LMP gene promoter hypermethylation is a mechanism for its down regulation in Kazak’s esophageal squamous cell carcinomas

Abnormal hypermethylation of CpG islands not only associated with tumor suppressor genes can lead to repression of gene expression, but also contribute to escape of the tumor from immune surveillance and contribute significantly to tumorigenesis. In the present study, we studied the hypermethylation of low molecular-weight protein (LMP) gene and its regulation on protein expression in biopsies from resected tissues from Kazak’s esophageal squamous cell carcinoma (ESCC) patients and their neighboring normal tissues. LMP2 and LMP7 genes promoter region methylation sequences were maped in esophageal cancer cell line Eca109 by bisulfite-sequencing PCR and quantitative detection of methylated DNA from 30 pairs of Kazak’s ESCC and adjacent normal tissues by MassARRAY (Sequenom, San Diego, CA, USA) and LMP2 and LMP7 protein expression were analyzed with immunohistochemistry. In Eca109, we identified 6 CG sites methylated from all of 22 CpG sites of LMP7 gene. However, no methylation was found for LMP2. The analysis of the data resulted from the quantitative analysis of single CpG site methylation by Sequenom MassARRAY platform, has shown that the methylation level between two groups CpG sites (CpG_5, CpG_9, CpG_20, CpG_21 and CpG_20) from CpG_1, CpG_2, CpG_3, CpG_4, CpG_5, CpG_6, CpG_7, CpG_8, CpG_9, CpG_10.11, CpG_12.13.14, CpG_15.16.17.18, CpG_19, CpG_20, CpG_21 and CpG_22 significant differences between ESCC and neighboring normal tissues. The analysis of methylation level of whole target CpG fragment indicated that the methylation level of LMP7 was significant higher in ESCC (0.0517 ± 0.0357) than in neighboring normal tissues (0.0380 ± 0.0214, P < 0.05). there was a tendency of decreasing the LMP7 proteins expression as the increasing the methylation level of LMP7 gene promoter regions (F = 7.69, P = 0.041). The LMP7 gene promoter methylation and protein downregulation were correlated at high extent in Kazakh’s ESCC patients, and may explain the epigenetic regulation on gene expression.     

Association of the Plasma and Tissue Riboflavin Levels with C20orf54 Expression in Cervical Lesions and Its Relationship to HPV16 Infection

Riboflavin deficiency can cause a variety of metabolic problems that lead to skin and mucosal disorders. Limited evidence suggests that high intake of riboflavin may reduce overall risks of cancer. However, association of this deficiency with cervical cancer and precancerous lesions are still not definitively known. In this study, we characterized the relationship between plasma and tissue riboflavin levels and C20orf54 protein expression in patients with cervical intraepithelial neoplasia (CIN) and cervical squamous cell carcinoma (CSCC) as well as the relationship of these levels with human papillomavirus virus 16, 18 (HPV16/18) infections. High-performance liquid chromatography (HPLC) was used to measure blood riboflavin levels in patients with CIN and CSCC, and an enzyme-linked immunosorbent assay (ELISA) was used to determine tissue riboflavin levels in patients with CSCC and matched normal mucous epithelia. The expression of C20orf54 in fresh CSCC and matched tissues were detected by qRT-PCR and western blot, respectively. And it was further confirmed by immunohistochemistry (IHC) with formalin-fixed, paraffin-embedded CIN and CSCC. An HPV genotyping chip was used to analyze HPV infection and typing. The results showed that patients with CIN and CSCC had decreased plasma riboflavin levels as compared with normal controls. There was also significantly decreased riboflavin in tissues from CSCC patients, when compared with normal cervical epithelia. C20orf54 expression were significantly up-regulated in CSCC compared to matched control on both mRNA and protein level. Tissue riboflavin levels were significantly lower in HPV16/18 positive tissue compared with HPV16/18-negative tissue, and an inverse association was found between tissue riboflavin levels and C20orf54 mRNA and protein expression in CSCC. Additionally, C20orf54 was significantly correlated with tumor stages. In conclusion, C20orf54 tend to play a protective role in Uyghur cervical carcinogenesis of which modulating riboflavin absorption, and it is also related with HPV infection.     

LncRNA C9orf139 can regulate the progression of esophageal squamous carcinoma by mediating the miR-661/HDAC11 axis

Increasing evidence has indicated that long non-coding RNAs (LncRNAs) play multiple functions in the development of cancer and function as indicators of diagnosis and prognosis. This aim of this study was to investigate the roles LncRNA C9orF139 had in the progression of esophageal squamous carcinoma (ESCC). We found C9orf139 was highly expressed in ESCC and knock down the expression of C9orf139 significantly suppressed cell proliferation, promoted apoptosis, and inhibited migration and invasion. C9orf139 was able to negatively regulate miR-661 expression. At the same time, HDAC11 expression was negatively regulated by miR-661. The C9orf139/miR-661/HDAC11 axis was further involved in regulating the expression of the NF-κB signaling pathway. The association between the C9orf139 knockdown and the reduced tumor growth and size was observed during in vivo study. C9orf139 is highly expressed in ESCC, and is thus qualified to be used as a potential diagnostic and prognostic marker for ESCC. Its promotion of ESCC progression is achieved by mediating the miR-661/HDAC11 axis.     

Adaptive regulation of riboflavin transport in heart: effect of dietary riboflavin deficiency in cardiovascular pathogenesis

Deficiency or defective transport of riboflavin (RF) is known to cause neurological disorders, cataract, cardiovascular anomalies, and various cancers by altering the biochemical pathways. Mechanisms and regulation of RF uptake process is well characterized in the cells of intestine, liver, kidney, and brain origin, while very little is known in the heart. Hence, we aimed to understand the expression and regulation of RF transporters (rRFVT-1 and rRFVT-2) in cardiomyocytes during RF deficiency and also investigated the role of RF in ischemic cardiomyopathy and mitochondrial dysfunction in vivo. Riboflavin uptake assay revealed that RF transport in H9C2 is (1) significantly higher at pH 7.5, (2) independent of Na⁺ and (3) saturable with a Km of 3.746 µM. For in vivo studies, male Wistar rats (110–130 g) were provided riboflavin deficient food containing 0.3 ± 0.05 mg/kg riboflavin for 7 weeks, which resulted in over expression of both RFVTs in mRNA and protein level. RF deprivation resulted in the accumulation of cardiac biomarkers, histopathological abnormalities, and reduced mitochondrial membrane potential which evidenced the key role of RF in the development of cardiovascular pathogenesis. Besides, adaptive regulation of RF transporters upon RF deficiency signifies that RFVTs can be considered as an effective delivery system for drugs against cardiac diseases.     

HLA-DRB1*1501 and HLA-DQB1*0301 alleles are positively associated with HPV16 infection–related Kazakh esophageal squamous cell carcinoma in Xinjiang China

Multiple determinant factors are involved in the occurrence and progression of esophageal squamous cell carcinoma (ESCC). Human papillomavirus (HPV) and human leukocyte antigen (HLA) polymorphism were identified as important factors. This study examined the associations between the development of Kazakh ESCC and the determinant factors including HLA-DRB1*0901, 1501; DQB1*0301, 0602; high-risk HPV infection in the area of Xinjiang, China. 200 Kazakh patients with ESCC and 150 controls were recruited, and polymerase chain reaction (PCR) was performed to detect HLA-DRB1*0901, 1501 and DQB1*0301,0602 using sequence-specific primers (SSPs). HPV16 was detected in esophageal specimens using PCR. HPV16 infection rate in Kazakh ESCC case group was 41 %, significantly higher than that of control group 14 % (OR = 3.62; 95 % CI, 2.15–6.09; P < 0.001). A positive association between ESCC and HLA-DRB1*1501 (OR = 2.46, P < 0.0125) or HLA-DQB1*0301 (OR = 3.34, P < 0.0125) alleles was observed. Similar tendencies were observed for HLA-DRB1*1501 (OR = 3.095, P < 0.0125) and HLA-DQB1*0301 (OR = 2.410, P < 0.0125) alleles with HPV16-positive ESCC. HLA-DRB1*1501, HLA-DQB1*0301 and DQB1*0602 were significantly associated with ESCC when the age was ≥55 years (P < 0.0125 for all), whereas only HLA-DQB1*0301 was significantly associated with ESCC when the age was <55 years (P < 0.0125). HLA-DRB1*1501 and HLA-DQB1*0301 were significantly associated with an increase in ESCC occurrence in females (P < 0.0125), whereas only HLA-DQB1*0301 was significantly associated with ESCC in males. Moreover, the occurrence of HLA-DQB1*0602 gene in poorly differentiated ESCC group (68.8 %) was slightly higher than that of well-differentiated squamous cell carcinoma group (31.2 %). The difference was not statistically significant (P > 0.0125). The study suggests that HLA-DRB1*1501 and HLA-DQB1*0301 may influence the immune response to specific tumor and HPV-encoded epitopes and affect the risk of Kazakh ESCC in XinJiang, China.     

Replication study of PLCE1 and C20orf54 polymorphism and risk of esophageal cancer in a Chinese population

Esophageal cancer is one of the most aggressive cancers in the world. Recent large-scale genome-wide association studies (GWAS) reported that functional genetic variations in the phospholipase C epsilon gene (PLCE1) were strongly associated with risk of esophageal squamous cell carcinoma (ESCC) and gastric cardia adenocarcinoma (GCA) in Chinese population. For C20orf54 rs13042395 genotype and risk of esophageal cancer, the results were inconsistent. We conducted a replication case-control study to evaluate the genetic effects of these two functional single nucleotide polymorphisms (SNPs) on the development of esophageal cancer. A total of 380 cases and 380 controls were recruited for this study. The genotypes were determined by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS). The variant alleles of the functional polymorphism, PLCE1 rs2274223 SNP was associated with the increased risk of esophageal cancer [adjusted odds ratio (OR) = 1.95, 95 % confidence interval (CI) = 1.05-3.59 for PLCE1 rs2274223 GG vs. AA]. However, there was no significant association between the C20orf54 rs13042395 genotype and esophageal cancer risk (adjusted OR = 0.99, 95 % CI = 0.63-1.57 for C20orf54 rs13042395 TT vs. CC). Stratified analyses indicated a significantly increased risk of esophageal cancer associated with the PLCE1 rs2274223 AG genotype was more evident among females, younger patients and never drinkers, compared with the PLCE1 rs2274223 AA genotypes. Stratified analyses also indicated a significantly increased risk of esophageal cancer associated with the PLCE1 rs2274223 GG genotype was more evident among never smokers and never drinkers compared with the PLCE1 rs2274223 AA genotypes. These findings indicated that functional polymorphisms PLCE1 rs2274223 might contribute to esophageal cancer susceptibility.     

The physiological role of riboflavin transporter and involvement of FMN-riboswitch in its gene expression in Corynebacterium glutamicum

Riboflavin is a precursor of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which work as cofactors of numerous enzymes. Understanding the supply system of these cofactors in bacteria, particularly those used for industrial production of value added chemicals, is important given the pivotal role the cofactors play in substrate metabolism. In this work, we examined the effect of disruption of riboflavin utilization genes on cell growth, cytoplasmic flavin levels, and expression of riboflavin transporter in Corynebacterium glutamicum. Disruption of the ribA gene that encodes bifunctional GTP cyclohydrolase II/3,4-dihydroxy-2-butanone 4-phosphate synthase in C. glutamicum suppressed growth in the absence of supplemental riboflavin. The growth was fully recovered upon supplementation with 1 μM riboflavin, albeit at reduced intracellular concentrations of FMN and FAD during the log phase. Concomitant disruption of the ribA and ribM gene that encodes a riboflavin transporter exacerbated supplemental riboflavin requirement from 1 μM to 50 μM. RibM expression in FMN-rich cells was about 100-fold lower than that in FMN-limited cells. Mutations in putative FMN-riboswitch present immediately upstream of the ribM gene abolished the FMN response. This 5′UTR sequence of ribM constitutes a functional FMN-riboswitch in C. glutamicum.     

Genetic variants at 4q21, 4q23 and 12q24 are associated with esophageal squamous cell carcinoma risk in a Chinese population

A recently published genome-wide association study (GWAS) in European populations identified several loci at 4q21, 4q23 and 12q24 that were associated with risk of upper aerodigestive tract (UADT) cancers, including esophageal squamous cell carcinoma (ESCC). In the current study, we conducted a case–control study in a Chinese population including 2,139 ESCC cases and 2,273 controls to evaluate the associations of six reported single nucleotide polymorphisms (SNPs) (rs1494961, rs1229984, rs1789924, rs971074, rs671 and rs4767364) with risk of ESCC. We found significant association with risk of ESCC for four SNPs, including rs1494961 in HEL308 at 4q21 [odds ratio (OR) = 1.15, 95 % confidence interval (CI) = 1.05–1.26], rs1229984 in ADH1B at 4q23 (OR = 1.24, 95 % CI = 1.13–1.36) and rs1789924 near ADH1C at 4q23 (OR = 1.20, 95 % CI = 1.03-1.39), and rs671 in ALDH2 at 12q24 (OR = 0.83, 95 % CI = 0.75–0.91). Combined analysis of these four SNPs showed a significant allele-dosage effect on ESCC risk for individuals with different number of risk alleles (P trend = 2.23 × 10^?11). Compared with individuals with “0–2” risk allele, those carrying “3”, “4” or “5 or more” risk alleles had 1.42-, 1.66-, or 1.76-fold risk of ESCC, respectively. Thus, our findings indicate that rs1494961 at 4q21, rs1229984 and rs1789924 at 4q23, and rs671 at 12q24 may be used as genetic biomarkers for ESCC susceptibility in Chinese population.     

Overexpression of S-adenosylhomocysteine hydrolase (SAHH) in esophageal squamous cell carcinoma (ESCC) cell lines: effects on apoptosis,migration and adhesion of cells

S-adenosylhomocysteine hydrolase (SAHH) is the sole enzyme that catalyses the hydrolysis of S-adenosylhomocysteine (SAH) in methylation reaction. Previous studies have shown that its inhibition or deficiency leads to several human disorders such as severe coagulopathy, hepatopathy and myopathy. However, the effects of SAHH on esophageal squamous cell carcinoma (ESCC) cells have not been explored so far. To determine whether SAHH is involved in carcinogenesis of the esophagus, we investigated the expression of SAHH in ESCC and normal esophageal epithelial cells and found that SAHH was downregulated in ESCC cells compared with normal esophageal epithelial cells (P < 0.05). The overexpressed SAHH in ESCC cells promoted cell apoptosis, inhibited cell migration and adhesion, but did not affect the cell proliferation and cell cycle. Furthermore, an interaction of SAHH with receptor of activated C kinase 1 (RACK1) protein was detected by coimmunoprecipitation and an increased RACK1, which is caused by overexpression of SAHH, was verified by Western blotting. The findings mentioned above demonstrate that SAHH can promote apoptosis, inhibit migration and adhesion of ESCC cells suggesting that it may be involved in carcinogenesis of the esophagus.     

Mast cells expressing interleukin 17 in the muscularis propria predict a favorable prognosis in esophageal squamous cell carcinoma

The proinflammatory cytokine interleukin 17 (IL-17) is considered to play a crucial role in diverse human tumors; however, its role in disease progression remains controversial. This study investigated the cellular source and distribution of IL-17 in esophageal squamous cell carcinoma (ESCC) in situ and determined its prognostic value. Immunohistochemistry, immunofluorescence and immunoelectron microscopy were used to identify IL-17-expressing cells in ESCC tissues, paying particular attention to their anatomic localization. Kaplan–Meier analysis and Cox proportional hazards regression models were applied to estimate overall survival in 215 ESCC patients with long-term follow-up (>10 years). The results showed that mast cells, but not T cells or macrophages, were the predominant cell type expressing IL-17 in ESCC tissues. Unexpectedly, these IL-17⁺ cells were highly enriched in the muscularis propria rather than the corresponding tumor nest (p < 0.0001). The density of IL-17⁺ cells in muscularis propria was inversely associated with tumor invasion (p = 0.016) and served as an independent predictor of favorable survival (p = 0.007). Moreover, the levels of IL-17⁺ cells in muscularis propria were positively associated with the density of effector CD8⁺ T cells and activated macrophages in the same area (both p < 0.0001). This finding suggested that mast cells may play a significant role in tumor immunity by releasing IL-17 at a previously unappreciated location, the muscularis propria, in ESCC tissues, which could serve as a potential prognostic marker and a novel therapeutic target for ESCC.     

Down-regulation of platelet-derived growth factor-D expression blockades NF-κB pathway to inhibit cell proliferation and invasion as well as induce apoptosis in esophageal squamous cell carcinoma

Substantial evidence has demonstrated that platelet-derived growth factor-D (PDGF-D) is tightly associated with the development and progression of tumors. However, its biological functions in esophageal squamous cell carcinoma (ESCC) remain to be delineated. In this study, we found that expressions of PDGF-D mRNA and protein in ESCC tissues and cells were significantly higher than that in normal esophageal epithelial tissues (P < 0.05), further investigation showed that PDGF-D protein level in EC1 cells was obviously higher than those in EC9706 and Eca109 cells (P < 0.05). Elevated PDGF-D level was closely associated with TNM staging, tumor differentiation and lymph node metastasis (P < 0.05), but not related to the patients’ age and gender (P > 0.05). In addition, down-regulation of PDGF-D expression markedly inhibited proliferation, reduced invasion and induced apoptosis in EC1 cells. More importantly, reduced PDGF-D level evoked the down-regulation of p65 and p-IκBα proteins and elevation of IκBα protein of NF-κB pathway, accompanied with the decreases of bcl-2 and MMP-9 protein expressions and increases of bax protein level and caspase-3 activities. Correctively, our data suggest that PDGF-D plays pivotal roles in the development and progression of ESCC, and combinations with PDGF-D and NF-κB pathway may be effective and feasible molecular targets for therapy of ESCC.     

Aberrant promoter methylation and reduced expression of p16 gene in esophageal squamous cell carcinoma from Kashmir valley: a high-risk area

Esophageal squamous cell carcinoma (ESCC) is one of the most prevalent cancer in Jammu and Kashmir region of India and has multi-factorial etiology involving dietary habits, genetic factors, and gene environmental interactions. Inactivation of the p16 gene expression by aberrant promoter methylation plays an important role in the progression of esophageal carcinoma. In the present investigation, we have studied the role of p16 promoter methylation in 69 histopathologically confirmed ESCC tissues and compared it with corresponding normal adjacent tissues for DNA methylation in the CpG island in the p16 promoter region by methylation-specific polymerase chain reaction (MSP) and p16 protein expression by immunoblotting. The results showed loss of p16 expression in 67% (46/69) of tumor tissues compared to only 3% in control tissues (2/69). Promoter methylation was observed in 52% (36/69) of tumor tissues and it gradually increased with the increasing severity of histological grades of the cancer (P = 0.0001). Loss of p16 expression with promoter methylation was observed in 26 of 36 cases (72%). Analysis of patients dietary habits revealed a strong association between promoter methylation and high consumption of hot salted tea (P < 0.05) which is a most favourite drink commonly consumed by Kashmiri people.     

Serum miR-1297: a promising diagnostic biomarker in esophageal squamous cell carcinoma

We aimed to value the diagnostic potential of serum miR-1297 in esophageal squamous cell cancer (ESCC). Its expression level was detected in 156 pairs of patients with ESCC and healthy volunteers using quantitative real-time polymerase chain reaction (qRT-PCR) method. It was statistically decreased in ESCC patients compared with healthy controls. AUC based on serum miR-1297 was 0.840?±?0.035 in discovery group and 0.837?±?0.034 in validation group. Further analysis on early-stage patients revealed that the AUC was 0.819?±?0.053 in discovery group and 0.814?±?0.044 in validation group. Its sensitivity and specificity were promising. In conclusion, serum miR-1297 can serve as an ideal indicator for the diagnosis of ESCC.     

Enhanced riboflavin production by recombinant Bacillus subtilis RF1 through the optimization of agitation speed

Dissolved oxygen is one of the most important bioprocess parameters that could affect cell growth and product formation, and it is easy to control by changing agitation speed. In this work, the effects of agitation speed on the performance of riboflavin production by recombinant Bacillus subtilis RF1 was investigated in fed-batch fermentation. The lower agitation speed (600 rpm) was beneficial for cell growth and riboflavin biosynthesis in the initial phase of fermentation process. While, during the later phase, higher agitation speed (900 rpm) was favor for cell growth and riboflavin biosynthesis. Thus, a two-stage agitation speed control strategy was proposed based on kinetic analysis, in which the agitation speed was controlled at 600 rpm in the first 26 h and then switched to 900 rpm to maintain high μ for cell growth and high q _p for riboflavin production during the entire fermentation process. However, it was observed that a sharp increase of agitation speed resulted in an adverse effect on cell growth and riboflavin synthesis within a short time. To avoid this phenomenon, a multi-stage agitation speed control strategy was set up based on the two-stage control strategy, the maximum concentration of riboflavin reached 9.4 g l^?1 in 48 h with the yield of 0.051 g g^?1 by applying this strategy, which were 20.5 and 21.4 % over the best results controlled by constant agitation speeds.     

The novel human MRC1 gene polymorphisms are associated with susceptibility to pulmonary tuberculosis in Chinese Uygur and Kazak populations

The MRC1 gene, encoding the human mannose receptor (MR), is a member of the C-type lectin receptors family. MR can recognize and bind to Mycobacterium tuberculosis by the extracellular structure, and play a role in antigen-presenting and maintaining a stable internal environment. This study aimed to investigate potential associations of SNPs in exon 7 of the MRC1 gene with pulmonary tuberculosis (TB). G1186A, G1195A, T1212C, C1221G, C1303T and C1323T were genotyped using PCR and DNA sequencing in 595 Chinese Uygur and 513 Kazak subjects. In the Uygur, the frequency of allele G (P = 0.031, OR = 1.29, 95 % CI = 1.02–1.62) and AA genotype (P = 0.033, OR = 1.64, 95 % CI = 1.04–2.60) for G1186A was lower in the pulmonary TB than healthy control and were significantly correlated with pulmonary TB. After adjustment for age and gender, G1186A was found to be additive models in association with pulmonary TB (P = 0.04, OR = 1.27, 95 % CI = 1.01–1.60). By calculating linkage disequilibrium, the frequency of haplotype GGTCCT (P = 0.032, OR = 0.75, 95 % CI = 0.57–0.97) and GGTCCC (P = 0.044, OR = 0.57, 95 % CI = 0.33–0.99) was significantly associated with pulmonary TB. No association was found between other SNPs and pulmonary TB. In the Kazak, all SNPs were not associated with pulmonary TB. Our results suggest that genetic factors play an important role in susceptibility to pulmonary TB at the individual level, and provide an experimental basis to clarify the pathogenesis of pulmonary TB.     

Epigenetic silencing of miR-203 in Kazakh patients with esophageal squamous cell carcinoma by MassARRAY spectrometry

Dysregulation of miR-203 by promoter methylation is associated with the development of various cancers. We aimed to explore the underlying link between promoter methylation and miR-203 expression in Kazakh esophageal squamous cell carcinoma (ESCC). MassARRAY® System spectrometry was used to quantitatively analyze the DNA methylation of 32 CpG sites within miR-203 in 99 Kazakh ESCC and 46 normal esophageal tissues (NETs) with similar population characteristics. We conducted real-time PCR to detect miR-203 expression levels and evaluated their association with methylation. Eleven CpG units within miR-203 promoter were frequently hypermethylated in ESCC compared with NETs (P < 0.05). The hypermethylation of several CpG units positively correlated with age, lower esophagus, constrictive type of ESCC, and moderately differentiated ESCC. Given the involvement of human papillomavirus (HPV) in etiology of ESCC was confirmed from our previous reports, herein we found that CpG units within miR-203 in HPV16-positive ESCC are more heavily methylated. Furthermore, miR-203 expression showed a nearly 4.5-fold decrease in ESCC than NETs (0.206 ± 0.336 vs. 0.908 ± 1.424, P < 0.001) and was significantly associated with lymph node metastasis (P = 0.012). The expression of miR-203 with 11 completely hypermethylated CpG units was approximately 6.5-fold lower than that with at least 1 unmethylated CpG unit (P < 0.001) and especially the CpG_15.16 and CpG_31.32 with higher methylation levels in ESCC tissues exhibited lower expression levels of miR-203, which indicated a reverse association between miR-203 methylation and expression. Hypermethylated miR-203 is a potential biomarker and targeted delivery of miR-203 could therefore serve as a preventive or therapeutic strategy for Kazakh ESCC.