S100P as a Shared Biomarker in Inflammatory Bowel Disease, Colorectal Cancer, and Pancreatic Adenocarcinoma: An Integrated Transcriptomic Analysis

Summary

This protocol integrates public transcriptomic datasets and colonic epithelial cell validation to identify S100P as a shared biomarker associated with inflammatory bowel disease, colorectal cancer, and pancreatic adenocarcinoma.

Abstract

Inflammatory bowel disease (IBD) is associated with an increased risk of colorectal cancer (CRC) and pancreatic adenocarcinoma (PAAD), yet the molecular features shared among these diseases remain incompletely understood. This study aimed to identify common genes and biological pathways associated with IBD, CRC, and PAAD through integrated transcriptomic analysis and experimental validation. Gene expression datasets for IBD, CRC, and PAAD were obtained from The Cancer Genome Atlas and Gene Expression Omnibus databases. Weighted gene co-expression network analysis and differential expression analysis were performed to identify disease-associated and shared genes. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (analyses were used to explore enriched biological functions and pathways. Immune cell infiltration was evaluated using Cell-type Identification by Estimating Relative Subsets of RNA Transcripts. Receiver operating characteristic analysis was performed to assess the diagnostic performance of common genes. Single-cell RNA sequencing analysis was conducted to examine the cellular distribution of S100P. In addition, the effects of S100P downregulation were evaluated in lipopolysaccharide (LPS)-stimulated colonic epithelial cells. A total of 162 disease-associated genes and four common genes were identified. Functional enrichment analyses indicated significant enrichment of immune- and inflammation-related pathways, including the interleukin-17 signaling pathway. Immune infiltration analysis revealed similar trends in several immune cell populations across IBD, CRC, and PAAD. Single-cell analysis showed elevated S100P expression in epithelial cells from all three diseases. Downregulation of S100P restored the proliferative capacity of LPS-stimulated colonic epithelial cells and reduced inflammatory cytokine expression. Integrated transcriptomic analysis identified S100P as a biomarker associated with IBD, CRC, and PAAD and highlighted shared immune-related features across these diseases.

Introduction

Inflammatory bowel disease (IBD) represents a spectrum of immune-related disorders that affect the gastrointestinal tract, including ulcerative colitis and Crohn’s disease¹. The etiology of IBD is highly complex, involving mucosal immune abnormalities, dysbiosis, and genetic susceptibility². IBD is a global health concern with increasing incidence rates and substantial economic burdens, and its growing prevalence has attracted considerable attention¹. Importantly, patients with IBD have a significantly increased risk of developing colorectal cancer (CRC)³ and pancreat....

Protocol

This study used publicly available, de-identified datasets from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO), as well as established commercial cell lines. No newly recruited human participants, identifiable patient information, or patient-derived samples were involved. All analyses were performed in accordance with relevant institutional guidelines and the terms of use of the public databases. Therefore, additional institutional ethical approval and informed consent were not required for this study.

Data Source
RNA-seq data for the IBD cohorts (GSE179285; platform: GPL6480 and GSE24287; platfo....

Results

DEGs in IBD, CRC, and PAAD
First, the IBD cohorts (GSE179285 and GSE24287), CRC cohorts (TCGA-CRC and GSE87211), and PAAD cohorts (GSE128735 and GSE62452) were integrated, and the integration was evaluated using PCA and gene expression density plots. The results showed that batch effects among the different datasets were effectively eliminated after integration (Figure 1A–F).

Discussion

Previous studies have highlighted the association of IBD, an immune-related gastrointestinal disorder, with several diseases, including neurodegenerative diseases¹⁷, multiple sclerosis¹⁸, amyotrophic lateral sclerosis¹⁹, endometriosis²⁰, rheumatoid arthritis², Hodgkin’s lymphoma²¹, CRC²², and PAAD²³. IBD is generally considere.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BXPC2 cell line	Cell Bank of the Chinese Academy of Sciences	N/A	Human pancreatic adenocarcinoma cell line
CIBERSORT	N/A	N/A	Immune cell infiltration analysis
clusterProfiler package	Bioconductor	v4.8.0	Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses
Dulbecco's Modified Eagle Medium (DMEM)	Thermo Fisher Scientific	11965092	Cell culture medium
Fetal bovine serum (FBS)	Thermo Fisher Scientific	A5256701	Cell culture supplement
FHC cell line	Cell Bank of the Chinese Academy of Sciences	N/A	Human normal colonic epithelial cell line
GEO dataset (GSE128735)	Gene Expression Omnibus (GEO)	N/A	Public gene expression dataset for pancreatic adenocarcinoma
GEO dataset (GSE154778)	Gene Expression Omnibus (GEO)	N/A	Public single-cell RNA sequencing dataset for pancreatic adenocarcinoma
GEO dataset (GSE166555)	Gene Expression Omnibus (GEO)	N/A	Public single-cell RNA sequencing dataset for colorectal cancer
GEO dataset (GSE179285)	Gene Expression Omnibus (GEO)	N/A	Public gene expression dataset for inflammatory bowel disease
GEO dataset (GSE214695)	Gene Expression Omnibus (GEO)	N/A	Public single-cell RNA sequencing dataset for inflammatory bowel disease
GEO dataset (GSE24287)	Gene Expression Omnibus (GEO)	N/A	Public gene expression dataset for inflammatory bowel disease
GEO dataset (GSE62452)	Gene Expression Omnibus (GEO)	N/A	Public gene expression dataset for pancreatic adenocarcinoma
GEO dataset (GSE87211)	Gene Expression Omnibus (GEO)	N/A	Public gene expression dataset for colorectal cancer
ggplot2 package	CRAN	v3.4.2	Data visualization
HCT116 cell line	Cell Bank of the Chinese Academy of Sciences	N/A	Human colorectal cancer cell line
ImageJ	National Institutes of Health (NIH)	v1.8.0	Colony counting
jetPRIME Transfection Reagent	Polyplus	101000046	Cell transfection
Lipopolysaccharide (LPS)	Beyotime Co., Ltd	S1735	Induction of an inflammation-mimicking IBD cell model
limma package	Bioconductor	v3.54.0	Differential expression analysis
NCM460 cell line	Cell Bank of the Chinese Academy of Sciences	N/A	Human normal colonic epithelial cell line
org.Hs.eg.db package	Bioconductor	v3.23.1	Gene annotation for enrichment analysis
PANC1 cell line	Cell Bank of the Chinese Academy of Sciences	N/A	Human pancreatic adenocarcinoma cell line
Penicillin–streptomycin	Thermo Fisher Scientific	15140-122	Antibiotic supplement for cell culture
pheatmap package	CRAN	v1.0.12	Heatmap visualization
pROC package	CRAN	v1.19.0.1	Receiver operating characteristic (ROC) analysis
PrimeScript RT Master Mix	Takara Bio	RR036A	Reverse transcription of RNA into cDNA
Primer sets for qRT-PCR	Tsingke Biotech Co., Ltd	N/A	Primer sequences reported in Reference 11
R software	R Foundation for Statistical Computing	v4.1.2	Statistical and bioinformatics analyses
Seurat package	CRAN	v4.0	Single-cell RNA sequencing data preprocessing and analysis
siS100P	Tsingke Biotech Co., Ltd	N/A	Small interfering RNA targeting S100P
SingleR package	Bioconductor	v2.6.0	Cell type annotation for single-cell RNA sequencing
STRING database	STRING Consortium	N/A	Protein-protein interaction analysis
SW1116 cell line	Cell Bank of the Chinese Academy of Sciences	N/A	Human colorectal cancer cell line
TB Green qPCR Mix	Takara Bio	RR430B	Quantitative real-time PCR
TCGA-CRC dataset	The Cancer Genome Atlas (TCGA)	N/A	Public colorectal cancer transcriptomic dataset
TRIzol reagent	Invitrogen	15596-026	Total RNA extraction
WGCNA package	CRAN	v1.73	Weighted gene co-expression network analysis