The EFNA4 gene is a potential prognostic biomarker in pancreatic cancer: a bioinformatics analysis.

Background: Pancreatic cancer is a highly aggressive malignancy with poor prognosis, and there is an urgent need to understand its molecular mechanisms for early diagnosis and treatment. Despite surgical resection being the only effective treatment, most patients are diagnosed at an advanced stage, missing the optimal window for therapy. Identifying novel biomarkers is crucial for prognostic assessment, treatment planning, and early intervention. Ephrin A4 (EFNA4), a member of the receptor tyrosine kinase family, is involved in vascular and epithelial development via regulation of cell migration and rejection. However, the role of EFNA4 in pancreatic cancer has not been reported. Therefore, our study aimed to clarify the role of EFNA4 in pancreatic cancer through bioinformatics analysis and vitro experiments. Methods: The expression of EFNA4 and its potential value as a diagnostic and prognostic biomarker in pancreatic cancer was analyzed using data from The Cancer Genome Atlas (TCGA) and the Gene Expression Profiling Interactive Analysis (GEPIA) database. According to the expression level of EFNA4, patients were divided into high expression group and low expression group, and the correlation between overall survival (OS) and disease-free survival (DFS) with different expression levels of EFNA4 and clinical parameters were analyzed. Subsequently, reverse-transcription quantitative polymerase chain reaction (RT-qPCR) was performed to detect EFNA4 expression. The proliferation, invasion, and cloning ability of the cells were detected via Cell Counting Kit 8 (CCK8), Transwell, and plate cloning assays, respectively. Results: EFNA4 is highly expressed in pancreatic cancer, and upregulation of EFNA4 is associated with poor prognosis. In this study, EFNA4 expression was correlated with T stage and TNM (tumor-node-metastasis) stage of pancreatic cancer, and the median survival time and progression-free survival (PFS) were worse in those with high EFNA4 expression (394 days) than in those with low expression (525 days) [hazard ratio (HR): 1.47, 95% confidence interval (CI): 1.00-2.16, P=0.047]. In addition, EFNA4 was also found to be involved in the regulation of signal pathways such as cell adhesion, cyclic AMP, insulin secretion, pancreatic secretion, and protein digestion and absorption. In vitro experiments demonstrated that EFNA4 knockdown significantly inhibited the proliferation, cloning ability, and invasiveness of the PANC-1 and SW1990 pancreatic cancer cell lines. Conclusions: The abnormal expression of EFNA4 in pancreatic cancer is associated with poor prognosis. Knockout of EFNA4 gene could significantly inhibit the proliferation and invasion of pancreatic cancer cells. Therefore, EFNA4 may be one of the molecular targets for poor prognosis of patients with pancreatic cancer.

Interference of EFNA4 suppresses cell proliferation, invasion and angiogenesis in hepatocellular carcinoma by downregulating PYGO2.

Hepatocellular carcinoma (HCC) is the most common type of liver cancer. Ephrin A4 (EFNA4) acts as an oncogene in multiple cancers but is little known in HCC. It is revealed that EFNA4 is highly expressed in patients with HCC and influences the proliferation of HCC cells; however, detailed regulatory mechanism of EFNA4 in HCC needs to be unveiled. Here, we discovered that EFNA4 was highly expressed in HCC cell lines. EFNA4 knockdown greatly suppressed cell proliferation, migration and invasion, as well as inhibiting angiogenesis in Huh7 cells. EFNA4 was demonstrated to interact with pygopus-2 (PYGO2) and positively regulate PYGO2 expression. Gene gain- and loss-of-function experiments revealed that the anti-tumor effect of EFNA4 knockdown was partly abolished by PYGO2 overexpression. Furthermore, EFNA4 knockdown blocked wnt/ß-catenin signaling in Huh7 cells, which was then abolished by PYGO2. In conclusion, this study further ensured the oncogenic role of EFNA4 in HCC, and disclosed that EFNA4 knockdown suppressed cell proliferation, invasion, angiogenesis, and wnt/ß-catenin signaling in HCC by downregulating PYGO2.

Neurotrophic factors in the posterodorsal medial amygdala of male and cycling female rats.

The posterodorsal medial amygdala (MePD) has a high concentration of receptors for gonadal hormones, is a sexually dimorphic region and dynamically controls the reproductive behavior of both males and females. Neurotrophic factors can promote dendritic spine remodeling and change synaptic input strength in a region-specific manner. Here, we analyzed the gene and protein expression of brain-derived neurotrophic factor (BDNF), insulin-like growth factor-I (IGF-1), polysialylated neural cell adhesion molecule (PSA-NCAM) and Ephrin-A4 in the MePD of adult males and females in diestrus, proestrus and estrus using real-time qPCR and fluorescent immunohistochemistry. The first approach showed their amplification except for Igf1 and the latter revealed that BDNF, IGF-1, PSA-NCAM and Ephrin-A4 are expressed in the MePD of the adult rats. Protein expression of these neurotrophic factors showed no differences between groups. However, proestrus females displayed a higher number of labelled puncta than males for BDNF expression and diestrus females for IGF-1 expression. In conjunction, results indicate that IGF-1 might be released rather than synthetized in the MePD, and the expression of specific neurotrophic factors varies specifically during proestrus. The dynamic modulation of BDNF and IGF-1 during this cyclic phase is coincident with synaptic changes and spine density remodeling in the MePD, the disinhibition of gonadotrophin secretion for ovulation and the display of sexual behavior.

Ephrin-A3 and ephrin-A4 contribute to microglia-induced angiogenesis in brain endothelial cells.

The association of microglia with brain vasculature during development and the reduced brain vascular complexity in microglia-deficient mice suggest the role of microglia in cerebrovascular angiogenesis. However, the underlying molecular mechanism remains unclear. Here, using an in vitro angiogenesis model, we found the culture supernatant of BV2 microglial cells significantly enhanced capillary-like tube formation and migration of brain microvascular endothelial cells (BMECs). The expression of angiogenic factors, ephrin-A3 and ephrin-A4, were specifically upregulated in BMECs exposed to BV2-derived culture supernatant. Knockdown of ephrin-A3 and ephrin-A4 in BMECs by siRNA significantly attenuated the enhanced angiogenesis and migration of BMECs induced by BV2 supernatant. Our further results indicated that the ability of BV2 supernatant to promote endothelial angiogenesis was caused by the soluble tumor necrosis factor α (TNF-α) released from BV2 microglial cells. Moreover, the upregulations of ephrin-A3 and ephrin-A4 in BMECs in response to BV2 supernatant were effectively abolished by neutralization antibody against TNF-α and TNF receptor 1, respectively. The present study provides evidence that microglia upregulates endothelial ephrin-A3 and ephrin-A4 to facilitate in vitro angiogenesis of brain endothelial cells, which is mediated by microglia-released TNF-α.