3.1Differentially expressed circRNA, miRNA and mRNA
The expression profiles of mRNA, miRNA and circRNA between 66 UC samples and 43 normal samples were calculated. The pre-processing of raw data revealed 1809 differentially expressed genes (DEGs), including 878 DEmRNAs, 881 DEcircRNAs and 50 DEmiRNAs (|logFC, log2 fold change| ≥2.0 and a P-value < 0.05). Of these, 499 DEmRNAs, 460 DEcircRNAs and 14 DEmiRNAs were up-regulated; A total of 379 DEmRNAs, 421 DEcircRNAs and 36 DEmiRNAs were down-regulated. The volcano map and heat map of the analyzed DEmRNAs (GSE36807) are shown in Figure 2 and Figure 3.
3.2 Establishment of miRNA-mRNA、circRNA-mRNA interactions
The TargetScan, miRDB as well as miRTarBase databases respectively predicted mRNAs by using 50 DEmiRNAs. The results obtained from the three databases were incorporated, a total of 5954 miRNA-mRNA pairs were acquired after removing duplicates. Then mRNAs in 5954 pairs were intersected with 878 DEmRNAs, successfully attained 285 pairs of miRNA-mRNA interactions, consisting of 13 DEmiRNAs and 209 DEmRNAs. Through the same procedure, starBase database successfully measured circRNAs by using 50 DEmiRNAs, harvesting 403 pairs of circRNA-mRNA interactions, which comprising 5 DEmiRNAs and 403 DEcircRNAs. The results received from the database were intersected with 881 DEcircRNAs, since no intersecting pairs of circRNA-mRNA were found, the ceRNA network was constructed using the 403 circRNA-mRNA pairs predicted by the starBase database.
3.3 Construction of Competing Endogenous RNAs Network
In order to better understand the role of DEcircRNAs in UC and to further elucidate the interaction between these DEcircRNAs and DEmiRNAs, we established a circRNA-miRNA-mRNA related competing endogenous RNAs network of UC. All circRNA-miRNA pairs and miRNA-mRNA pairs were integrated, and Cytoscape 3.7.2 was further adopted for constructing and visualizing the circRNA-miRNA-mRNA network. As shown in Figure 4, the circRNA-associated ceRNA network was composed of 403 circRNA nodes, 5 miRNA nodes, 138 mRNA nodes and 559 edges.
3.4 The key circRNA–miRNA–mRNA subnetwork
For further identification of hub gene as well as their relevanted networks, Cytoscape 3.7.2 plug-in CytoHubba was utilized for calculation and visualization of all node degree values in the circRNA-associated ceRNA network. We selected the top 10 nodes ranked by the degree value, nodes selected were far fewer than the top 25 % initial nodes in the ceRNA network, which represented more accuracy. The top 10 nodes including hsa-miR-342-3p, hsa-miR-199a-5p, hsa-miR-650, hsa-miR-142-3p, hsa-miR-483-3p, ZNF652, INO80D, ANK3, TSPAN6 and PCDH17. The key network formed by the top 10 nodes is shown in Figure 5. To be specific, hsa-miR-342-3p, hsa-miR-199a-5p, hsa-miR-650, hsa-miR-142-3p and hsa-miR-483-3p were five of the top-ranked nodes, it was regretful that hsa-miR-650 and hsa-miR-483-3p were not interconnected with the key subnetwork, therefore, we got rid of them, and 3 new key subnetworks were reconstructed. 3 miRNAs served as the center of subnetwork were significant in regulating transcription, 3 key subnetworks of circRNA-hsa-miR-342-3p-mRNA, circRNA-hsa-miR-199a-5p-mRNA, circRNA-hsa-miR-142-3p-mRNA were extracted and shown in Figure 6 (A-C). The key subnetwork of circRNA-hsa-miR-342-3p-mRNA comprised 1 miRNA node, 50 mRNA nodes as well as 89 circRNA nodes (Figure 6A), The key subnetwork of circRNA-hsa-miR-199a-5p-mRNA consisted of 1 miRNA node, 43 mRNA nodes and 81 circRNA nodes (Figure 6B), The key subnetwork of circRNA-hsa-miR-142-3p-mRNA was composed of 1 miRNA node, 44 mRNA nodes along with 62 circRNA nodes (Figure 6C).
3.5 Functional Enrichment Analysis of Differentially Expressed mRNAs
Analysis about the functions of 138 DEmRNAs which came from the circRNA-associated ceRNA network were carried out. The results elucidated that enrichment of 167 GO categories occurred in the biological process, including 115 biological processes (BP), 36 cell components (CC) and 16 molecular functions (MF) with a threshold value of P＜0.05. The top 5 most significant GO terms in each section are shown in Table 1, Figure 7. On the biological process (BP) level, the DEmRNAs were principally participated in positive regulation of angiogenesis, cellular response to fluid shear stress, semaphorin-plexin signaling pathway involved in axon guidance, wound healing, spreading of cells, negative regulation of cell proliferation. Those in cellular component (CC), in general, were affiliated with the parts of adherens junction, postsynaptic density, filopodium, actin cytoskeleton, GABA-ergic synapse. The majority of DEmRNAs enriched in molecular function (MF) were associated with long-chain fatty acid-CoA ligase activity, xenobiotic-transporting ATPase activity, arylsulfatase activity, efflux transmembrane transporter activity, protein binding. Subsequently, KEGG pathway enrichment analysis of all DEmRNAs in the circRNA-associated ceRNA network were implemented. The total 14 KEGG pathway terms are demonstrated in Table2. The genes were significantly enriched in 7 pathways, comprising with pathways in cancer, cAMP signaling pathway, Metabolic pathways, PPAR signaling pathway, TNF signaling pathway, PI3K-Akt signaling pathway, AGE-RAGE signaling pathway in diabetic complications, as shown in the Figure 8. As mentioned above, these results proved that DEmRNAs played a crucial role in a number of biological processes and functions, such as inflammation response and immune response, which were crucial for the occurrence and development of UC.
Table1. The top 5 GO terms in each section are obtained by analyzing DEmRNAs.
BP, biological process; CC, cellular component; MF, molecular function. Enrichment score: the enrichment score of GO, which is accounted by -log 10 (P-value).
Table2. The total 14 KEGG pathway terms are obtained by analyzing DEmRNAs. KEGG, Kyoto Encyclopedia of Genes and Genomes.
3.6 Building of protein-protein interaction (PPI) Network
We input DEmRNAs within the circRNA-associated ceRNA network into the STRING database for building a PPI network, which including 71 nodes and 150 edges, shown in Figure 9. Thereafter, this PPI network was analyzed by the CytoHubba plug in Cytoscape 3.7.2. According to the degree value obtained by topological analysis, nodes whose degree value >5 in the PPI network were further selected as the core targets, genes totaling 15 were selected, including CD44, HIF1A, MMP2, LOX, PTGS2, CAV1, VCAM1, EDN1, IL1A, HGF, COL1A2, PCSK9, CYR61, ACSL1, ADAMTS5. We further reconstructed a PPI network graphic of the 15 core targets based on the ceRNA network, and the results are shown in Figure 10. The core PPI network consisted of 15 nodes and 69 edges, nodes were represented by circular, nodes from small to large represented the degree value from low to high and color from light to dark, the higher the value, the more important the target. Among them, CD44 was the highest degree value (degree=19), followed by HIF1A (degree= 16) and MMP2 (degree=15). Thus, these 3 targets were worthy to be the most signifcant core targets in the ceRNA network associated with UC and considered conducting further research.