Comprehensive analysis of lncRNA-miRNA-mRNA regulatory networks for microbiota-mediated colorectal cancer associated with immune cell infiltration

Figures & data

Figure 1. The flow chart of bioinformatics analysis. Caco-2, human epithelial colorectal adenocarcinoma cells; LGG, Lactobacillus rhamnosus GG; B.caccae, Bacteroides caccae; CRC, Colorectal cancer; CR, Colorectum; Fn, Fusobacterium nucleatum; GDC, Genomic Data Commons Data Portal

Table 1. The expression of 8 DEmiRNAs in probiotic environment from TCGA and GEO database

miRNA	TCGA (colorectal cancer vs normal)		GEO (with probiotics VS without probiotics)			Role
	LogFC	FDR	LogFC	P.value	Group Set
hsa-mir-429	4.114057	1.62E-19	−3.31806	0.01654	LGG VS control	Oppose
hsa-mir-141	5.414177	7.61E-42	3.056937	0.021687	LGG+ B.caccae VS control	Support
hsa-mir-140	−1.31675	3.01E-08	3.03569	0.022295	LGG+ B.caccae VS control	Opposite
hsa-mir-22	2.181122	9.85E-14	2.735955	0.033153	LGG+ B.caccae VS control	Support
hsa-mir-132	−1.35959	9.53E-08	2.810566	0.030002	LGG+ B.caccae VS control	Oppose
hsa-mir-454	6.098412	3.47E-22	3.601023	0.010924	LGG+ B.caccae VS control	Support
			3.342003	0.016062	LGG VS control	Support
hsa-mir-153	5.705182	3.50E-05	−2.77244	0.03157	LGG+ B.caccae VS control	Oppose
hsa-mir-143	3.623069	4.96E-08	3.097131	0.02177	LGG VS control	Support

Control group, without bacteria; FDR, false discovery rate; FC, fold change; Role, the role of probiotics on colorectal carcinogenesis and tumor progression; LGG, Lactobacillus rhamnosus GG; B.caccae, Bacteroides caccae

Table 2. The expression of 4 DEmiRNAs in pathogenic environment from TCGA and GEO database

miRNA	TCGA (colorectal cancer vs normal)		GEO (with pathobionts VS without pathobionts)			Role
	LogFC	FDR	LogFC	P.value	Group Set
hsa-mir-223	3.057351	2.23E-07	1.398719	0.030564	CR tissue + Fn VS control	Support
hsa-mir-32	3.841335	6.25E-22	−1.490555	0.003800	CRC tissue + Fn VS control	Oppose
hsa-mir-96	5.457990	5.53E-18	2.366778	0.009546	CRC tissue + Fn VS control	Support
hsa-mir-106a	4.314718	3.32E-07	1.2774166	0.020353	CR tissue + Fn VS control	Support

FDR, false discovery rate; FC, fold change; Role, the role of pathogenic on colorectal carcinogenesis and tumor progression; CR, Colorectum; Fn, Fusobacterium nucleatum

Figure 2. Overview of lncRNA-miRNA-mRNA competing endogenous RNA (ceRNA) network in probiotics-mediated CRC. Red represents upregulation, and green represents downregulation. LncRNAs, miRNAs, and mRNAs in the networks are represented as diamonds, round rectangles, and circles, respectively. Bar plots show the key RNAs that have the top interaction number in the whole network

Figure 3. Overview of lncRNA-miRNA-mRNA competing endogenous RNA (ceRNA) network in pathobionts-mediated colorectal cancer. Red represents upregulation, and green represents downregulation. LncRNAs, miRNAs, and mRNAs in the networks are represented as diamonds, round rectangles, and circles, respectively. Bar plots show the key RNAs that have the top interaction number in the whole network

Figure 4. GO functional analysis (a) and KEGG pathway (b) analysis of differentially expressed RNAs in microbiota-mediated colorectal cancer

Table 3. Univariate and multivariate analysis

Variables	Univariate analysis			Multivariate analysis
	HR	95% CI	pvalue	HR	95% CI	pvalue
Age	1.02	1.00–1.05	0.02	1.14	1.09–1.19	0.00
Gender	0.95	0.61–1.50	0.84	2.21	0.91–5.33	0.08
Stage	2.12	1.64–2.73	0.00	1.84	0.90–3.76	0.10
AJCC-T	2.51	1.60–3.95	0.00	1.01	0.68–3.31	0.31
AJCC-N	2.10	1.60–2.74	0.00	2.48	1.15–5.35	0.02
AJCC-M	1.92	1.48–2.50	0.00	3.08	1.49–6.36	0.00
FRMD6-AS2	1.04	1.00–1.07	0.03	1.35	1.15–1.58	0.00
LINC00461	1.04	1.02–1.05	0.00	0.92	0.82–1.04	0.17
DIRC3	1.02	1.00–1.03	0.01	1.21	1.08–1.37	0.00
LIFR-AS1	1.02	1.00–1.04	0.03	0.81	0.68–0.95	0.01
NAALADL2-AS2	1.02	1.01–1.03	0.00	1.18	0.74–1.87	0.50
LINC00402	1.01	1.00–1.03	0.03	1.08	0.99–1.17	0.10
ADAMTS9-AS2	1.01	1.00–1.01	0.01	0.96	0.90–1.01	0.13
MRPL23-AS1	1.01	1.00–1.01	0.04	1.03	1.01–1.06	0.00
LHX1	1.01	1.01–1.02	0.00	1.02	0.96–1.08	0.56
RBM20	1.00	1.00–1.01	0.01	1.00	0.99–1.01	0.94

AJCC, the classification system developed by the American Joint Committee on Cancer

Figure 5. Overall survival analysis of RNAs in the ceRNA network of microbiota-mediated colorectal cancer. (a), (b) Kaplan-Meier survival curves of prognostic DELs both in probiotics-mediated ceRNA network and pathobionts-mediated ceRNA network. (c), (d) Kaplan-Meier survival curves of prognostic DELs only in proniotics-mediated ceRNA network

Figure 6. Protein-protein interaction network of differentially expressed RNAs in microbiota-mediated colorectal cancer, the nodes represent proteins, and the edges demonstrate the predicted functional associations between them, line thickness indicates the strength of data support

Figure 7. The expressions of 12 hub genes were determined using GEPIA. The expressions of genes are expressed as relative gene expression using transformed log2 (TPM+1) Value (Y-axis) of tumor (red bar) and normal (black bar) samples and displayed as a whisker plot. * p-value <0.05. GEIPIA, gene expression profiling interactive analysis

Table 4. Results of connectivity map analysis

Rank	Cmap name	Mean	n	Enrichment	p
1	Netilmicin	0.591	4	0.898	0.00010
2	Pentoxyverine	−0.62	4	−0.854	0.00082
3	Meclofenamic acid	0.499	5	0.779	0.00116
4	Timolol	0.559	4	0.832	0.00119
5	Ciclopirox	0.583	4	0.813	0.00237
6	Rimexolone	−0.601	4	−0.811	0.00251
7	Zuclopenthixol	0.561	4	0.803	0.00284
8	Doxylamine	−0.537	5	−0.731	0.00284
9	Prestwick-1082	0.601	3	0.885	0.00310
10	Minaprine	−0.571	5	−0.726	0.00330

Cmap, Connectivity Map

Figure 8. Three-dimensional diagram of the three most significant candidate drugs. (a) Pentoxyverine (b) Rimexolone (c) Doxylamine

Figure 9. Integrative analysis between hub identified signature with humor-infiltrating immune cells

Data availability statement:

The datasets generated during the current study are available in the Gene Expression Omnibus repository: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE79383 and https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE122182, and the NCI’s Genomic Data Commons Data Portal: http://portal.gdc.cancer.gov.