Identification of DEcircRNAs, DEmiRNAs and DEGs in human CRC
A total of 412 DEGs (including 82 upregulated and 330 downregulated) were screened out between 473 CRC and 41 normal samples with the standard of logFC > 2 and adj.P.Val < 0.05 (Fig. 1A). 260 DEcircRNAs (including 253 upregulated and 7 downregulated) were altered significantly between 23 CRC and 23 normal samples by log2FC > 1 and adj.P.Val < 0.05 (Fig. 1B). In terms of the DEmiRNAs, 200 DEmiRNAs reached the inclusion criteria in 450 CRC and 8 normal samples including 82 upregulated and 108 downregulated miRNAs (Fig. 1C). The top 10 DEcircRNAs, DEmiRNAs and DEGs are presented in Table 1.
Table 1
Top 10 DEcircRNAs, DEmiRNAs and DEGs in human CRC
|
Symbol
|
Log Fold Change
|
P value
|
Type
|
circRNAs
|
hsa_circ_0043278
|
7.35193499
|
1.42E-07
|
Up
|
hsa_circ_0072088
|
3.72357337
|
6.43E-06
|
Up
|
hsa_circ_0006174
|
6.97297226
|
2.11E-07
|
Up
|
hsa_circ_0000512
|
6.69347647
|
2.83E-07
|
Up
|
hsa_circ_0000518
|
4.77979328
|
2.11E-06
|
Up
|
hsa_circ_0000511
|
4.62226461
|
2.49E-06
|
Up
|
hsa_circ_0000519
|
6.75758135
|
2.64E-07
|
Up
|
hsa_circ_0005273
|
5.26030064
|
1.27E-06
|
Up
|
hsa_circ_0000520
|
6.94732137
|
2.17E-07
|
Up
|
hsa_circ_0000515
|
5.22840906
|
1.32E-06
|
Up
|
miRNAs
|
hsa-mir-374a
|
7.33005053
|
2.26E-72
|
Up
|
hsa-mir-486-2
|
-6.0682766
|
1.35E-32
|
Down
|
hsa-mir-142
|
6.04852788
|
1.06E-29
|
Up
|
hsa-mir-486-1
|
-6.028201
|
3.42E-31
|
Down
|
hsa-mir-135b
|
5.86491219
|
5.52E-22
|
Up
|
hsa-mir-21
|
5.80174888
|
5.68E-114
|
Up
|
hsa-mir-19b-2
|
5.74040834
|
1.35E-40
|
Up
|
hsa-mir-19a
|
5.558011
|
1.99E-26
|
Up
|
hsa-mir-139
|
-5.4424734
|
2.64E-52
|
Down
|
hsa-mir-328
|
-5.4243365
|
3.50E-51
|
Down
|
mRNAs
|
AQP8
|
-6.9967508
|
3.94E-82
|
Down
|
CA1
|
-6.4593573
|
3.89E-111
|
Down
|
GUCA2A
|
-6.2382921
|
1.29E-60
|
Down
|
GUCA2B
|
-6.2006141
|
3.68E-109
|
Down
|
CLCA4
|
-6.1925953
|
1.30E-69
|
Down
|
ZG16
|
-6.1848044
|
2.71E-52
|
Down
|
SLC26A3
|
-5.9938727
|
2.32E-41
|
Down
|
CD177
|
-5.894624
|
1.03E-81
|
Down
|
TMIGD1
|
-5.6845743
|
6.53E-129
|
Down
|
MS4A12
|
-5.6317548
|
1.05E-68
|
Down
|
Functional enrichment analysis of GO and KEGG analysis of DEGs
DEGs were functionally classified into biological process (BP), cellular component (CC) and molecular function (MF categories). In the BP category, the top 9 most enriched terms were “regulation of protein processing”, “protein activation cascade”, “regulation of acute inflammatory response” and “complement activation”. In the CC category, the top 4 most enriched terms were “extracellular matrix”, “collagen-containing extracellular matrix”, “blood microparticle” and “apical part of cell”. In the MF category, the 3 most enriched terms were “antigen binding”, “receptor regulator activity” and “receptor ligand activity” (Fig. 2A). In addition, almost 16 KEGG pathways were significantly enriched in our analysis. The top 3 most enriched terms were “cytokine-cytokine receptor interaction”, “Mineral absorption” and “Steroid hormone biosynthesis” (Fig. 2B).
PPI network construction and survival analysis
A total of 412 DEGs (82 upregulated and 330 downregulated) were used to construct the PPI networks, which included 226 nodes and 478 edges. The combined minimum required interaction score༞0.5 was considered statistically significant (Fig. 3). 5 genes (CXCL8, TIMP1, CXCL1, SPP1,CXCL12) were confirmed as hub genes and next were taken for survival analysis.
Survival analysis of hub genes
The prognostic value of the 5 hub genes was assessed in CRC patients using Kaplan-Meier analysis and log-rank test. The results indicated that CRC patients with high expression of TIMP1 showed worse overall survival (P = 0.004). In contrast, the other 4 hub genes (CXCL8, CXCL1, SPP1,CXCL12) were not related to the overall survival of CRC patients (p > 0.05) (Fig. 4).
CeRNAs regulatory network construction
Not only CircRNA-miRNA but also miRNA-mRNA interactivity was taken into account, an integrated ceRNAs network was constructed. In order to clearly show the interaction in ceRNAs,the regulatory network contained some well-described biomarkers, for example, hsa-miR-671-5p༌hsa-miR-17-3p༌hsa-miR-328-3p and TIMP1. The information in this ceRNAs network is particularly crucial for searching the potential biomarkers for CRC. For instance༌hsa-miR-671-5p interacted with TIMP1, while mediated by hsa-circ-0002191. Hsa-miR-17-3p interacted with TIMP1, while mediated by hsa-circ-0023397(Fig. 5).
The expression levels of DEmiRNAs and DEGs in human CRC tissue
To identify the authenticity and feasibility of CeRNAs regulatory network,some vital DEmiRNAs and DEGs are evaluated in CRC and normal tissues. We found that TIMP1 overexpressed in CRC tissue compared to normal tissue (P༜0.001 ). In contrast, the expression level of hsa-miR-671-5p༌hsa-miR-17-3p and hsa-miR-328-3p was significantly decreased in CRC tissue (Fig. 6).