[The association between the whole blood riboflavin level and the occurrence, development and prognosis of esophageal squamous cell carcinoma].

Objective: To investigate the association between the whole blood riboflavin level and the occurrence, development and prognosis of esophageal squamous cell carcinoma (ESCC) in China. Methods: From March 2014 to September 2018, ESCC patients from three hospitals (the Affiliated Hospital of Medical College of Shantou University, Shantou Central Hospital in Southern Chaoshan area and First Affiliated Hospital of Zhengzhou University in Northern Taihang Mountain) were selected as a case group; non-esophageal patients who had a physical examination were selected as a control group. The case and control group were paired by age (±5 years) and a 1:1 ration. A total of 1 528 subjects were enrolled including 764 patients in the case group and 764 patients in the control group. About 3-5 ml venous blood samples were collected, and the erythrocyte glutathione reductase activity coefficient (GRAC) was measured to assess the whole blood riboflavin level. A multivariate conditional logistic regression model was used to analyze the association between the GRAC and the risk of ESCC. The association between the GRAC and the prognosis of ESCC was analyzed by using Cox proportional risk regression model based on 288 patients with complete survival data. They were divided into two groups, the high GRAC group (GRAC≥7.87) group and the low GRAC group (GRAC<7.87) according to the strongest correlation between the total survival time, survival outcome and GRAC (GRAC=7.87). Results: Among the 1 528 patients, 958 patients were from Southern Chaoshan area, including 479 patients in the case group with an average age about (59.90±9.34) years and 479 patients in the control group with an average age about (59.55±8.77) years. Other 570 patients were from Northern Taihang Mountain area, including 285 patients in the case group with an average age (58.39±5.19) years and 285 patients in the control group with an average age about (58.74±4.57) years. The multivariate conditional logistic regression showed that the OR (95%CI) of the GRAC and the risk of ESCC was 1.009 (0.998-1.019). The Cox proportional hazard regression model analysis showed that the HR (95%CI) of the high GRAC group was 1.712 (1.034-2.824) compared with the low GRAC group in the 50-70 years group. Conclusion: The whole blood riboflavin level might not be associated with the occurrence of ESCC. The high whole blood riboflavin level would be more beneficial to the prognosis of ESCC patients aged 50-70 years.

MicroRNA-377 suppresses initiation and progression of esophageal cancer by inhibiting CD133 and VEGF.

Esophageal cancer is one of the most lethal cancers worldwide with poor survival and limited therapeutic options. The discovery of microRNAs created a new milestone in cancer research. miR-377 is located in chromosome region 14q32, which is frequently deleted in esophageal squamous cell carcinoma (ESCC), but the biological functions, clinical significance and therapeutic implication of miR-377 in ESCC are largely unknown. In this study, we found that miR-377 expression was significantly downregulated in tumor tissue and serum of patients with ESCC. Both tumor tissue and serum miR-377 expression levels were positively correlated with patient survival. Higher serum miR-377 expression was inversely associated with pathologic tumor stage, distant metastasis, residual tumor status and chemoradiotherapy resistance. The roles of miR-377 in suppressing tumor initiation and progression, and the underlying molecular mechanisms were investigated. Results of in vitro and in vivo experiments showed that miR-377 overexpression inhibited the initiation, growth and angiogenesis of ESCC tumors as well as metastatic colonization of ESCC cells, whereas silencing of miR-377 had opposite effects. Mechanistically, miR-377 regulated CD133 and VEGF by directly binding to their 3' untranslated region. Moreover, systemic delivery of formulated miR-377 mimic not only suppressed tumor growth in nude mice but also blocked tumor angiogenesis and metastasis of ESCC cells to the lungs without overt toxicity to mice. Collectively, our study established that miR-377 plays a functional and significant role in suppressing tumor initiation and progression, and may represent a promising non-invasive diagnostic and prognostic biomarker and therapeutic strategy for patients with ESCC.

Integrative analyses of transcriptome sequencing identify novel functional lncRNAs in esophageal squamous cell carcinoma.

Long non-coding RNAs (lncRNAs) have a critical role in cancer initiation and progression, and thus may mediate oncogenic or tumor suppressing effects, as well as be a new class of cancer therapeutic targets. We performed high-throughput sequencing of RNA (RNA-seq) to investigate the expression level of lncRNAs and protein-coding genes in 30 esophageal samples, comprised of 15 esophageal squamous cell carcinoma (ESCC) samples and their 15 paired non-tumor tissues. We further developed an integrative bioinformatics method, denoted URW-LPE, to identify key functional lncRNAs that regulate expression of downstream protein-coding genes in ESCC. A number of known onco-lncRNA and many putative novel ones were effectively identified by URW-LPE. Importantly, we identified lncRNA625 as a novel regulator of ESCC cell proliferation, invasion and migration. ESCC patients with high lncRNA625 expression had significantly shorter survival time than those with low expression. LncRNA625 also showed specific prognostic value for patients with metastatic ESCC. Finally, we identified E1A-binding protein p300 (EP300) as a downstream executor of lncRNA625-induced transcriptional responses. These findings establish a catalog of novel cancer-associated functional lncRNAs, which will promote our understanding of lncRNA-mediated regulation in this malignancy.

Circulating levels of autoantibodies against L1-cell adhesion molecule as a potential diagnostic biomarker in esophageal squamous cell carcinoma.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is a common malignant disease worldwide, especially in China. We aimed to determine the level of autoantibodies against L1CAM in patients with ESCC. METHODS: Levels of circulating autoantibodies against L1CAM antigens were determined by an enzyme-linked immunosorbent assay in cohort 1 (191 patients with ESCC and 94 normal controls) and validated in cohort 2 (47 patients with ESCC and 47 normal controls). Receiver-operating characteristics were employed to calculate diagnostic accuracy. Cumulative survival time was calculated by the Kaplan-Meier method and analyzed by the log-rank test. RESULTS: In cohorts 1 and 2, levels of autoantibodies against L1CAM were all significantly higher in sera of patients with ESCC compared to normal controls (P < 0.05). Detection of autoantibodies against L1CAM provided a sensitivity of 26.2%, a specificity of 90.4%, and an area under the curve (AUC) of 0.603 (95% CI 0.535-0.672) in diagnosing ESCC in cohort 1, and a sensitivity of 27.7%, a specificity of 91.5%, and an AUC of 0.628 (95% CI 0.516-0.741). Similar results were observed in the diagnosis of early stage ESCC (25.2% sensitivity, 90.4% specificity, and an AUC of 0.611 (95% CI 0.533-0.689) in cohort 1, and 33.3% sensitivity, 91.5% specificity, and an AUC of 0.636 (95% CI 0.439-0.832) in cohort 2). Moreover, positive rates of autoantibodies against L1CAM had no statistical correlation with clinical outcome of ESCC (P > 0.05). CONCLUSIONS: Our results suggest that circulating autoantibodies against L1CAM is a potential biomarker for the early detection of ESCC.

Chronic periodontitis and the risk of erectile dysfunction: a systematic review and meta-analysis.

The objective of this study is to evaluate the association between chronic periodontitis (CP) and the risk of erectile dysfunction (ED). Electronic search using PubMed, Embase and the Cochrane Library was carried out for observational studies, longitudinal, cohort, case-control and epidemiological studies on humans, published up to December 2015. Manual searches were also performed. Odds ratios (ORs) and corresponding 95% confidence intervals (CIs) were used to estimate the association between CP and the risk of ED. Methodological quality assessment was carried out using the Newcastle-Ottawa Quality Assessment Scale. Four case-control studies and one cross-sectional studies involving 213, 006 participants were included. Based on the random-effects model, analyses of all studies showed that CP was associated with an increased risk of ED (OR=2.28, 95% CI: 1.50-3.48). There was heterogeneity among the studies (P<0.001, I2=97.8%). Estimates of total effects were generally consistent with the sensitivity and subgroup analyses. In conclusion, our meta-analysis suggested that there was a significant association between CP and the risk of ED. Further epidemiological studies are needed to better estimate the key risk factors for periodontitis and their interaction effects.

Association of mu-opioid receptor expression with lymph node metastasis in esophageal squamous cell carcinoma.

The mu-opioid receptor (MOR), a membrane-bound G protein-coupled receptor, is the main target for opioids in the nervous system. MOR1 has been found in several types of cancer cells and reported to be involved in tumor progression and metastasis. However, the expression and clinical significance of MOR1 in esophageal squamous cell carcinoma (ESCC) remain unclear. In our study, the expression of MOR1 was confirmed in ESCC cell lines (KYSE180, KYSE150, and EC109) by Western blot. MOR1 was also detected on tissue microarrays of ESCC samples in 239 cases using immunohistochemical staining. We found that MOR1 was mainly located in the cytoplasm and occasionally occurred in the membrane or nucleus of ESCC cells. Moreover, results indicated that MOR1 expression in the cytoplasm was associated with lymph node metastasis (R = 0.164, P = 0.008, Kendall's tau-b-test). No more associations were found between MOR1 expression status and other clinical parameters. However, no statistical significant differences were found between MOR1 expression in the cytoplasm, nucleus/membrane, and the overall survival of ESCC patients (P = 0.848; P = 0.167; P = 0.428, respectively, log-rank test). Our results suggest that the cytoplasmic MOR1 may be a high-risk factor for lymph node metastasis of ESCC patients. We also hypothesize that MOR1 agonists used in ESCC patients should be prudent, and opioid receptor antagonists may be novel therapeutic drugs for ESCC patients.

Evaluating the susceptibility of mitochondrial DNA germline mutations in Chinese cancer patients.

It has been suggested that impairment of mitochondrial oxidative phosphorylation (OXPHOS) is a common character in cancer cells, urging attention to variation on mitochondrial DNA (mtDNA) that encodes 13 units of the OXPHOS. However, most of mtDNA somatic mutations in cancer were suggested to result from the relaxed functional constrains and thus the byproducts of tumorigenesis. MtDNA germline mutations present not only in the cancer tissue but also in the normal tissue. However, it remains unclear whether the cancerous mtDNA germline mutations suffered similar selective constraints as the somatic mutations did. To address this question, we collected 153 whole mitochondrial genomes (including 20 newly obtained genomes in this study) from the normal tissues of cancer patients and compared with a number of 561 whole mtDNA sequences from the general population in China. Different from the observations on cancerous mtDNA somatic mutations, our results revealed that the germline mutations showed no significant difference between the cancer patients and the general population in either the sub-haplogroup composition, mutation pattern or the potential pathogenicity of the private mutations. It then seems that regulation on cellular OXPHOS level, triggered by mtDNA variation to some extent, exerts little influence on the susceptibility of cancer, which echoes the opinion that aerobic glycolysis, not mitochondrial respiration, plays the key role in generating energy in cancer cells, thus suggesting the role of most mtDNA mutations in cancer likely being overestimated.

Invasive and prognostic significance of pRB in esophageal squamous cell carcinoma: a meta-analysis.

This paper investigates the association between protein retinoblastoma (pRB) loss and the T,N stage and prognosis in esophageal squamous cell carcinomas (ESCCs) using meta-analysis. We conducted a meta-analysis of 16 studies, comprising 1,117 patients to clarify this issue. All the studies searched by the electronic literature PubMed and http://www.KJEBM.com, which had been published during the period from January 1996 to January 2012 according to the inclusion criteria. Summary odds ratios (OR) were calculated using fixed or random-effects models. The summary odds ratios (ORs) for pRB inactive were 0.64 (95% confidence interval [CI]:0.45-0.91, P = 0.01) for T1/T2 versus T3/T4 tumors; summary OR = 0.69 (95% CI:0.51-0.94, P = 0.02) for N0 versus N1 tumors. The association between pRB loss and prognosis was examined in nine studies, and the summary hazard ratio was 1.39 (95% CI:1.11-1.74, P = 0.004). pRB inactive was significant associated with T3/T4 tumors and N1 stage as well as adverse prognosis for ESCCs. It appears warranted to prospectively validate that pRB loss may be used for subdividing the T,N stage evaluation of patients with ESCCs, and these patients may be the preponderant people for individualized treatment or target therapy.

Expression of cysteine-rich 61 is correlated with poor prognosis in patients with esophageal squamous cell carcinoma.

INTRODUCTION: Cysteine-rich 61 (Cyr61), a secreted protein belonged to the CCN family, was involved in the progression of many cancers. The purpose of this study was to explore the clinical significance of Cyr61 expression in esophageal squamous cell carcinoma (ESCC). MATERIALS AND METHODS: Cyr61 expression was detected on tissue microarrays of ESCC samples in 372 cases by using immunohistochemical staining. Survival analysis was assessed by the Kaplan-Meier analysis. Relative risk was evaluated by the multivariate Cox proportional hazards model. RESULTS: The staining pattern of Cyr61 was heterogeneous and varied from negative to intense expression in a cytoplasmic distribution. Kaplan-Meier analysis revealed that expression of Cyr61 was related to poor survival of ESCC patients (P = 0.001). Further analysis revealed that Cyr61 high-expression was related to poorer overall survival of patients in stage I/II (P = 0.001); but did not effect the overall survival of patients in stage III/IV. Univariate and multivariate analysis suggested that Cyr61 expression status was an independent prognostic factor for ESCC (P = 0.001). DISCUSSION: Cyr61 might play important roles in the progression of ESCC. Cyr61 is a new biomarker to predict the prognosis of ESCC patients.

Connective tissue growth factor expression in precancerous lesions of human esophageal epithelium and prognostic significance in esophageal squamous cell carcinoma.

Connective tissue growth factor (CTGF, CCN2), a secreted protein, is involved in the development and progression of esophageal squamous cell carcinoma (ESCC). However, it remains unclear how CTGF expression affects the progression of ESCC. Our study implicated differences of CTGF protein status in precancerous lesions, and retrospectively examined the associations of CTGF mRNA and protein levels with clinical prognosis in ESCC patients. Here immunohistochemistry and the quantitative real-time real-time reverse transcription polymerase were performed for predicting the CTGF protein status and mRNA levels in ESCC patients, respectively. Different degrees of CTGF protein status presented in normal human esophageal epithelium and precancerous lesions, and CTGF protein was highly expressed in ESCCs. Survival analysis showed that CTGF protein status was significantly related to poor survival of ESCC patients (P= 0.024), while no significant difference was observed between CTGF mRNA levels and the survival of ESCC patients (P= 0.196). Multivariate Cox analysis demonstrated that CTGF protein status was the independent factor in prognosis of ESCC patients. In that way, CTGF protein status might elevate the progression of ESCC, and would be significant for the diagnosis of precancerous lesions or early ESCC.

Autophagic and apoptotic cell death in amniotic epithelial cells.

The aim of this paper is to determine if autophagic cell death is associated with apoptosis and whether it participates in the process of term amniotic rupture. Forty pieces of fresh term amnions, including twenty from a position near the margin of the placentas and twenty from the margin of the naturally ruptured part of the placentas in term gestation were collected, respectively. The amnions were examined by scanning electron microscopy (SEM) and amniotic epithelial (AE) cells were examined by transmission electron microscopy (TEM). Autophagic and apoptotic cell death (PCD) were assayed by laser scanning confocal microscopy (LSCM) or flow cytometry using monodansylcadaverin (MDC) and propidium iodide (PI) stain. BCL(2) and BAX were examined by immunoblotting. Under SEM the amniotic epithelia appeared normal in the position near the placenta. They had an atrophied appearance in the margin of their natural broken parts. In the AE cells PCD was divided into three subtypes by TEM: autophagic cell death with positive stains of MDC and PI; apoptotic cell death; and the mixed type. Quantitative detection showed that there were more death cells, including autophagic and apoptotic, in the AE cells near the ruptured parts than near the placentas. An increased expression of BAX and a decreased expression of BCL(2) protein in the AE cells near the broken margin were observed. Apoptotic and autophagic cell death by the intrinsic pathway are the basic event in the AE cell and they are involved in the cause of membrane rupture of the human amnion in term gestation.

Role of fascin in the proliferation and invasiveness of esophageal carcinoma cells.

Fascin, an actin-bundling protein, induces membrane protrusions and increases cell motility in various transformed cells. The overexpression of fascin in esophageal squamous cell carcinoma (ESCC) has been described only recently, but the roles and mechanism still remained unclear. Here, by using RNA interference (RNAi), we have stably silenced the expression of the fascin in EC109 cells, an ESCC cell line. Down-regulation of fascin resulted in a suppression of cell proliferation and as well as a decrease in cell invasiveness. Furthermore, we revealed that fascin might have functions in regulating tumor growth in vivo. The effect of fascin on cell invasiveness correlated with the activation of matrix metalloproteases such as MMP-2 and MMP-9. We examined that fascin down-expression also led to a decrease of c-erbB-2 and beta-catenin at the protein level. These results suggested that fascin might play crucial roles in regulating neoplasm progression of ESCC.