Esophageal Cancer-related Gene 4 Expression Research Articles

A potential link between aberrant expression of ECRG4 and atrial fibrillation.

Esophageal cancer-related gene-4 (ECRG4), a 148-amino acid propertied and new tumor suppressor, is initially cloned from the normal esophageal epithelium. ECRG4 was found to be expressed not only in esophageal tissues but also in cardiomyocytes. Previous studies demonstrated that ECRG4 is constitutively expressed in esophageal epithelial cells, and its degree of downregulation is directly proportional to prognosis in patients with esophageal cancer. In the heart, ECRG4 shows greater expression in the atria than in the ventricles, which accounts for its heterogeneity. Downregulation of ECRG4 expression level correlates with esophageal cancer, as well as myocardial injuries and arrhythmias. As a result, this review summarizes the possible susceptibility gene, ECRG4 and its associated molecular mechanisms in cancer patients with atrial fibrillation and myocardial injury. The review begins by describing ECRG4's biological background, discusses its expression in the cardiovascular system, lists the clinical and animal research related to the downregulation of ECRG4 in atrial fibrillation, and focuses on its potential role in atrial fibrillation. Downregulation of ECRG4 may increase the risk of atrial fibrillation by affecting ion channels, MMPs expression and inflammatory response. We will then discuss how ECRG4 can be used in the treatment of tumors and arrhythmias, and provide a novel possible strategy to reduce the occurrence of perioperative cardiovascular adverse events in patients with tumors such as esophageal cancer and gastric cancer.

Esophageal cancer-related gene 4 inhibits gastric cancer growth and metastasis by upregulating Krüppel-like factor 2 expression.

There are a large number of people suffering from gastric cancer (GC) worldwide, so the study of biomarkers for GC is urgently needed. This study aimed to investigate the role of esophageal cancer-related gene 4 (ECRG4) in the growth, metastasis, and prognosis of GC and the possible underlying mechanism. The expression of ECRG4 was detected in GC tissues by quantitative polymerase chain reaction (PCR), Western blot, and immunohistochemistry. The relationships between ECRG4 expression and clinicopathological parameters of patients with GC were statistically analyzed, and Kaplan-Meier prognosis and survival curves of the patients were plotted. ECRG4 was overexpressed in the human gastric adenocarcinoma cell line (AGS) and human GC cell line 27 (HGC27), and the in vivo effects of ECRG4 overexpression on the growth, invasion, and metastasis of GC were analyzed and verified in nude mice. To identify the downstream transcription factors potentially regulated by ECRG4, ribonucleic acid (RNA) sequencing and differential gene expression analysis were performed on ECRG4-overexpressing cells. Quantitative PCR, Western blot, and immunohistochemistry were used to detect the expression of the downstream transcription factors targeted by ECRG4 in GC. The ECRG4 mRNA and protein expression levels were low in GC tissues and were associated with a poor prognosis. Least absolute shrinkage and selection operator (LASSO) Cox regression and Kaplan-Meier survival analyses showed that patients with low ECRG4 expression had worse prognosis and survival. Overexpression of ECRG4 inhibited the proliferation, metastasis, and invasion of GC cells. RNA sequencing analysis showed that overexpression of ECRG4 induced the upregulation of Krüppel-like factor 2. Our findings show that ECRG4 promotes GC progression via Krüppel-like factor 2 signaling and highlight ECRG4 as a potential GC biomarker and therapeutic target.

Brain endothelial cells-derived extracellular vesicles overexpressing ECRG4 inhibit glioma proliferation through suppressing inflammation and angiogenesis.

Esophageal cancer related gene-4 (ECRG4) has been shown to be a candidate tumor suppressor in many tumors, but its role in glioma remains poorly understood. This study aimed to explore whether extracellular vesicles (EVs) derived from brain endothelial cells which overexpressed ECRG4 have anti-tumor effect on gliomas in vivo and in vitro, as well as the possible mechanism. A constructed lentivirus expressing the ECRG4 gene was transfected into the hCMEC/D3 cell line. The EVs were isolated from the cells and characterized by Western blot with exosome markers of CD9, CD63, CD81, Alix. RT-PCR and Western blot were performed to verify ECRG4 expression. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and clone formation assays were applied to detect the proliferation of glioma cells incubated with EVs expressing the ECRG4 (ECRG4-exo). The level of inflammatory cytokines and angiogenesis related factors, including nuclear factor kappa-B (NF-κB), interleukin (IL)-1β, IL-6, IL-8, monocyte chemoattractant protein-1 (MCP-1), hypoxia-inducible factor 1-alpha (HIF-1α), vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor 2 (VEGFR2) levels were detected by ELISA. The T98G cell xenograft mouse model was established and treated with ECRG4-EV. The tumor volume and weight were recorded. p38-MAPK, p-p38-MAPK proteins were determined by Western blot in tumor tissues. As a result, EVs can be internalized into U87MG and T98G cells. ECRG4-EV inhibited U87MG and T98G cell proliferation. ECRG4-EV also inhibited the expression of factors involved in inflammation and angiogenesis. In addition, ECRG4-EVs suppressed tumor growth and decreased the production of inflammatory cytokines through inactivation of p38-MAPK signal pathway. In conclusion, ECRG4-EVsuppresses glioma proliferation through modulating the inflammation and angiogenesis.

MicroRNA-196b promotes gastric cancer progression by targeting ECRG4.

Gastric cancer is one of the most common malignant tumors. MicroRNA-196b (miR-196b) has been demonstrated to play important roles in human cancers. However, its functions in gastric cancer progression were still largely unknown. In this study, the expression of miR-196b was determined by quantitative real-time PCR. Esophageal cancer-related gene 4 (ECRG4) level was examined by western blot assay and immunohistochemistry staining assay. Cell proliferation was evaluated by Cell Counting Kit-8 (CCK-8) assay and colony formation assay. Cell migration and invasion were analyzed by transwell assay. The association between miR-196b and ECRG4 was analyzed by dual-luciferase reporter assay. The functional role of miR-196b in vivo was analyzed by murine xenograft assay. As a result, we found the expression of miR-196b was elevated and the protein expression of ECRG4 was reduced in gastric cancer tissues and cells. MiR-196b inhibition suppressed gastric cancer cell proliferation, migration and invasion. ECRG4 was a target of miR-196b and its protein expression was negatively regulated by miR-196b. Moreover, ECRG4 overexpression showed similar effects with miR-196b inhibition on the malignant behaviors of GC cells and ECRG4 knockdown reversed the effects of miR-196b inhibition on gastric cancer cell proliferation, migration and invasion. In addition, miR-196b inhibition suppressed tumor volume and weight in vivo. In conclusion, downregulation of miR-196b inhibited gastric cancer progression by modulating ECRG4 expression, indicating that miR-196b might be a potential therapeutic target for gastric cancer.

ECRG4 acts as a tumor suppressor gene frequently hypermethylated in human breast cancer.

Human breast cancer is a malignant form of tumor with a relatively high mortality rate. Although esophageal cancer-related gene 4 (ECRG4) is thought to be a possible potent tumor suppressor gene that acts to suppress breast cancer, its precise role in this disease is not understood. Herein, we assess the correlation between ECRG4 expression and DNA methylation, probing the potential epigenetic regulation of ECRG4 in breast cancer. We analyzed ECRG4 promoter methylation via methylation-specific PCR (MSPCR), bisulfite sequencing, and a promoter reporter assay in human breast cancer cell lines and samples. Gene expression was assessed by quantitative real-time PCR (qPCR), while protein levels were assessed by Western blotting. CCK8 assays were used to quantify cell growth; Esophageal cancer-related gene 4 wound healing assays were used to assess cellular migration, while flow cytometry was used to assess apoptosis and cell cycle progression. Apoptosome formation was validated via CO-IP and Western blotting. We found that human breast cancer samples exhibited increased methylation of the ECRG4 promoter and decreased ECRG4 expression. Remarkably, the down-regulation of ECRG4 was highly associated with promoter methylation, and its expression could be re-activated via 5-aza-2′-deoxycytidine treatment to induce demethylation. ECRG4 overexpression impaired breast cancer cell proliferation and migration, and led to G0/G1 cell cycle phase arrest. Moreover, ECRG4 induced the formation of the Cytc/Apaf-1/caspase-9 apoptosome and promoted breast cancer cell apoptosis. ECRG4 is silenced in human breast cancer cells and cell lines, likely owing to promoter hypermethylation. ECRG4 may act as a tumor suppressor, inhibiting proliferation and migration, inducing G0/G1 phase arrest and apoptosis via the mitochondrial apoptotic pathway.

Downregulation and DNA methylation of ECRG4 in gastric cancer.

BackgroundEsophageal cancer-related gene 4 (ECRG4) is a novel candidate tumor suppressor gene. Our study investigated the expression and function of ECRG4 in gastric cancer and highlighted the role of DNA hypermethylation at the promoter in silencing the ECRG4 expression.MethodsThe GSE63089 data set was obtained from the Gene Expression Omnibus and analyzed for differentially expressed genes. Carcinoma and para-carcinoma tissues of 102 patients with gastric cancer were collected from January 2010 to July 2011. Immunohistochemistry, real-time polymerase chain reaction (PCR), and western blot analyses were performed to evaluate the expression of ECRG4. After measuring the change in the level of ECRG4 expression, CCK-8, Transwell, and flow cytometric cell cycle assays were performed. In addition, methylation-specific PCR was performed to detect the methylation state of ECRG4, and 5-aza-2′-deoxycytidine was used for demethylation of ECRG4. All statistical analyses were performed using the SPSS 17.0 software.ResultsWe found that ECRG4 expression was downregulated in gastric cancer, and this was closely related to lymph node metastasis. After ECRG4 was silenced using a specific small interfering RNA, the BGC-823 cell line became highly aggressive and proliferative. In addition, we verified whether downregulation of ECRG4 was highly correlated with DNA methylation of the ECRG4 promoter and found that the demethylating agent 5-aza-2′-deoxycytidine could effectively enhance ECRG4 expression.ConclusionThe aberrant expression of ECRG4 is associated with hypermethylation in the promoter region and plays an important role in the malignancy of gastric cancer. Therefore, ECRG4 may be a potential biomarker for molecular diagnosis of gastric cancer, and the use of 5-Aza-dC to reverse the hypermethylation of ECRG4 may be a new approach to the treatment of gastric cancer.

Downregulation of esophageal cancer-related gene 4 promotes proliferation and migration of hepatocellular carcinoma.

Esophageal cancer-related gene 4 (ECRG4) is a candidate tumor suppressor gene, which is involved in cell apoptosis, migration, infection and inflammation responsiveness; however, its expression level and clinical significance in hepatocellular carcinoma (HCC) remains unclear. In the present study, the authors aim to evaluate the clinical significance and potential role of ECRG4 in HCC. Level of ECRG4 protein expression in HCC and peripheral tissues was investigated in tissue specimens obtained from 56 consecutive HCC patients by immunohistochemistry. Cell proliferation, cell migration and invasion regulations were examined by MTT curves, flow cytometry, Transwell assays and western blotting. ECRG4 expression was weak positive in normal liver cells but was downregulated in HCC cells in vivo or in vitro. A decreased expression of ECRG4 was associated with the age of the patients, metastasis and Ki-67 proliferation index. However, decreased ECRG4 expression was not associated with differentiation, tumor size, the presence of portal vein tumor thrombosis, satellite lesions, tumor relapse or mortality. Further investigations revealed that ectopic expression of ECRG4 inhibited cell proliferation, migration and invasion and promoted cell apoptosis in SMMC-7721 cells, which was mediated by the regulation of BAX and B cell lymphoma-2, in addition to the upregulation of epithelial-mesenchymal transition markers. In conclusion, the results of the present study indicated that ECRG4 was downregulated in HCC and served important roles in promoting cell proliferation and migration.

Downregulated ECRG4 is correlated with lymph node metastasis and predicts poor outcome for nasopharyngeal carcinoma patients.

Esophageal cancer-related gene 4 (ECRG4) is a new candidate tumor suppressor gene. In this retrospective study, we evaluated ECRG4 protein expression in patients with nasopharyngeal carcinoma (NPC) under curative treatment and examined its association with pathological features and clinical outcomes as a possible biomarker for diagnosis and prognosis of NPC. We enrolled 122 patients with a first diagnosis between January 2001 and December 2003. Tumor tissue and control tissue from biopsies underwent immunohistochemical staining for ECRG4. ECRG4 expression was analyzed by clinicopathological variables. After Kaplan-Meier survival analysis, we used Cox proportional hazards regression to estimate the predictive effect of ECRG4 expression on overall survival. ECRG4 protein level was lower in NPC than control tissue (P<0.01). It was inversely related to node status (P<0.001) and clinical stage (P=0.027). ECRG4 expression was associated with overall survival, and downregulated ECRG4 expression was an independent prognostic factor of poor survival (hazard ratio = 0.677, 95% confidence interval 0.463-0.989, P=0.044). A significant NPC patients showed downregulated ECRG4 expression, which is correlated with lymph node metastasis. The marker could be an independent prognostic factor for NPC patients. The precise function of ECRG4 in the progression of NPC, especially for lymphatic metastasis, deserves further investigation, which would bring a new target for personalized therapy.

Monitoring Neutrophil-Expressed Cell Surface Esophageal Cancer Related Gene-4 after Severe Burn Injury.

We identified recently esophageal cancer related gene-4 (ECRG4) as a candidate cytokine that is expressed on the surface of quiescent polymorphonuclear leukocytes (PMNs) and shed in response to ex vivo treatment with lipopolysaccharide. To investigate the potential biologic relevance of changes in cell surface ECRG4 in human samples, we performed a pilot study to examine a population of burn patients in whom blood could be analyzed prospectively. We hypothesized that cutaneous burn injury would alter cell surface expression of ECRG4 on PMNs. Patients admitted with more than 20% total burn surface area (TBSA) (n = 10) had blood collected at the time of admission and weekly thereafter. For comparison, blood was obtained from a control group of healthy human volunteers (n = 4). We used flow cytometry to measure changes in ECRG4(+) PMNs from patients during recovery from injury. Esophageal cancer related gene-4 expression at each time point was compared with the patient's clinical status based on a Multiple Organ Dysfunction (MOD) score. Esophageal cancer related gene-4 was detected on the PMN surface of cells collected from healthy volunteers, however, within 48 h of admission after burn injury (n = 10 patients), the number of PMNs with cell surface ECRG4 was decreased. Esophageal cancer related gene-4 expression in PMNs was re-established over the course of patient recovery, unless complications occurred. In this case, the decrease in cell surface ECRG4(+) PMNs preceded the clinical diagnosis of infectious complications and was reflected by increased organ injury scores. From a small sample set, we were able to determine that PMN cell surface ECRG4 expression was decreased after burn injury and returned to baseline during recovery from injury. Although larger studies are needed to define the role of ECRG4 in human PMNs further, this report is the first assessment of cell surface ECRG4 protein in a patient population to support analogous findings in animal studies.

Downregulated ECRG4 is associated with poor prognosis in renal cell cancer and is regulated by promoter DNA methylation.

Esophageal cancer-related gene 4 (ECRG4) has been proposed as a putative tumor suppressor gene in several tumors. However, the role and regulation of ECRG4 in the pathogenesis of human renal cancer remain largely unknown. Our current study revealed that expression of ECRG4 is downregulated in renal cell lines and renal cancer tissues. ECRG4 expression was significantly associated with histological grade of tumors (p < 0.001), primary tumor stage (p = 0.017), and distant metastasis (p = 0.017). Low expression of ECRG4 was an independent prognostic indicator for survival of renal cancer patients. Silencing of ECRG4 expression in renal cell lines was associated with its promoter methylation. Moreover, ectopic expression of ECRG4 markedly inhibited cell proliferation and invasion in renal cancer cell lines. These results indicated that ECRG4 is frequently silenced by the methylation of promoter in renal cell cancers. ECRG4 may be a tumor suppressor in renal cancer and serve as a prognostic marker.

A preliminary study of the effect of ECRG4 overexpression on the proliferation and apoptosis of human laryngeal cancer cells and the underlying mechanisms.

Human esophageal cancer-related gene 4 (ECRG4) is a potential tumor suppressor gene isolated from human esophageal epithelial cells. Studies have shown that ECRG4 effectively inhibits the proliferation of tumor cells and induces apoptosis. However, the role of ECRG4 in laryngeal cancer has not yet been clearly defined. In this study, a human laryngeal cancer cell line stably overexpressing ECRG4 was established. The effect of ECRG4 on the proliferation and apoptosis of laryngeal cancer cells and the associated mechanisms were investigated. The Hep-2 human laryngeal carcinoma cell line exhibited a low basal level of ECRG4 expression and was selected for the present study. The eukaryotic expression plasmid pcDNA3.1-ECRG4 was constructed and introduced into Hep-2 cells by transfection reagents. Western blot analysis, reverse transcription-quantitative polymerase chain reaction and immunofluorescence staining confirmed high-level expression of ECRG4. The 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and colony formation assay showed that ECRG4 over-expression suppressed the proliferative capacity of laryngeal cancer cells in vitro. Cell cycle analysis showed that ECRG4 induced cell cycle arrest at the G0/G1 phase. Flow cytometric analysis and Hoechst staining demonstrated that overexpres-sion of ECRG4 significantly induced apoptosis. Western blot analysis confirmed that Bcl-2-associated X protein, cleaved-caspase-3 and cleaved-poly (ADP-ribose) polymerase were upregulated in the apoptotic process, whereas B-cell lymphoma 2 was downregulated. In conclusion, overexpression of ECRG4 inhibited laryngeal cancer cell proliferation and induced cancer cell apoptosis. Therefore, ECRG4 exhibits potential as an effective target in gene therapy for laryngeal cancer.

ECRG4 expression in normal rat tissues: expression study and literature review.

The Esophageal Cancer Related Gene 4 (ECRG4) is a highly conserved tumour suppressor gene encoding various peptides (augurin, CΔ16 augurin, ecilin, argilin, CΔ16 argilin) which can be processed and secreted. In the present work, we examined ECRG4 expression and location in a wide range of rat organs and reviewed the available literature. ECRG4 mRNA was identified in all examined tissues by quantitative PCR (qPCR). ECRG4 immunoreaction was mainly cytoplasmic, and was detected in heart and skeletal muscles, smooth muscle cells showing only weak reactions. In the digestive system, ECRG4 immunostaining was stronger in the esophageal epithelium, bases of gastric glands, hepatocytes and pancreatic acinar epithelium. In the lymphatic system, immunoreactive cells were detectable in the thymus cortex, lymph node medulla and splenic red pulp. In the central and peripheral nervous systems, different neuronal groups showed different reaction intensities. In the endocrine system, ECRG4 immunoreaction was detected in the hypothalamic paraventricular and supraoptic nuclei, hypophysis, thyroid and parathyroid glands, adrenal zona glomerularis and medulla and Leydig cells, as well as in follicular and luteal cells of the ovary. In the literature, ECRG4 has been reported to inhibit cell proliferation and increase apoptosis in various cell types. It is down-regulated, frequently due to hypermethylation, in esophageal, prostate, breast and colon cancers, together with glioma (oncosuppressor function), although it is up-regulated in papillary thyroid cancer (oncogenic role). ECRG4 expression is also higher in non-proliferating cells of the lymphatic system. In conclusion, our identification of ECRG4 in many structures suggests the involvement of ECRG4 in the tumorigenesis of other organs and also the need for further research. In addition, on the basis of the location of ECRG4 in neurons and endocrine cells and the fact that it can be secreted, its role as a neurotransmitter/neuromodulator and endocrine factor must be examined in depth in the future.

The tumor-promoting function of ECRG4 in papillary thyroid carcinoma and its related mechanism.

This study aimed to explore the tumor-promoting function of esophageal cancer-related gene 4 (ECRG4) in the papillary thyroid cancer and its related mechanism. ECRG4 Messenger RNA (mRNA) and protein expression analysis in papillary thyroid cancer tissues was performed by quantitative real-time PCR (Q-RT-PCR), Western blot, and immunohistochemistry methods. Ten pairs of fresh samples from the papillary thyroid carcinoma patients were analyzed for ECRG4 promoter CpG island methylation status by bisulfite sequencing analysis. We also transfected ECRG4 into papillary thyroid cancer cell lines W3 and K1 with lentivirus and analyzed ECRG4 functions through evaluating the changes of the proliferation activity, the cell cycle, and the cell apoptosis rate of these transformed cells. We found that ECRG4 expression was upregulated in most papillary thyroid cancer samples (70.0%, 28 out of 40 papillary thyroid cancer samples) on the protein level, and the ECRG4 mRNA level was also enhanced in tumor tissues compared to their matched nontumor tissues. CpG islands around the ECRG4 promoter region were demethylated in the papillary thyroid cancer samples. At the same time, the upregulated expression of ECRG4 in papillary thyroid cancer cell lines W3 and K1 could promote both the proliferation activity and the cell cycle transition from the G1 phase into the G2 but could not affect the cell apoptosis rate. The expression of ECRG4 is frequently upregulated in a papillary thyroid carcinoma through the demethylation mechanism of CpG islands in the gene promoter region, and the ECRG4 has a tumor-promoting function through inducing the cell cycle transition from the G1 phase to the G2 in papillary thyroid carcinoma cells.

Overexpression of ECRG4 enhances chemosensitivity to 5-fluorouracil in the human gastric cancer SGC-7901 cell line

The aim of this study was to examine the effects of esophageal cancer-related gene 4 (ECRG4) expression levels on chemotherapeutic sensitivity of gastric cancer cells. A SGC-7901 cell system with tetracycline-inducible ECRG4 expression (SGC-7901/ECRG4) was successfully established. ECRG4 mRNA and protein expression levels were detected using quantitative reverse transcription polymerase chain reaction and Western blotting, respectively. Chemosensitivity to 5-fluorouracil (5-FU) was examined by cell proliferation assay and cell apoptosis assay. ECRG4 mRNA and protein expression levels were significantly upregulated in SGC-7901/ECRG4 cells induced with tetracycline. Compared with control cells, the growth inhibition rate of cells with ECRG4 overexpression was significantly increased when treated with 5-FU. Treatment with 5 μmol/l 5-FU resulted in 15.2 % apoptotic cells, whereas such treatment after overexpression of ECRG4 resulted in 44.5 % apoptotic cells. In conclusion, overexpression of ECRG4 enhanced the chemosensitivity of gastric cancer SGC-7901 cells to 5-FU through induction of apoptosis.

ECRG4 is a negative regulator of caspase-8-mediated apoptosis in human T-leukemia cells

We previously established Fas-resistant variant clones from the human T-cell leukemia lines Jurkat and SUP-T13. Comparative gene expression analysis of the Fas-resistant and Fas-sensitive clones revealed several genes that were aberrantly expressed in the Fas-resistant clones. One of the genes, esophageal cancer-related gene 4 (ECRG4), contained a VDAC2-like domain that might be associated with apoptotic signals. In the present study, we examined the subcellular localization and function of ECRG4 in Fas-mediated apoptosis. By confocal fluorescence microscopy, ECRG4-EGFP fusion protein was detected in mitochondria, endoplasmic reticulum and the Golgi apparatus in gene-transfected HeLa cells. Overexpression of ECRG4 in Fas-sensitive Jurkat cells inhibited mitochondrial membrane permeability transition, leading to resistance against Fas-induced apoptosis. Tumor necrosis factor-alpha-induced apoptosis was also suppressed in ECRG4-overexpressing Jurkat cells. Immunoprecipitation assay demonstrated that ECRG4 is associated with procaspase-8. The inhibitory mechanism included the inhibition of caspase-8 activity and Bid cleavage. Since ECRG4 expression is downregulated in activated T cells, our results suggest that ECRG4 is a novel antiapoptotic gene which is involved in the negative regulation of caspase-8-mediated apoptosis in T cells.

The candidate tumor suppressor gene ECRG4 inhibits cancer cells migration and invasion in esophageal carcinoma

BackgroundThe esophageal cancer related gene 4 (ECRG4) was initially identified and cloned in our laboratory from human normal esophageal epithelium (GenBank accession no.AF325503). ECRG4 was a new tumor suppressor gene in esophageal squamous cell carcinoma (ESCC) associated with prognosis. In this study, we investigated the novel tumor-suppressing function of ECRG4 in cancer cell migration, invasion, adhesion and cell cycle regulation in ESCC.MethodsTranswell and Boyden chamber experiments were utilized to examined the effects of ECRG4 expression on ESCC cells migration, invasion and adhesion. And flow cytometric analysis was used to observe the impact of ECRG4 expression on cell cycle regulation. Finally, the expression levels of cell cycle regulating proteins p53 and p21 in human ESCC cells transfected with ECRG4 gene were evaluated by Western blotting.ResultsThe restoration of ECRG4 expression in ESCC cells inhibited cancer cells migration and invasion (P < 0.05), which did not affect cell adhesion capacity (P > 0.05). Furthermore, ECRG4 could cause cell cycle G1 phase arrest in ESCC (P < 0.05), through inducing the increased expression of p53 and p21 proteins.ConclusionECRG4 is a candidate tumor suppressor gene which suppressed tumor cells migration and invasion without affecting cell adhesion ability in ESCC. Furthermore, ECRG4 might cause cell cycle G1 phase block possibly through inducing the increased expression of p53 and p21 proteins in ESCC.

Esophageal cancer related gene 4 (ECRG4) is a marker of articular chondrocyte differentiation and cartilage destruction

With the aim of identifying novel genes regulating cartilage development and degeneration, we screened a cartilage-specific expressed sequence tag database. Esophageal cancer related gene 4 (ECRG4) was selected, based on the criteria of ‘chondrocyte-specific’ and ‘unknown function.’ ECRG4 expression was particularly abundant in chondrocytes and cartilage, compared to various other mouse tissues. ECRG4 is a secreted protein that undergoes cleavage after secretion. The protein is specifically expressed in chondrocytes in a manner dependent on differentiation status. The expression is very low in mesenchymal cells, and dramatically increased during chondrogenic differentiation. The ECRG4 level in differentiated chondrocytes is decreased during hypertrophic maturation, both in vitro and in vivo, and additionally in dedifferentiating chondrocytes induced by interleukin-1β or serial subculture, chondrocytes of human osteoarthritic cartilage and experimental mouse osteoarthritic cartilage. However, ectopic expression or exogenous ECRG4 treatment in a primary culture cell system does not affect chondrogenesis of mesenchymal cells, hypertrophic maturation of chondrocytes or dedifferentiation of differentiated chondrocytes. Additionally, cartilage development and organization of extracellular matrix are not affected in transgenic mice overexpressing ECRG4 in cartilage tissue. However, ectopic expression of ECRG4 reduced proliferation of primary culture chondrocytes. While the underlying mechanisms of ECRG4 expression and specific roles remain to be elucidated in more detail, our results support its function as a marker of differentiated articular chondrocytes and cartilage destruction.

Expression of esophageal cancer related gene 4 (ECRG4), a novel tumor suppressor gene, in esophageal cancer and its inhibitory effect on the tumor growth in vitro and in vivo

The ECRG4 gene was initially identified and cloned in our laboratory from human normal esophageal epithelium (GenBank accession no. AF325503). We revealed the expression of ECRG4 protein was downregulated in 68.5% (89/130) ESCC samples using tissue microarray. The low ECRG4 protein expression was significantly associated with regional lymph node metastasis, primary tumor size, and tumor stage in ESCC (p < 0.05). ECRG4 mRNA expression was downregulated in ESCC due to the hypermethylation in the gene promoter. The treatment with 5-aza-2'-deoxycytidine, which is a DNA methyltransferase inhibitor restored ECRG4 mRNA expression in ESCC cells. The result indicated that promoter hypermethylation may be 1 main mechanism leading to the silencing of ECRG4. The high expression of ECRG4 in patients with ESCC was associated with longer survival compared with those with low ECRG4 expression by Kaplan-Meier survival analysis (p < 0.05). ECRG4 protein was an independent prognostic factor for ESCC by multivariable Cox proportional hazards regression analysis (p < 0.05). The restoration of ECRG4 expression in ESCC cells inhibited cell proliferation, colony formation, anchorage-independent growth, cell cycle progression and tumor growth in vivo (p < 0.05). The transfection of ECRG4 gene in ESCC cells inhibited the expression of NF-kappaB and nuclear translocation, in addition to the expression of COX-2, a NF-kappaB target gene, was attenuated. Taken together, ECRG4 is a novel candidate tumor suppressor gene in ESCC, and ECRG4 protein is a candidate prognostic marker for ESCC.