Circ DENND4C inhibits pyroptosis and alleviates ischemia-reperfusion acute kidney injury by exosomes secreted from human urine-derived stem cells.

Acute kidney injury (AKI) is a disease characterised by acute onset, high mortality, and poor prognosis, and is mainly caused by ischemia-reperfusion (I/R). Human urine-derived stem cells (USCs) exhibit antioxidant, anti-inflammatory, and anti-apoptotic cytoprotective effects. Previously, we found that exosomes from USCs had the ability to inhibit apoptosis and protect kidneys from I/R injury. This study aimed to investigate the role of USC-derived exosomes (USC-Exos) in reducing pyroptosis and alleviating I/R-AKI. Models of HK-2 cells hypoxia-reoxygenation (H/R) and I/R kidney injury was established in Sprague Dawley rats to simulate AKI in vitro and in vivo. USC-Exos were isolated using ultracentrifugation and identified via electron microscopy and western blotting. USC-Exos were co-cultured with HK-2 cells and injected into rats via the tail vein. The expression of pyroptosis-related molecules (GSDMD, caspase-1, and NLRP-3) was verified using PCR and western blotting. Changes in renal function were reflected in the serum creatinine, urea, and cystatin C levels. The degree of renal injury was determined using haematoxylin and eosin and immunohistochemical staining. The levels of IL-1ß and IL-18 were detected using enzyme-linked immunosorbent assay (ELISA) to verify the role of USC-Exos in pyroptosis. Differentially expressed circRNAs in I/R rat kidneys were screened by transcriptome sequencing, and a dual-luciferase experiment was used to verify the interaction between upstream and downstream molecules. Ischemia-reperfusion resulted in significantly impaired renal function and expression of pyroptosis molecules, and significantly increased concentrations of inflammatory factors. These effects were reversed by injecting USC-Exos. Circ DENND4C was the most significantly decreased circRNA in I/R rat renal tissue, and knock-down of circ DENND4C can aggravate AKI in vivo and in vitro. DAVID(http://david.abcc.ncifcrf.gov) website showed that miR 138-5p/FOXO3a is a potential downstream target of circ DENND4C. Knock-down of circ DENND4C in HK-2 cells resulted in increased expression of miR 138-5p and increased miR 138-5p can reverse the regulation of FOXO3a. Dual-luciferase assay verified the reverse interaction between circ DENND4C, miR 138-5p, and FOXO3a. Exosomes promote cell proliferation and inhibit the activation of NLR family pyrin domain containing 3 through the circ DENND4C/miR 138-5p/FOXO3a pathway, thereby reducing pyroptosis and AKI. Circ DENND4C may be a potential therapeutic target for AKI.

Prediction of lymph node metastasis in early esophageal cancer.

BACKGROUND: Given the poor prognosis of patients with lymph node metastasis, estimating the lymph node status in patients with early esophageal cancer is crucial. Indicators that could be used to predict lymph node metastasis in early esophageal cancer have been reported in many recent studies, but no recent studies have included a review of this subject. AIM: To review indicators predicting lymph node metastasis in early esophageal squamous cell carcinoma (ESCC) and early esophageal adenocarcinoma (EAC). METHODS: We searched PubMed with "[early esophageal cancer (Title/Abstract)] and [lymph node (Title/Abstract)]" or "[early esophageal carcinoma (Title/Abstract)] and [lymph node (Title/Abstract)]" or "[superficial esophageal cancer (Title/Abstract)] and [lymph node (Title/Abstract)]." A total of 29 studies were eligible for analysis. RESULTS: Preoperative imaging (size), serum markers (microRNA-218), postoperative pathology and immunohistochemical analysis (depth of invasion, tumor size, differentiation grade, lymphovascular invasion, neural invasion, expression of PIM-1 < 30%) were predictive factors for lymph node metastasis in both early ESCC and EAC. Serum markers (thymidine kinase 1 ≥ 3.38 pmol/L; cytokeratin 19 fragment antigen 21-1 > 3.30 ng/mL; stathmin-1) and postoperative pathology and immunohistochemical analysis (overexpression of cortactin, mixed-lineage leukaemia 2, and stanniocalcin-1) were predictive for lymph node metastasis in early ESCC. Transcription of CD69, myeloid differentiation protein 88 and toll-like receptor 4 and low expression of olfactomedin 4 were predictive of lymph node metastasis in early EAC. A total of 6 comprehensive models for early ESCC, including logistic regression model, nomogram, and artificial neural network (ANN), were reviewed. The areas under the receiver operating characteristic curve of these models reached 0.789-0.938, and the ANN performed best. As all these models relied on postoperative pathology, further models focusing on serum markers, imaging and immunohistochemical indicators are still needed. CONCLUSION: Various factors were predictive of lymph node metastasis in early esophageal cancer, and present comprehensive models predicting lymph node metastasis in early ESCC mainly relied on postoperative pathology. Further studies focusing on serum markers, imaging and immunohistochemical indicators are still in need.

Progress, Key Issues, and Future Prospects for Li-Ion Battery Recycling.

The overuse and exploitation of fossil fuels has triggered the energy crisis and caused tremendous issues for the society. Lithium-ion batteries (LIBs), as one of the most important renewable energy storage technologies, have experienced booming progress, especially with the drastic growth of electric vehicles. To avoid massive mineral mining and the opening of new mines, battery recycling to extract valuable species from spent LIBs is essential for the development of renewable energy. Therefore, LIBs recycling needs to be widely promoted/applied and the advanced recycling technology with low energy consumption, low emission, and green reagents needs to be highlighted. In this review, the necessity for battery recycling is first discussed from several different aspects. Second, the various LIBs recycling technologies that are currently used, such as pyrometallurgical and hydrometallurgical methods, are summarized and evaluated. Then, based on the challenges of the above recycling methods, the authors look further forward to some of the cutting-edge recycling technologies, such as direct repair and regeneration. In addition, the authors also discuss the prospects of selected recycling strategies for next-generation LIBs such as solid-state Li-metal batteries. Finally, overall conclusions and future perspectives for the sustainability of energy storage devices are presented in the last chapter.

Multiple rectal neuroendocrine tumors: An analysis of 15 cases and literature review.

Multiple neuroendocrine tumors (M-NETs) are rare in the rectum and there is no consensus on their characteristics and treatments. Here, we report 15 cases of rectal M-NETs and review the previous literature. We discuss the clinical characteristics, endoscopic features and pathological features of rectal M-NETs, aiming to analyze the treatments and follow-up strategies in combination with these characteristics. We retrospectively reviewed and analyzed the data of 15 patients with rectal M-NETs who were diagnosed and treated at Beijing Friendship Hospital, Capital Medical University. Their clinical data, endoscopic findings, pathological features and treatments were analyzed. Follow-up evaluations and literature review were performed. In all, 14 male (93.3%) and 1 female (6.7%) were recruited. The average age at diagnosis was 55.7 years. The clinical manifestations include asymptomatic in 9 patients (60.0%), defecation habits changes in 2 patients (13.3%), anal distension in 2 patients (13.3%), and abdominal distension in 2 patient (13.3%). The largest tumor diameter ≤10mm was found in 13 patients (86.7%) and >10mm in 2 patients (13.3%). All of the lesions originated from the mucous or submucosa layer. WHO grades were all NET G1. The number of tumors diagnosed by pathology in 13 patients was consistent with that observed by endoscopy, while more lesions were observed by pathology than endoscopy in two patients. Lymph node metastasis occurred in 1 patient (6.7%), and vascular or lymphatic invasion occurred in 9 patients (60.0%). Among the 13 patients with the largest tumor diameter being ≤10mm, lymphovascular invasion occurred in 8 patients (61.5%). And among the 2 patients with the largest tumor diameter of >10mm, lymphovascular invasion occurred in 1 patient (50.0%). 14 patients underwent endoscopic resection and 1 underwent surgical excision. Postoperative follow-up was achieved in 13 patients and no recurrence or metastasis was found. The true number of rectal M-NETs may be more than seen under endoscopy. Rectal M-NETs is associated with a high risk of metastasis; therefore, treatment and surveillance strategies should be more radical than single lesion.

Disposable versus reusable gastroscopes: a prospective randomized noninferiority trial.

BACKGROUND AND AIMS: Disposable gastroscopes have recently been developed to eliminate the risk of infection transmission from contaminated reusable gastroscopes. We compared the performance of disposable and reusable gastroscopes in patients undergoing gastroscopy. METHODS: Patients requiring gastroscopy were randomized to either the disposable or reusable digital gastroscope group. The primary endpoint was the success rate of photographing customary anatomic sites, with a noninferiority margin of -8%. Secondary endpoints were technical performance factors such as gastroscope imaging quality, maneuverability, gastroscopy completion rate, device failure/defect rate, operating time, and safety. Data were analyzed using the Newcombe-Wilson score method and Fisher exact 2-tailed t test. RESULTS: Of 110 patients, 55 were treated using disposable gastroscopes and 55 using reusable gastroscopes. The success rate for capturing images of customary anatomic sites was 100% in both groups. The average imaging quality score was significantly lower (37.02 ± 3.09 vs 39.47 ± 1.92, P < .001) and the operating time significantly longer (P < .001) in the disposable gastroscope group. No significant differences in maneuverability, gastroscopy completion rate, device failure/defect rate, operating time, or safety were found between the 2 groups. CONCLUSIONS: Given the overall safety profile and similar technical performance, disposable gastroscopes represent an alternative to reusable gastroscopes for routine examination, bedside first aid, and some certain circumstances.

Protective Effects of Taraxasterol against Deoxynivalenol-Induced Damage to Bovine Mammary Epithelial Cells.

Deoxynivalenol (DON), a mycotoxin produced by Fusarium graminearum, is one of the most prevalent contaminants in livestock feed and causes very large losses to animal husbandry every year. Taraxasterol, isolated from Taraxacum officinale, has anti-inflammatory, antioxidative stress, and antitumor effects. In the present study, bovine mammary epithelial cells (MAC-T) were used as a model, and different concentrations of taraxasterol (0, 1, 5, 10, and 20 µg/mL) were used to protect against DON-induced cell damage. The results showed that taraxasterol at a concentration of 10 µg/mL significantly increased cell viability. Analysis of lactate dehydrogenase (LDH) levels indicated that taraxasterol substantially decreased LDH release caused by DON. Taraxasterol effectively alleviated the depletion of glutathione (GSH), the increase in the lipid peroxidation of malondialdehyde (MDA), the reduction in total superoxide dismutase (T-SOD) activity, and the decrease in total antioxidant capacity (T-AOC) induced by DON. The results further showed that taraxasterol reduced the accumulation of reactive oxygen species (ROS). Taraxasterol was found to relieve endoplasmic reticulum (ER) stress by suppressing the expression of glucose-regulated protein 78 kDa (GRP78), activating transcription factor 6 (ATF6), activating transcription factor 4 (ATF4) and the transcription factor C/EBP homologous protein (CHOP), and reducing cell apoptosis by suppressing the expression of caspase-3 and Bcl2-associated X (BAX) and upregulating the expression of the antiapoptotic protein B-cell lymphoma-2 (Bcl-2). Our research results indicate that taraxasterol could alleviate DON-induced damage to MAC-T cells.

Polydatin Protects Bovine Mammary Epithelial Cells Against Zearalenone-Induced Apoptosis By Inhibiting Oxidative Responses and Endoplasmic Reticulum Stress.

Zearalenone (ZEA) is a mycotoxin of the Fusarium genus that can cause endoplasmic reticulum (ER) stress and Apoptosis in bovine mammary epithelial cells (MAC-T). Polydatin (PD), a glycoside purified from Polygonum cuspidatum, has antioxidant properties. This study aimed to explore whether PD can alleviate ZEA-induced damage on bovine mammary epithelial cells (MAC-T). We found that incasing the concentration of ZEA (0, 7.5, 15, 30, 60, 90, 120, and 240 µM) gradually decreased the cell viability. PD treatment alone at 5, 10, and 20 µM did not affect cell viability. Follow-up studies then applied 30 µM of ZEA and 5 µM of PD to treat cells; the results showed that the ZEA + PD treatment group effectively reduced cell oxidative damage compared with the ZEA treatment group. The qPCR analysis showed that ZEA treatment significantly up-regulated the expression of ER stress-related genes, relative to the control. However, adding PD significantly down-regulated the expression of ER stress-related genes. The cell apoptosis detection results showed that, compared with the ZEA treatment group, the ZEA + PD treatment group down-regulated the Bax gene and up-regulated the Bcl-2 gene expressions, which reduced the cell apoptosis rate and Caspase-3 activity. Taken together, these results indicate that PD reduces ZEA-induced apoptosis by inhibiting oxidative damage and ER stress.

ECT2 Increases the stability of EGFR and Tumorigenicity by Inhibiting Grb2 Ubiquitination in Pancreatic Cancer.

The poor prognosis of patients with pancreatic ductal adenocarcinoma (PDAC) is associated with the invasion and metastasis of tumor cells. Epithelial cell transforming 2 (ECT2) is a guanine nucleotide exchange factor (GEF) of the Rho family of GTPases. It has also been reported that upregulation of ECT2 in pancreatic cancer, but the role and mechanism of ECT2 have not been previously determined. We found that ECT2 was significantly elevated in PDAC tissues and cells, correlated with more advanced AJCC stage, distant metastases, and overall survival of patients with PDAC. Inhibition and overexpression tests showed that ECT2 promoted proliferation, migration and invasion in vitro, and promoted tumor growth and metastasis in vivo. We determined that ECT2 was involved in the post-translational regulation of Grb2. ECT2 inhibited the degradation of Grb2 through deubiquitination. Furthermore, knockdown of ECT2 downregulated EGFR levels by accelerating EGFR degradation. EGF stimulation facilitated the formation of ECT2-Grb2 complex. Overall, our findings indicated that ECT2 could be used as a promising new therapeutic candidate for PDAC.

Deoxynivalenol induces oxidative stress, inflammatory response and apoptosis in bovine mammary epithelial cells.

Deoxynivalenol (DON) is a toxic secondary metabolite produced by Fusarium graminearum. It is one of the most common feed contaminants that poses a serious threat to the health and performance of dairy cows. This study investigated the in vitro cytotoxicity of DON on bovine mammary epithelial cells (MAC-T). DON at different concentrations (0.25, 0.3, 0.5, 0.8, 1 or 2 µg/ml) inhibited the growth of MAC-T cells after 24 hr of exposure (p < .001). DON at 0.25 µg/ml increased lactate dehydrogenase (LDH) leakage (p < .05); decreased glutathione (GSH) levels (p < .001), total superoxide dismutase (T-SOD) activity and total antioxidant capacity (T-AOC; p < .01); and increased malondialdehyde (MDA) concentration (p < .01) in MAC-T cells after 24 hr of exposure. We also observed that DON increased reactive oxygen species (ROS) levels in cells incubated for 9, 15 and 24 hr (p < .001). DON at 0.25 µg/ml triggered oxidative damage in MAC-T cells. Furthermore, it induced an inflammatory response in the cells incubated for 9, 15 and 24 hr (p < .05) by increasing the mRNA expression levels of nuclear factor kappa B, myeloid differentiation factor 88 (MyD88), tumour necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), IL-6, cyclooxygenase-2 and IL-8. We further examined the effect of DON on apoptosis. DON prevented normal proliferation of MAC-T cells by blocked cell cycle progression in 24 hr (p < .001). In addition, the apoptosis rate measured using annexin V-FITC significantly increased (p < .05) with increase in the mRNA expression level of Bax (p < .01) and increase in the Bax/Bcl-2 ratio (p < .01) in cells incubated for 24 hr. In summary, DON exerts toxic effects in MAC-T cells by causing oxidative stress, inducing an inflammatory response, affecting cell cycle and leading to apoptosis.

Polyparasitism of Rhabditis Axei and Enterobius Vermicularis in a Child from Beijing, China.

BACKGROUND: Rhabditis (Rhabditellae) axei is a common species in soil, which has been reported repeatedly in human urine and the digestive system. Humans exposed to sewage or mistakenly polluted sewage is the cause of larvae infecting the digestive tract or via the urethra. We reported a patient infected with Rhabditis axei and Enterobius Vermicularis. The migration of the nematodes caused true signs of hematuria, diarrhea, and high eosinophilia. METHODS: Stool and urine are collected to detect parasite eggs and genotype. Specimens are sent for polymerase chain reaction (PCR)-based species identification. Amplification of the 18S ribosomal RNA gene was performed by PCR as described [1]. RESULTS: Morphological features and PCR amplification of the 18S ribosomal RNA gene confirmed Rhabditis axei and Enterobius vermicularis as the pathogen of infection. CONCLUSIONS: Herein, we presented a case that confirmed Rhabditis axei and Enterobius vermicularis infection in humans can be associated with high eosinophilia.

A study on relationship between elderly sarcopenia and inflammatory factors IL-6 and TNF-α.

BACKGROUND: This report aims to study the relationship between sarcopenia of elderly in community and inflammatory factors IL-6 and TNF-α. METHODS: A total of 441 elders who undertook physical examinations were included into this study. The age of these subjects were >60, in which 235 subjects were male and 206 subjects were female. According to the diagnostic standards of sarcopenia set by EWGSOP and AWGS, these subjects were divided into two groups: sarcopenia, and non-sarcopenia groups. The living habits, disease status, biochemical indexes, and levels of IL-6 and TNF-α of these subjects were investigated. RESULTS: The morbidity rate of sarcopenia was 17.02% in male subjects and 18.9% in female subjects. In elderly subjects >80 years old, morbidity rate was 25.3% in male subjects and 35.1% in female subjects. The history of smoking in patients with sarcopenia was long, and their regular exercise history was short (P < 0.01). Furthermore, differences in handgrip strength (HG), fat-free mass (FFM), bone mineral content (BMC), plasma albumin (ALB) and serum creatinine (Cr), and body fat content (FAT) in patients between the sarcopenia and non-sarcopenia groups were statistically significant (P < 0.05). Moreover, differences in IL-6 and TNF-α levels between these two groups were statistically significant (P < 0.05). In addition, BMI was positively correlated to TNF-α levels, and ALB was negatively correlated to IL-6; while BMI and VFA were positively correlated to TNF-α levels, and SMM, HDL-C, Hb, HG were negatively correlated to IL-6 level (P < 0.05). Multiple linear regression analysis suggested plasma ALB and BMI were the independent risk factors of TNF-α, while VFA was the independent risk factor of IL-6. CONCLUSIONS: The onset of sarcopenia was associated with poor exercise habits, disease history, and nutritional status. The emergence of sarcopenia was accompanied by increased levels of inflammation factors TNF-α and IL-6. Plasma albumin, BMI, and VFA were inflammatory factor predictors of TNF and IL-6.

AHNAK2 is a potential prognostic biomarker in patients with PDAC.

BACKGROUND: AHNAK nucleoprotein 2 (AHNAK2) belongs to the AHNAK protein family. The studies of AHNAK2 are limited. A recent study reported that AHNAK2 might be a biomarker for pancreatic ductal adenocarcinoma (PDAC); however, tissue-based experiments have not been conducted. The aim of this study was to determine the tissue expression of AHNAK2 and to find the correlation between AHNAK2 and overall survival rate in PDAC. RESULTS: AHNAK2 is highly expressed in PDAC (n=79) compared with adjacent normal tissues (n=64, P<0.001). Overexpression of AHNAK2 showed a significant relationship with a lower overall survival rate (P=0.033) in PDAC patients. The predictive value of increased expression of AHNAK2 remains relevant in patients with AJCC grade above II (n=43, P=0.006) or lymph node metastasis (n=32, P=0.004). Cox regression analysis showed that AHNAK2 expression (P=0.003) and pathology grade (P<0.001) are independent prognostic factors for PDAC. The nomogram model was performed to predict the 1- and 3-year survival rates based on Cox regression. The C-index was 0.61. The calibration curves were also made to show the association between the observed and predicted probability of the overall survival rates. MATERIALS AND METHODS: AHNAK2 expression was performed in tissue microarrays by immunohistochemistry. The overall survival rate analysis was performed using the Kaplan-Meier method, Cox proportional hazards regression, and a nomogram model. CONCLUSIONS: AHNAK2 is overexpressed in PDAC tissues and is an independent prognostic factor in patients with PDAC.

Berberine inhibits EGFR signaling and enhances the antitumor effects of EGFR inhibitors in gastric cancer.

Cetuximab plus chemotherapy for advanced gastric cancer (GC) shows an active result in phase 2 trials. Unfortunately, Combination of cetuximab does not provide enough benefit to chemotherapy alone in phase 3 trials. Studies have demonstrated that berberine can suppress the activation of EGFR in tumors. In this study, we evaluated whether berberine could enhance the effects of EGFR-TKIs in GC cell lines and xenograft models. Our data suggest that berberine could effectively enhance the activity of erlotinib and cetuximab in vitro and in vivo. Berberine was found to inhibit growth in GC cell lines and to induce apoptosis. These effects were linked to inhibition of EGFR signaling activation, including the phosphorylation of STAT3. The expressions of Bcl-xL and Cyclind1 proteins were decreased, whereas the levels of cleavage of poly-ADP ribose polymerase (PARP) were considerably increased in the cell lines in response to berberine treatment. These results suggest a potential role for berberine in the treatment of GC, particularly in combination with EGFR-TKIs therapy. Berberine may be a competent therapeutic agent in GC where it can enhance the effects of EGFR inhibitors.

[Analysis of cytochrome P450 2E1 RsaI/PstI and DraI polymorphisms in workers exposed to benzene].

OBJECTIVE: To investigate the cytochrome P450 2E1 (CYP2E1) RsaI/PstI and DraI polymorphisms in workers exposed to benzene. METHODS: A cross-sectional survey was carried out. A total of 71 workers exposed to benzene were included in observation group and the same number of people without occupational benzene exposure were included in control group. Blood samples from the two groups were collected and genotyping for CYP2E1 RsaI/PstI and DraI were conducted using the polymerase chain reaction-restriction fragment length polymorphism. RESULTS: There were no significant differences in CYP2E1 DraI genotype and allele distributions between the observation group and the control group (χ² = 2.374, P > 0.05; χ² = 2.113, P > 0.05). Significant differences in CYP2E1 RsaI/PstI genotype and allele distributions between the two groups were observed (χ² = 9.129, P < 0.01; χ² = 6.028, P < 0.05). CONCLUSION: Mutations at CYP2E1 RsaI/PstI can enhance the expression of CYP2E1 and this suggests individuals with the mutated gene have increased susceptibility to chronic benzene poisoning.

Aberrant methylation of the 3q25 tumor suppressor gene PTX3 in human esophageal squamous cell carcinoma.

AIM: To identify the novel methylation-silenced gene pentraxin 3 (PTX3) in esophageal squamous cell carcinoma (ESCC). METHODS: PTX3 mRNA expression was examined in six human ESCC cell lines, one human immortalized normal esophageal epithelial cell line, primary ESCC tumor tissue, and paired adjacent nontumor tissue using reverse transcription polymerase chain reaction (RT-PCR). Semi-quantitative immunohistochemistry was used to examine cellular localisation and protein levels. Methylation specific PCR and bisulphite genomic sequencing were employed to investigate the methylation of the candidate gene. RESULTS: In the majority of ESCC cell lines, we found that PTX3 expression was down-regulated due to gene promoter hypermethylation, which was further confirmed by bisulphite genomic sequencing. Demethylation treatment with 5-aza-2'-deoxycytidine restored PTX3 mRNA expression in ESCC cell lines. Methylation was more common in tumor tissues (85%) than in adjacent nontumor tissues (25%) (P < 0 .01). CONCLUSION: PTX3 is down-regulated through promoter hypermethylation in ESCC, and could potentially serve as a biomarker of ESCC.

Epigenetic inactivation of the SFRP1 gene in esophageal squamous cell carcinoma.

INTRODUCTION: The secreted frizzled-related protein 1 (SFRP1) gene, as a Wnt signaling modulator, is frequently inactivated by promoter methylation in many tumors including gastric cancer, breast cancer, oral squamous cell carcinoma, and esophageal adenocarcinoma. However, the role of SFRP1 in esophageal squamous cell carcinoma (ESCC) is not clear. In this study, we investigated the epigenetic inactivation of the SFRP1 gene in ESCC. METHODS: Nine ESCC cell lines, two immortalized human esophageal epithelial cell lines, twenty ESCC tissues, and paired adjacent nontumor tissues were analyzed in the study. Methylation-specific polymerase chain reaction (PCR), bisulfite sequencing, reverse-transcription PCR, immunohistochemistry, and chromatin immunoprecipitation assay were used to detect SFRP1 promoter methylation, expression of the SFRP1 gene, and histone modification in the SFRP1 promoter region. RESULTS: The SFRP1 promoter was found to be highly methylated in 95% (19/20) of the ESCC tissues and in nine ESCC cell lines, compared with 65% (13/20) of the paired nontumor tissues. Moreover, we confirmed that complete methylation of the SFRP1 gene promoter was correlated with its greatly reduced expression level. After individual treatment with 5-aza-2'-deoxycytidine (DAC) and trichostatin A (TSA), the messenger RNA (mRNA) level of the SFRP1 gene was not obviously rescued in the EC9706 cell line. Combined incubation with DAC and TSA can, however, substantially increase the SFRP1 mRNA expression level in the EC9706 cell line. Chromatin immunoprecipitation assay showed that acetylated histone H3 and H4 were found in the SFRP1 promoter region. CONCLUSION: Promoter hypermethylation of SFRP1 is a frequent event in ESCC. Promoter methylation and histone acetylation may cooperatively regulate expression of the SFRP1 gene.

Identification of GPX3 epigenetically silenced by CpG methylation in human esophageal squamous cell carcinoma.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is one of the most common causes of cancer mortality in the gastrointestinal tract. Promoter hypermethylation of tumor suppressor genes contributes to gene inactivation during development of ESCC. AIM: To identify novel methylation-silenced genes in ESCC. METHODS: Genome-wide microarrays were applied to search for genes that were markedly upregulated after treatment with 5-aza-2'-deoxycytidine (5-Aza-dC) and that were markedly decreased in tumor tissue compared with paired adjacent nontumor tissue. Reverse-transcription polymerase chain reaction (PCR), immunohistochemistry, methylation-specific PCR, and bisulfite genomic sequencing were employed to investigate expression and methylation of candidate genes in five human ESCC cell lines, two human immortalized normal esophageal epithelial cell lines, primary ESCC tumor tissues, and paired adjacent nontumor tissues. RESULTS: GPX3 was selected as a novel candidate hypermethylated gene in ESCC through microarray analysis. In most ESCC cell lines, GPX3 messenger RNA (mRNA) expression was downregulated and the CpG island of GPX3 promoter was methylated. Demethylation treatment with 5-Aza-dC restored GPX3 mRNA expression. Methylation of GPX3 promoter was more frequent in ESCC tumor tissues (71.4%) than in adjacent nontumor tissues (10.7%) (P < 0.001), and methylation of GPX3 promoter correlated significantly with GPX3 mRNA downregulation. Finally, GPX3 protein expression was also significantly lower in ESCC tumor tissues than in adjacent nontumor tissues. CONCLUSION: GPX3 is downregulated through promoter hypermethylation in ESCC, which may be a potential biomarker of ESCC.