Construction of ceRNA Network to Reveal Potential Biomarkers in Crohn’s Disease and Validation in a TNBS Induced Mice Model

Figures & data

Figure 1 Flowchart of this work.

Table 1 The Sequences of Primers

human-PTGS2-F	CGGTGAAACTCTGGCTAGACAG
human -PTGS2-R	GCAAACCGTAGATGCTCAGGGA
human -GAPDH-F	ACCCACTCCTCCACCTTTGA
human -GAPDH-R	CTGTTGCTGTAGCCAAATTCGT
mouse-PTGS2-F	CAGGACTCTGCTCACGAAGG
mouse-PTGS2-R	ATCCAGTCCGGGTACAGTCA
mmu-miR-429-3p-R	GTCGTATCGACTGCAGGGTCCGAGGTATTCGCAGTCGATACGACACGGCA
mmu-miR-429-3p-F	GCCGGCTAATACTGTCTGGTAA
mouse-GAPDH-F	GCAAGTTCAACGGCACAG
mouse-GAPHD-R	GCCAGTAGACTCCACGACAT

Figure 2 DEmRNA screening and enrichment analysis. (A) Volcano plot of DEmRNA. (B) Top 10 GO enrichment. (C) Top 10 KEGG enrichment. (D) KEGG chord plot of top 10 terms.

Table 2 The DEmiRNAs

Upregulated			Downregulated
Gene Symbol	Fold Change	P.Adjust	Gene Symbol	Fold Change	P.Adjust
KIF9-AS1	1.59	0.000391	CDKN2B-AS1	−1.52	0.00366
LINC00907	1.13	0.0009
IFITM4P	1.9	1.85E-10
MIR4435-2HG	1.25	0.0000517
WFDC21P	1.19	0.000138
AC100861.1	1.07	0.00102
BDNF-AS	1.11	0.00103
USP30-AS1	1.09	0.0000213
MIR3142HG	1.34	5.45E-08
CYTOR	1.05	0.000445

Figure 3 mRNA modules analyzed by WGCNA. (A) Determination of soft-thresholding power. (B) Clustering dendrogram. (C) The module-trait relationships.

Figure 4 Identification of candidate overlapping mRNA. (A) Intersection of DEmRNAs, WGCNA modules, and miRwalk-predict. (B) Heatmap of overlapping mRNA.

Figure 5 The overlapping mRNA function annotation. (A) PPI network. (B) hub gene identified via cytohubba. (C) KEGG enrichment. (D) GO enrichment. (E) GO chord plot of top 10 term. (F) KEGG chord plot of top 10 term. Lollipop chart.

Table 3 The DElncRNAs

Upregulated			Downregulated
Gene Symbol	Fold Change	P.Adjust	Gene Symbol	Fold Change	P.Adjust
hsa-miR-423-5p	1.00551	0.00225	hsa-miR-196b-3p	−1.70768	1.37E-05
hsa-miR-1307-3p	1.002718	0.00662	hsa-miR-196b-5p	−3.21552	3.53E-05
hsa-miR-345-5p	1.020774	0.0102	hsa-miR-489-3p	−1.39583	0.00072
			hsa-miR-338-5p	−1.56735	0.00129
			hsa-miR-30b-5p	−1.03646	0.00256
			hsa-mir-429	−1.211	0.00335
			hsa-miR-376c-3p	−1.13185	0.00881
			hsa-miR-26b-5p	−1.96192	0.00895
			hsa-miR-29b-3p	−1.56409	0.0198
			hsa-miR-30e-3p	−1.11327	0.0205
			hsa-miR-31-3p	−1.3858	0.0206
			hsa-miR-30a-3p	−1.67973	0.0214
			hsa-miR-30e-5p	−1.1215	0.0282
			hsa-miR-141-3p	−1.37106	0.0314
			hsa-miR-29c-3p	−1.79602	0.0422
			hsa-miR-337-5p	−1.041	0.0461

Figure 6 DElncRNA screening. (A) Volcano plot of DElncRNA. (B) Determination of soft-thresholding power. (C) Clustering dendrogram. (D) The module-trait relationships. (E) Intersection of DElncRNA, WGCNA, and LncBase-predict.

Figure 7 The construction of (A) ceRNA network, red color represents upregulate and blue represents downregulate, circle shape represents mRNAs, triangle represents miRNAs, and rhombus represents lncRNAs. (B) Sankey diagram of final ceRNA network.

Figure 8 Validation and immune infiltration analysis. (A) and (B) ROC of mRNAs in the ceRNA network. (C) The differential of immune infiltration between CD and normal controls. (D) Correlation matrix of immune cell proportions and PTGS2. (E) Lollipop chart of correlation between immune cell and PTGS2. *p<0.05, **p<0.01, ***p<0.001.

Abbreviation: ns, not significant.

Figure 9 The relative expression validated via qRT-PCR. (A) The relative expression of PTGS2 in TNBS-induced mice and control mice. (B) The relative expression of mmu-mir-429 in TNBS-induced mice and control mice. (C) The relative expression of PTGS2 in HCT116 cells transfected with NC, si-MIR3142HG, and mir-429 inhibitor. ***p<0.001.