Identification of DEGs in ESCC
After analyzing with GEO2R with adj. P value < 0.01, |log FC| > 1, DEGs (1504 in GSE161533, 1680 in GSE20347 and 972 in GSE77861) were identified. The intersection of three gene sets of DEGs contains 230 genes as shown in venn diagram (Fig.1), which consists of 144 upregulated genes and 86 downregulated genes between normal and ESCC tissues.
GO and KEGG enrichment analysis of DEGs
To annotate the DEGs, GO and KEGG enrichment analysis were performed using DAVID, with P value<0.05 considered significant. GO analysis results (Fig.2) showed that changes in biological processes (BP) were mainly enriched in oxidation-reduction process, positive regulation of cell proliferation, cell-cell adhesion and inflammatory response. Changes in cellular components (CC) of DEGs were significantly enriched in cytoplasm, extracellular exosome, cytosol and extracellular space. Changes in molecular function (MF) of DEGs were enriched in calcium ion binding, protein homodimerization activity, cadherin binding involved in cell-cell adhesion and actin binding. And KEGG analysis showed that DEGs mainly enriched in transcriptional misregulation in cancer and p53 signaling pathway.
PPI network construction and hub genes identification
Prediction of the functional interaction was conducted by STRING online and the PPI network of DEGs was constructed by Cytoscape (Fig.3). Subsequently, 14 hub genes were identified with MCODE score > 10 (Fig.4).
Hub gene analysis
Among all hub genes, only ESPL1 is down-regulated. GO and KEGG analysis network of hub genes was performed using ClueGO (Fig.5). Result showed that hub genes were mainly enriched in positive regulation of mitotic cell cycle phase transition, regulation of cytokinesis and DNA replication origin binding. Subsequently, we conducted an extensive literature search on the hub genes.
AURKA, which is significantly overexpressed in various cancers including ESCC 54 has been reported to contribute to the malignant development of ESCC 55 and Jin et al. have revealed part of the mechanism underlying this process 56. Ke et al. showed that downregulated CDC6 functioned as downstream molecule of RYBP in the inhibition of cell proliferation in ESCC 57. An association between overexpression of DTL and detrimental outcome in basal-like and luminal breast cancer and non-small cell lung adenocarcinomas but not esophagus-stomach cancer was found in another bioinformatic analysis 58. The gene ECT2 encoding guanine nucleotide exchange factor (GEF) functions as an oncogene in a wide spectrum of cancers 59. Sun et al. 60 showed that ECT2 promoted proliferation and metastasis of ESCC via the RhoA-ERK signaling pathway. And the overexpression of ECT2 often suggests a poor outcome in cancers, especially in breast cancer 61,62 , gastric cancer 63, non-small-cell lung cancer 64,65, and ESCC 65. ESPL1, the dysregulation of which plays an important role in the development of aneuploidy 66, is a candidate oncogene in luminal B breast cancers 67. Studies have revealed the significant association between high expression of FOXM1 and poor outcome of ESCC 68,69. GTSE1 was found to promote malignant behavior in hepatocellular carcinoma (HCC) 70, confer to cisplatin resistance in gastric cancer cells 71 and to be involved in breast cancer progression 72. Overexpression of KIF14 in ESCC was validated in a study, in which KIF14 was a downstream gene regulated by the miR-375/MMP13 axis 73. The overexpression of MCM10 in ESCC has been confirmed in an experiment using semiquantitative reverse transcription-PCR 74. MCM2, of which the diagnostic value has been confirmed in different types of cancers 75-77 was found to be a more reliable and useful marker than Ki67 in assessing tumor growth and tumor aggressiveness in patients with ESCC 78 and in screening patients at high risk of ESCC in mass surveys 79. RFC4 has been found to be overexpressed in several cancers 80, while study about its role in ESCC remains blank. RRM1 has been found to be an oncogene in lung cancer 81, the overexpression of which is involved in tumor progression 82 and is transforming to the therapeutic target 83,84. A large-scale, long-term follow-up retrospective analysis 85 showed that TOP2A expression was not only associated with perineural invasion and poorer differentiation, but it could be also an independent prognostic factor. Additionally, as TOP2A is a specific marker for the use of chemotherapeutic drugs such as anthracycline, therapy targeting TOP2A protein may be an appropriate way of individualized treatment and improving the prognosis of ESCC patients. Studies 86,87 using immunohistochemical analysis confirmed that UBE2C protein expression was upregulated in all ESCC cases, but absent in the histologically normal tumor surrounding tissues, pointing out its role as a diagnostic biomarker for ESCC. Besides, high expression of UBE2C is a marker of poor prognosis in ESCC 87.
Table2. Fuction and clinical significance of 14 hub genes with MCODE score ≥10. “→” indicates “was significantly associated with”