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Keywords: Cyclin E; Nuclear transcription factor Y subunit alpha; Oncogene; Gastric cancer
The incidence of gastric cancer is increasing in developing countries. This imposes a significant finan-cial burden for patients and the healthcare system. Surgical treatment, chemotherapy, and targeted-therapy have been employed in gastric cancer treatment, but the treatment outcome and patients’ post-treatment sur-vival remain disappointing, especially for patients with late stage carcinoma. Cancer cells are characterized by uncontrolled hyper-proliferation. Targeting the cell proliferation machinery or the signal transduction network promoting cell proliferation has been pro-posed as possible therapeutic options for cancer man-agement. Regulation of the 68528-80-3 is frequently altered in cancer by different genetic and epigenetic causes, and the de-regulated cell cycle progression is crucial in cell proliferation and cancer development, in which cyclins and cyclin dependent kinases are direct promoters.1 Targeting the cell cycle has been accepted in-principle as a potential therapeutic option.2e5 However, the molecular mechanisms of altered cell cycle machineries that promote gastric cancer pro-gression remain unclear. r> Gene amplification and overexpression of cyclin E have been recently linked to gastric cancer develop-ment and poor prognosis.6,7 During the progression of the cell cycle, cyclin E binds to and activates cyclin-dependent kinase 2, which promotes G1/S entry by phosphorylating the appropriate substrates. Cyclin E overexpression has been reported in gastric can-cer,8e11 but the molecular mechanism of the up-regulation remains unclear. In this study, we aimed to discover possible mechanisms that may be involved in regulating cyclin E expression by identifying and investigating genes that are potentially co-expressed with cyclin E in gastric cancer. This was done by querying The Cancer Genome Atlas (TCGA)
stomach adenocarcinoma sequencing data with different bioinformatics approaches.
Materials and methods
Bioinformatics analysis pipeline
We used the cBioPortal online platform12,13 to query the TCGA stomach adenocarcinoma (2017, provisional) sequencing dataset (http://www.cbioportal.org/study? id¼stad_tcga#summary). A total of 415 tumor samples (from the TCGA database) with messenger RNA (mRNA) next-generation sequencing data were used. Co-expressed genes predicted by cBioPortal online analysis with Pearson correlation coefficient 0.4 were
selected for gene ontology (GO) enrichment annotation using the PANTHER online platform (Ver. 7).14,15 Protein
interactions were predicted using the STRING online analysis platform (Ver. 10.5)16 with minimum required interaction score adjusted to 0.15 to obtain the maximal interactions. The acquired protein interaction network was subjected to topological structural analysis using Cytoscape software (Ver. 3.5.1)17 using the default set-tings. Genes with a high degree of connection were sub-jected to transcription factor enrichment prediction using FunRich software (Ver. 3)18 using the default settings.
This research was approved by the ethical board of Henan Tumor Hospital (No. 2018132). Twenty-two gastric cancer patients were enrolled. Informed con-sent was obtained from each patient. Their clinical-pathological records are summarized in Table 1. Gastric cancer biopsies and non-cancerous adjacent biopsies were obtained from these patients. The tissue samples were analyzed by Western blotting to detect cyclin E and nuclear transcription factor Y subunit
Clinical and pathological information of 22 patients with gastric cancer.
Clinical-pathological characteristic n (%)
Highly differentiated 5 (22.7) Moderately differentiated 10 (45.5) Poorly differentiated 7 (31.8) TNM stage
TNM: Tumor Node Metastasis.
alpha (NF-YA) protein expression using beta-actin protein as the loading control. Primary antibodies against cyclin E (ab71535), NF-YA (ab23471), beta-actin (ab16039), and correlating secondary antibody (ab205718) were purchased from Abcam Trading Company Ltd. (Cambridge, United Kingdom). Gastric cancer patients were grouped into cyclin E high/low and NF-YA high/low groups based on the gray scale analysis of Western blotting results. Cyclin E or NF-YA expression higher or lower than the average was considered high or low expression, respectively. The NF-YA and cyclin E expression levels in gastric cancer specimens from patients with different TNM stages are presented in Table 2.