeled in grey. (B–G) Volcano plots depicting dysregulated mRNAs. Pink and blue dots represent upregulated and downregulated mRNAs, respectively (log2FC >|0.58| and adjusted p-value <0.05). Non-significant mRNAs are displayed in grey. Dysregulated mRNAs associated with selected processes are labeled in dark red or blue. (H) Selected KEGG signaling pathways significantly enriched among dysregulated mRNAs (log2FC >|0.58| and adjusted p-value <0.05). The enrichment score for each dysregulated gene annotated to the corresponding KEGG pathway is presented. p-values greater than 0.05 are labeled in grey.

FIGURE 3

GSEA analysis shows activation of abnormality of the autonomic nervous system (ANS) and aganglionic megacolon in iCD. (A,B) Gene Set Enrichment Analysis (GSEA) of abnormality of the autonomic nervous system-related gene sets in iCD and iUC specimens vs. niBD. (C) Top 20 core enrichment genes associated with abnormality of the autonomic nervous system. (D,E) GSEA of aganglionic megacolon-related gene sets in iCD and iUC specimens vs. niBD. (F) Top 20 core enrichment genes associated with aganglionic megacolon.

FIGURE 4

Validation by real-time qPCR of genes overactivated or downregulated in colon specimens of iCD (n = 5) and iUC (n = 5) compared to those from niBD (n = 5). Graphs display interleaved box and whisker plots representing the range from minimum to maximum values. For RNA sequence values expressed in FPKM and qPCR values expressed in 2^−ΔΔCt, statistical analysis was performed using Fisher’s Least Significant Difference (LSD) test.Nonsignificant p-values (>0.05) are denoted. Asterisks (*) indicate statistical significance levels: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

FIGURE 5

Baseline information of specimen sections prepared with non-inflamed and inflamed colon from CD and UC patients. (A) Comparison of disease onset ages between CD and UC patients. (B) Gender distribution in CD and UC patients. (C) Variation in depth scores of inflammatory infiltration observed in CD and UC patients. (D) Proportion of CD patients exhibiting periganglionitis.

FIGURE 6

Abundant expression of SEMA3D and PDE1A proteins in layers of muscularis propria and mucosa in inflamed CD specimens. (A) Immunohistochemistry (IHC) images showing representative colon specimens from patients with CD and UC, including non-inflammatory and inflammatory samples. Staining demonstrates expression of SEMA3D and PDE1A at a magnification of ×20. The scale bar corresponds to 40 μm. (B,C) Graphs presenting the quantification of staining (percentage of DAB-positive tissue) in niBD, iUC, and iCD cohorts (niBD, n = 4; iUC, n = 7; iCD, n = 15). Statistical analysis was performed using Fisher’s LSD test. Nonsignificant p-values (>0.05) are denoted. Asterisks (*) indicate statistical significance levels: *p ≤ 0.05, **p ≤ 0.01.