The edge information plays an important role in the identification of hub genes in a PPI network. We used the parameter“degree” to calculate the edge counts of every single gene in a PPI network. The top 5% degree genes are shown in Table 3, which were assessed as hub genes. Thirteen genes in the PPI network were selected as hub genes in ESCC. These hub genes might play crucial roles in ESCC.
To further understand the regulatory relationship between 26 significant DEMs and hub genes, the miRNA-mRNA regulation network was established and is shown in Figure 5. There were no hub genes that could be the target genes of hsa-miR-339-3p, hsa-miR-375 or hsa-miR-196b-3p. MiR-183-5p was with the highest connectivity target genes, targeting 7 hub genes (upregulated: MMP9, TOP2A, AURKA, and MCM2; downregulated: IVL, FOS, and CYP2C9). Five hub genes could be target genes of hsa-miR-9-3p and hsa-miR-130b-3p. Moreover, several genes were predicted to be common targets of different miRNAs. For instance, FOS was predicted as a common target of hsa-miR-133a-3p, hsa-miR-34b-5p, hsa-miR-34c-5p, hsa-miR-133b, hsa-miR-150-5p, hsa-miR-130b-3p, hsa-miR-431-5p, hsa-miR-382-5p, hsa-miR-424-5p, hsa-miR-196a-3p, hsa-miR-183-5p, and hsa-miR-497-5p.
We investigated the miRNAs expression level of 26 significant DEMs in the TCGA dataset, and found that 23 miRNAs were consistent with the results from GEO databases. However, the expression levels of hsa-miR-203a, hsa-miR-182-3p, and hsa-miR-1-3p were not significantly different between the ESCC samples and normal ESCC tissues. Using the TCGA ESCA dataset, we assessed the relationship of miRNAs with the clinical TNM stages. Detailed results of the significant DEMs are presented in Table 4.
Based on the TCGA survival data, survival analysis using the Kaplan–Meier method and the log-rank test was conducted. The results indicated that hsa-miR-9-3p, hsa-miR-34c-3p and FOS were related to overall survival (OS) in ESCC patients (Fig. 6). Higher expression of the transcript was associated with the poor OS in patients with ESCC.
ESCC is one of the most lethal cancers and is a public health issue of great concern worldwide. Despite many advances in diagnosis and treatment, there is still no effective treatment for ESCC, and survival remains very poor. Thus, the molecular mechanisms that are involved in the progress of ESCC have not been clarified. Therefore, it is crucial to study the mechanism and identify the molecular targets for diagnosis and treatment. In the present study, we identified 26 significant DEMs, 96 upregulated DEGs, and 184 downregulated DEGs. The results of the functional enrichment analysis indicated that significant DEGs were related to the ECM-receptor interaction and cytochrome P450 pathways in ESCC. Thirteen genes in the PPI network were selected as hub genes in ESCC. Importantly, hsa-miR-9-3p, hsa-miR-34c-3p and FOS were validated and were found to be correlated with tumor stages and survival, which meant they could not only regulate cellular process but could also be of valuable in clinical practice.
As was suggested by the functional enrichment analysis of significant DEGs, we found that the DEGs were mainly enriched in biological processes including, the ECM-receptor interaction and the cytochrome P450 metabolic pathways, and so on. ECM-receptor interaction was a chief contributor of cancer progression. Cytochrome P450 levels showed significant differences between ESCC patients and healthy subjects and may contribute to the development of ESCC. The genetic polymorphism RsaI/PstI in cytochrome P450 2E1 (CYP2E1) was a risk factor for EC. It demonstrated that CYP2E1 was a genetic determinant in the development of ESCC. CYP2C9 expression was relevant for high Ki-67 labeling indices in EC. Moreover, inhibition of cytochrome P450 2C9 (CYP2C9) could affect the tumor cell proliferation in early EC development.
A growing body of evidence suggests that miRNAs have significant roles in human tumorigenesis, tumor progression and metastasis. MiRNAs are unique in their ability to regulate mRNA, which is more than 60% of protein-coding genes. The aberrant expression of miRNAs in ESCC causes destruction to the miRNA-regulated mRNA networks and can function as tumor suppressors or oncogenes. Therefore, the identification of the miRNA-mRNA regulatory network is important for further research concerning ESCC. Compared with normal samples, a microarray analysis identified 26 significant DEMs in our results. MiR-18a/b,[35,36] miR-503, miR-9, miR-133a/b,[39,40] miR-34b/c,[41,42] miR-375, miR-203a, miR-150, miR-130b, miR-7, miR-382, miR-424, miR-146b, miR-196a, miR-183,[15,52], and miR-1 were reported in ESCC studies, and mostly involved in ESCC pathogenesis. MiR-18a overexpression was positively correlated with the stage that promoted the expression Cyclin D1 regulating PTEN-PI3K-AKT-mTOR signaling axis in ESCC cells. MiR-150 plays an oncogenic role in ESCC. MiR-150 promots ESCC cell migration and invasion by directly targeting ZEB1, SPOCK1 and Gli1.[45,54,55] MiR-133a, miR-133b and miR-375 are known diagnostic and prognostic markers that are associated with tumor suppressor miRNAs. MiR-133a, miR-133b and miR-375 are frequently downregulated in ESCC and are closely related to advanced clinical stage, tumor metastasis and poor prognosis. MiR-133a can suppress the migration and invasion of ESCC cells by targeting Sox4 and the EMT process. MiR-133b can regulate metastases of ESCC by affecting the MAPK/ERK and PI3K/AKT signaling pathways by targeting EGFR. MiR-375 is involved in the development and progression of ESCC by repressing metadherin expression.[39,40,43] In this study, higher expression levels of hsa-miR-9-3p and hsa-miR-34c-3p were found to be associated with poor OS in patients with ESCC. It was reported that miR-9 induces epithelial-mesenchymal transition (EMT) in ESCC, which is a key event in tumor metastasis. High plasma miR-9 concentrations are significantly correlated with poor tumor differentiation.[38,56] MiR-34b and miR-34c are located on intron 1 and exon 2 of the common primary transcript. MiR-34b and miR-34c expression levels were significantly higher in ESCC in the corresponding normal samples. Inhibiting the expression of miR-34b or miR-34c in ESCC cells may suppress cell growth in vitro. However, it may inactivate the p53-miR-34 pathway. Taken together, these significant DEMs provide potential biomarkers and molecular mechanisms for the diagnosis and therapy of ESCC.
In this study, 26 DEMs and 280 DEGs that consisted of 96 upregulated and 184 downregulated genes were identified. Hsa-miR-9-3p, hsa-miR-34c-3p and FOS were found to be related to OS in ESCC patients. The higher expression levels of the transcript were associated with poor OS in patients with ESCC. The present study provides potential biomarkers and molecular mechanisms for the diagnosis and therapy of ESCC and provides valuable clues for further research.
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