Screening and prioritization for DEGs
An available Expression profiling data set (GSE36701) in NCBI was selected in this study to identify differentially expressed genes in the comparisons between IBS-D patients and the healthy volunteers. Then the differentially expressed genes were further filtered for functional associations with neurotransmitters/mediators of pain. Each array was normalized by quantile, and then the DEGs analysis was performed (P-value <0.05, FC≥1.2 or ≤0.8). Compared with the HVs, we find out 9 upregulated genes (CCKBR, CCL13, ACPP, BDKRB2, GRPR, SLC1A2, NPFF, P2RX4, and TRPA1) and 4 downregulated genes (TLX2, MRGPRX3, CXCR1 and PAX2) that may have relevance in the initiation and development of visceral pain. The most significant upregulated or downregulated genes specially linked to pain transmission are illustrated in Table 1 and Table 2.
Table 1 Upregulated genes specially involved in pain transmission in IBS-D patients in comparison with HVs.
ID
|
P Value
|
Gene symbol
|
Gene title
|
FC
|
206407_s_at
|
1.98E-04
|
CCL13
|
C-C motif chemokine ligand 13
|
1.58
|
205870_at
|
1.51E-03
|
BDKRB2
|
bradykinin receptor B2
|
1.38
|
217590_s_at
|
1.62E-02
|
TRPA1
|
transient receptor potential cation channel subfamily A member 1
|
1.23
|
204393_s_at
|
4.26E-03
|
ACPP
|
acid phosphatase, prostate
|
1.55
|
208389_s_at
|
1.11E-02
|
SLC1A2
|
solute carrier family 1 member 2
|
1.31
|
204088_at
|
1.41E-02
|
P2RX4
|
purinergic receptor P2X4
|
1.23
|
207929_at
|
2.62E-02
|
GRPR
|
gastrin releasing peptide receptor
|
1.37
|
206402_s_at
|
4.65E-02
|
NPFF
|
neuropeptide FF-amide peptide precursor
|
1.25
|
234475_x_at
|
1.70E-02
|
CCKBR
|
cholecystokinin B receptor
|
1.77
|
HVs: healthy volunteers; FC: fold change
Table 2 Downregulated genes specially involved in pain transmission in IBS-D patients in comparison with HV.
ID
|
P Value
|
Gene symbol
|
Gene title
|
FC
|
207410_s_at
|
2.27E-02
|
TLX2
|
T-cell leukemia homeobox 2
|
0.65
|
1553293_at
|
1.24E-02
|
MRGPRX3
|
MAS related GPR family member X3
|
0.71
|
206228_at
|
2.58E-02
|
PAX2
|
paired box 2
|
0.76
|
207094_at
|
7.11E-03
|
CXCR1
|
C-X-C motif chemokine receptor 1
|
0.57
|
HVs: healthy volunteers; FC: fold change
Functional Enrichment Analysis
Functional analysis was carried out with GO biological process and KEGG pathways. Genes differentially were classified according to their putative Gene Ontology (GO) based on similarity with known genes recorded in public databases (Fig. 1). To further understand the functions of the DEGs, we represented significant pathways based on KEGG databases (Fig. 2). The upregulated DEGs were mainly associated with Glutamatergic synapse, Dilated cardiomyopathy, Arginine biosynthesis. The downregulated DEGs were linked to Circadian entrainment, Alcoholism, Aldosterone-regulated sodium reabsorption, Maturity onset diabetes of the young, cGMP-PKG signaling pathway, Dopaminergic synapse, Morphine addiction, Systemic lupus erythematosus, Glutamatergic synapse, Glycine, serine and threonine metabolism, Retrograde endocannabinoid signaling, Renin secretion.
PPI Network
A PPI network of the proteins encoded by identified DEGs was constructed by STRING. To identify the key modules of the PPI network, module clustering was then performed through the MCODE plugin of Cytoscape (Fig. 3). The modules showed that several pain or itch related genes like GRPR, NPFF, CCKBR, BDKRB2, CXCR1 and CCL13 genes (Red Rectangle, Fig. 3) were also clustered and most of these genes also occurred in GO terms or KEGG pathways enriched above. Finally, we identified that GRPR, NPFF and TRPA1 as potential biomarkers for abdominal pain of IBS patients.