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  • br Statistical analyses br Statistical analyses were perform

    2020-08-12


    Statistical analyses
    Statistical analyses were performed using Graphpad Prism (Ver. 7). Patients’ survival was compared by KaplaneMeier curve analysis (log-rank test) using SPSS software ver. 19.0 (IBM, New York, NY, USA). Student’s t-test was adopted for statistical analysis of Western blotting results. A P-value < 0.05 was considered statis-tically significant.
    Results
    Analysis of Phenylacetic Acid E co-expression genes in gastric cancer 
    Table 2
    NF-YA and cyclin E expression in gastric cancer specimens from patients with different TNM stages (n ¼ 22).
    Classification NF-YA
    Cyclin E
    Low High
    Low High
    TNM stage
    NF-YA: nuclear transcription factor Y subunit alpha; TNM: Tumor Node Metastasis.
    cBioPortal online platform to predict cyclin E co-expression genes among all 415 samples within the stomach adenocarcinoma (TCGA provisional) sample set. After filtering the co-expression gene prediction results, 78 predicted genes, including 73 protein coding genes and 5 non-coding genes with Pearson correlation coefficient 0.4 were selected. Their ex-pressions were considered to be correlated with cyclin E expression. The top 15 protein coding genes with their gene symbols, chromosome locations, Pearson correlation, and Spearman correlation coefficients are summarized and listed in Table 3. To better understand the involvement of these predicted genes in cellular composition and function, we annotated them in the GO database using the PANTHER online classification system (Fig. 1). The annotation results suggested that these genes that were presumably co-expressed with cyclin E participated mostly in mitosis and regulation
    Table 3
    Top 15 genes with highest Pearson correlation coefficient in 78 genes predicted to co-express with cyclin E.
    Gene symbol Cytoband Pearson Co. Spearman Co.
    To identify genes co-existing with cyclin E and to Pearson Co.: Pearson correlation coefficient; Spearman Co.: identify potential oncogenes in gastric cancer, we used Spearman correlation coefficient.
    Fig. 1. Gene Ontology (GO) enrichment analysis of 78 predicted cyclin E co-expressing genes. GO terms of cellular components (red), molecular functions (green), and biological processes (blue) are plotted in different colors.
    of the cell cycle, consistent with the role of cyclin E in proliferation. We next surveyed the interaction (including predicted association) network among the 73 protein coding genes using the STRING analysis platform and further analyzed the topological structure of the predicted interaction network using Cytoscape software (Ver. 3.5.1). Nineteen genes at the central of the predicted interaction network were identified (Fig. 2 and Table 4). The majority have been previ-ously suggested to promote the progression of gastric cancer or have been associated with poor prognosis. Considering the potential co-expression of these 19 genes with cyclin E and their involvement in gastric 
    cancer progression, we performed a transcription fac-tor enrichment prediction using FunRich software (Fig. 3). The enrichment prediction revealed tran-scription factor NF-YA as the only one significant result (P < 0.05). It was the most highly related to the 19 queried genes.
    High expression of cyclin E and NF-YA is associated with gastric cancer progression and poor prognosis
    Based on the previous results, we hypothesized that cyclin E and NF-YA may be related to the pro-motion of the progression of gastric cancer. As a
    Fig. 2. Predicted proteineprotein interaction network within the 78 predicted cyclin E co-expressing genes.
    proof-of-concept, we examined cyclin E and NF-YA protein expression levels in 22 pairs of gastric can-cer specimens and non-cancerous counterparts. Sig-nificant up-regulation and co-expression (P < 0.001) of cyclin E and NF-YA in gastric cancer were revealed by Western blotting (Fig. 4). The results of Fig. 4A and B showed that cyclin E and NF-YA were highly expressed in gastric cancer tissues compared with adjacent tissues. Fig. 4C showed that high expression of NF-YA tended to be associated with increased cyclin E expression. Analysis of specimen donors’ clinical records also revealed that the high expression of NF-YA tended to be associated with increased cyclin E expression, both of which were 
    associated with the progression of gastric cancer (Table 2). We further compared the survival curves of patients with different cyclin E and NF-YA expression levels based on the Western blotting analysis results by KaplaneMeier curve analysis (Fig. 5). Gastric cancer patients with high cyclin E or high NF-YA expression level clearly showed relatively low over-all survival compared to patients with low cyclin E or NF-YA expression (P < 0.05).