Molecular dynamics based improvement of the solubilizing self-cleavable tag Zbasic-ΔI-CM application in the preparation of recombinant proteins in Escherichia coli.

Zbasic-ΔI-CM is a novel intein-based self-cleavable tag we developed to accelerate the soluble expression of recombinant proteins in Escherichia coli (E. coli). Previously we found that intein activity could be interfered by its flanking exteins, and thus reducing the production efficiency and final yield. In this work, we used CXC-chemokine 9 (CXCL9) as a model C-extein, which fusion with Zbasic-ΔI-CM showed high intein activity. When the fusion protein got soluble expression, CXCL9 was released immediately and purified directly from cell lysis supernatant. The results demonstrated that Zbasic-ΔI-CM tag had successfully mediated the efficient production of high-quality CXCL9 with reduced time and resources consumption in comparison with inclusion bodies expression. Molecular dynamics simulations suggested that the improved cleavage activity of Zbasic-ΔI-CM upon fusion with CXCL9 may be due to the higher dynamics of the first half loop and stabilization of the second half loop of intein. Our results proved that the self-cleavable Zbasic-ΔI-CM mediated soluble expression could be a feasible process for cytokines like CXCL9, thus of attractive potentials for production of therapeutic proteins using E. coli expression system.

A single nucleotide polymorphism in the Epstein-Barr virus genome is strongly associated with a high risk of nasopharyngeal carcinoma.

BACKGROUND: Epstein-Barr virus (EBV) commonly infects the general population and has been associated with nasopharyngeal carcinoma (NPC), which has a high incidence in certain regions. This study aimed to address how EBV variations contribute to the risk of NPC. METHODS: Using logistic regression analysis and based on the sequence variations at EBV-encoded RPMS1, a multi-stage association study was conducted to identify EBV variations associated with NPC risk. A protein degradation assay was performed to characterize the functional relevance of the RPMS1 variations. RESULTS: Based on EBV-encoded RPMS1 variations, a single nucleotide polymorphism (SNP) in the EBV genome (locus 155391: G>A, named G155391A) was associated with NPC in 157 cases and 319 healthy controls from an NPC endemic region in South China [P < 0.001, odds ratio (OR) = 4.47, 95% confidence interval (CI) 2.71-7.37]. The results were further validated in three independent cohorts from the NPC endemic region (P < 0.001, OR = 5.20, 95% CI 3.18-8.50 in 168 cases vs. 241 controls, and P < 0.001, OR = 5.27, 95% CI 4.06-6.85 in 726 cases vs. 880 controls) and a non-endemic region (P < 0.001, OR = 7.52, 95% CI 3.69-15.32 in 58 cases vs. 612 controls). The combined analysis in 1109 cases and 2052 controls revealed that the SNP G155391A was strongly associated with NPC (P(combined) < 0.001, OR = 5.27, 95% CI 4.31-6.44). Moreover, the frequency of the SNP G155391A was associated with NPC incidence but was not associated with the incidences of other EBV-related malignancies. Furthermore, the protein degradation assay showed that this SNP decreased the degradation of the oncogenic RPMS1 protein. CONCLUSIONS: Our study identified an EBV variation specifically and significantly associated with a high risk of NPC. These findings provide insights into the pathogenesis of NPC and strategies for prevention.

Evaluation study of single-anastomosis duodenal-ileal bypass with sleeve gastrectomy in the treatment of Chinese obese patients based on efficacy and nutrition.

To evaluate the efficacy and nutrition of single-anastomosis duodenal-ileal bypass with sleeve gastrectomy (SADI-S) in Chinese obese patients in the first postoperative year. Clinical data of 66 obese patients who underwent SADI-S surgery at China-Japan Union Hospital of Jilin University from November 2018 to May 2022 were retrospectively collected. The weight, body mass index (BMI), percentage of excess weight loss (%EWL), and percentage of total weight loss (%TWL) were recorded at 3, 6, and 12 months after surgery. Moreover, metabolic disease remission and nutrient deficiencies were assessed at 1 year postoperatively. Overall, 66 patients (38 males and 28 females) were recruited, with a mean age of 35 (18-61) years and an average preoperative BMI of 42.94 kg/m2. Before surgery, 38 patients had type 2 diabetes mellitus (T2DM), 46 patients had hyperuricemia (HUA), 45 patients had hypertension (HTN), 35 patients had hyperlipidemia, 12 patients had hypercholesterolemia, 12 patients had hyper-low-density lipoproteinemia, and 14 patients had gastroesophageal reflux disease symptoms (GERD). All patients had undergone a DaVinci robotic or laparoscopic SADI-S surgery, and none converted to laparotomy or died. Four patients developed postoperative complications and were cured and discharged after conservative treatment or surgical treatment. At 3, 6 and 12 months, the average %EWL was 62.07 ± 26.56, 85.93 ± 27.92, and 106.65 ± 29.65%, %TWL was 22.67 ± 4.94, 32.10 ± 5.18, and 40.56 ± 7.89%, respectively. Fasting blood glucose (FBG), glycated hemoglobin (HbA1c), uric acid (UA), triglycerides (TG), blood pressure (BP), and other indexes were significantly lower after one year post-surgery compared with the preoperative period (P < 0.05). The remission rates of T2DM, HUA, HTN, hypertriglyceridemia, hypercholesterolemia, and hyper-low-density lipoproteinemia 1 year after surgery were 100, 65.2, 62.2, 94.3, 100, and100%, respectively. One year after surgery, the remission rate of GERD was 71.4% (10/14), the rate of new occurrence of GERD was 12.1% (8/66), and the overall incidence rate was 18.2% (12/66). Except for vitamin B12(vit B12), the other nutrient indexes were significantly decreased after 1 year of surgery relative to levels before surgery (P < 0.05). The deficiency rates for vitamin A (vit A), vitamin E (vit E), zinc ion (Zn), and folic acid (FA) were higher (45.5, 25.8, 24.2, and 16.7%, respectively); however, there were no related clinical symptoms. SADI-S had significant effects on weight loss and metabolic disease remission. The main nutrient deficiencies after SADI-S were vit A, vit E, Zn, and FA deficiencies. The long-term efficacy and safety of SADI-S warrant further follow-up.

Efficacy and safety of single-anastomosis duodenal-ileal bypass with sleeve gastrectomy for the treatment of Chinese T2D patients with obesity.

BACKGROUND: Bariatric surgery is the most effective treatment for obese T2D. As one of the most effective bariatric surgeries, SADI-S was recently introduced in China, and there is limited evidence of its efficacy and safety in the treatment of obese T2D. The aim of this study is to investigate the safety and efficacy of SADI-S in the treatment of obese T2D in China. METHODS: The clinical data of 32 obese T2D patients who underwent SADI-S was included in this study. Changes in weight-related indicators, diabetes-related indicators, and patient nutritional outcomes were analyzed. RESULTS: SADI-S was conducted successfully in all of the 32 cases without conversion to laparotomy or death. The incidence of surgical complications was 15.6% (5/32). The major complication rate was 6.3% (2/32). At 1 year after surgery, the BMI (kg/m2) significantly decreased from 40.8 ± 7.4 to 23.9 ± 2.9 (P < 0.05) and the mean HbA1c significantly decreased from 8.5% (6.4-11.5) to 5.0% (3.8-5.6) (P < 0.05). At 2 years after surgery, the BMI (kg/m2) significantly decreased to 24.9 ± 2.4 (P < 0.05) and the mean HbA1c significantly decreased to (4.8 ± 0.4)% (P < 0.05). The %TWL was (40.4 ± 6.5)% and (42.9 ± 4.9)% at 1 year and 2 years, respectively. The complete remission rates for T2D were both 100% at 1 year and 2 years. Triglyceride levels were significantly improved compared with preoperative, from (3.2 ± 3.0) mmol/L to (1.0 ± 0.3) mmol/L (P < 0.05), but there was no significant difference in other nutritional outcomes. CONCLUSION: The SADI-S has excellent curative effect in the treatment of Chinese obese T2D, but the operation is challenging and the complication rate is high. Its long-term efficacy and safety require further study.

Based on Propensity Matching Scores: Comparison of the Efficacy of Two Kinds of Bariatric Surgery for Obese Type 2 Diabetes.

PURPOSE: To compare the efficacy of OADS and SG in treating obese T2D in China. MATERIALS AND METHODS: We included 99 obese Chinese patients with T2D undergoing OADS or SG with a 1-year postoperative follow-up from January 2014 to October 2021. Using the propensity score matching (PSM) method, patients from both groups were matched 1:1. Outcomes for losing weight, controlling diabetes, and nutrition were then determined. RESULTS: There were 32 patients in each group after using the PSM method, and there was no statistically significant difference between the two groups in terms of all the baseline indicators (P > 0.05). When comparing weight loss outcomes, the OADS group outperformed the SG group in terms of change in BMI and %TWL, with statistically significant differences [15.0 (8.1-26.6) kg/m2, 10.0 (4.10-23.5) kg/m2 P = 0.001; 38.5% ± 6.7%, 29.5% ± 9.4%, P = 0.000]. When comparing the efficacy of diabetes, the OADS group outperformed the SG group in terms of HbA1C and complete remission of diabetes, with statistically significant differences [5.1 (3.8-5.6)%, 5.4 (4.3-7.9)%, P = 0.001; 100%, 75%, P = 0.005]. Besides, the incidence of postoperative zinc deficiency in the OADS group was significantly higher than in the SG group (P = 0.019) and there was no significant difference in other postoperative nutritional outcomes between the two groups. CONCLUSION: Although OADS and SG are both effective in the treatment of obese T2D, OADS performs better. Besides, the long-term efficacy of both needs to be recorded at subsequent follow-up.

LncRNA NORAD defects deteriorate the formation of age-related macular degeneration.

Long noncoding RNAs (lncRNAs) play important roles in the development of age-related macular degeneration (AMD). However, the effect of long non-coding RNA activated by DNA damage (NORAD) on AMD remains unknown. This study aimed to investigate the effect of NORAD on RPE cell senescence and degeneration. Irradiated adult retinal pigment epithelial cell line-19 (ARPE-19) and sodium iodate-treated mice were used as in vitro and in vivo AMD models. Results showed that irradiation-induced AMD characteristics of ARPE-19 and NORAD-knockdown aggravated cell cycle arrest in the G2/M phase, cell apoptosis and cell senescence along with the increased expression of phosphorylated P53 (p-P53) and P21. AMD factors C3, ICAM-1, APP, APOE, and VEGF-A were also increased by NORAD-knockdown. Moreover, NORAD-knockdown increased irradiation-induced reduction of mitochondrial homeostasis factors, (i.e., TFAM and POLG) and mitochondrial respiratory chain complex genes (i.e., ND1 and ND5) along with mitochondrial reactive oxygen species (ROS). We also identified a strong interaction of NORAD and PGC-1α and sirtuin 1 (SIRT1) in ARPE-19; that is, NORAD knockdown increases the acetylation of PGC-1α. In NORAD knockout mice, NORAD-knockout accelerated the sodium iodate-reduced retinal thickness reduction, function impairment and loss of retinal pigment in the fundus. Therefore, NORAD-knockdown accelerates retinal cell senescence, apoptosis, and AMD markers via PGC-1α acetylation, mitochondrial ROS, and the p-P53-P21signaling pathway, in which NORAD-mediated effect on PGC-1α acetylation might occur through the direct interaction with PGC-1α and SIRT1.

The Therapeutic Effect of Coriolus versicolor Fruiting Body on STZ-Induced ICR Diabetic Mice.

Coriolus versicolor is a natural drugs which has many pharmacological effects such as antitumor and enhanced immune activity. This paper studies the therapeutic effect of Coriolus versicolor fruiting body (CVFB) on streptozotocin (STZ)-induced Institute of Cancer Research (ICR) diabetic mice, the STZ solution was administered intraperitoneally at a dose of 150 mg/kg after fasting the mice, and ICR mice with fasting blood glucose >16.7 mmol/l were selected for research. Metformin was the positive control, and the dose of CVFB powder (1000 mg/kg, 2000 mg/kg, and 4000 mg/kg) for 28 consecutive days by gavage. The serum and liver of mice were collected for relevant index content testing. The results showed that CVFB can control or reduce the fasting blood glucose of mice and accelerate the rate of glucose metabolism, can reduce the levels of total cholesterol (T-CHO), triglyceride (TG), and high-density lipoprotein cholesterol (HDL-C) in mice, and regulate the abnormal symptoms of blood lipid metabolism commonly found in diabetes. It can increase the activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) antioxidant enzymes and enhance the ability of antioxidative stress in diabetic mice. In the H&E staining and apoptosis experiments of pancreatic tissue, CVFB can greatly reduce the inflammatory factors present in islets, increase the islet cells, and reduce the apoptotic rate caused by diabetes. All data confirmed the therapeutic effect of CVFB on diabetic ICR mice. The present study provides a scientific basis for the development of drugs for the prevention and treatment of diabetes, it is of great significance to the in-depth study of Coriolus versicolor.

Decarboxylative oxidation-enabled consecutive C-C bond cleavage.

The selective cleavage of C-C bonds is of fundamental interest because it provides an alternative approach to traditional chemical synthesis, which is focused primarily on building up molecular complexity. However, current C-C cleavage methods provide only limited opportunities. For example, selective C(sp3)-C(sp3) bond cleavage generally relies on the use of transition-metal to open strained ring systems or iminyl and alkoxy radicals to induce ß-fragmentation. Here we show that by merging photoredox catalysis with copper catalysis, we are able to employ α-trisubstituted carboxylic acids as substrates and achieve consecutive C-C bond cleavage, resulting in the scission of the inert ß-CH2 group. The key transformation relies on the decarboxylative oxidation process, which could selectively generate in-situ formed alkoxy radicals and trigger consecutive C-C bond cleavage. This complicated yet interesting reaction might help the development of other methods for inert C(sp3)-C(sp3) bond cleavage.

A single nucleotide polymorphism in the matrix metalloproteinase 2 promoter is closely associated with high risk of nasopharyngeal carcinoma in Cantonese from southern China.

Matrix metalloproteinase 2 (MMP2) has been shown to play an important role in several steps of cancer development. The -1306C/T polymorphism of the MMP2 gene displays a strikingly lower promoter activity than the T allele, and the CC genotype in the MMP2 promoter has been reported to associate with the development of several cancers. To assess the contribution of the MMP2 -1306C/T polymorphism to the risk of nasopharyngeal carcinoma (NPC), we conducted a case-control study and analyzed MMP2 genotypes in 370 patients with NPC and 390 frequency-matched controls using real-time PCR-based TaqMan allele analysis. We found that subjects with the CC genotype had an increased risk (OR = 1.55, 95% CI = 1.05-2.27) of developing NPC compared to those with the CT or TT genotypes. Furthermore, we found that the risk of NPC was markedly increased in subjects who were smokers (OR = 15.04, 95% CI = 6.65-33.99), heavy smokers who smoked ≥ 20 pack-years (OR = 18.66, 95% CI = 7.67-45.38), or young (<60 years) at diagnosis (OR = 1.52, 95% CI = 1.01-2.29). Our results provide molecular epidemiological evidence that the MMP2 -1306C/T promoter polymorphism is associated with NPC risk, and this association is especially noteworthy in heavy smokers.

A hemolysin secretion pathway-based novel secretory expression platform for efficient manufacturing of tag peptides and anti-microbial peptides in Escherichia coli.

BACKGROUND: Although Escherichia coli has been widely used for the expression of exogenous proteins, the secretory expression in this system is still a big obstacle. As one of the most important secretion pathways, hemolysin A (HlyA) system of E. coli can transport substrates directly from the cytoplasm to extracellular medium without the formation of any periplasmic intermediate, making it an ideal candidate for the development of the secretory production platform for exogenous proteins. RESULTS: In this work, we developed a novel production platform, THHly, based on the HlyA secretion system, and explored its applications in the efficient preparation and quick detection of tag peptides and anti-microbial peptides. In this novel platform the signal sequence of HlyA is fused to the C-terminal of target peptide, with Tobacco Etch Virus (TEV) protease cleavage site and 6*His tag between them. Five tag peptides displayed good secretory properties in E. coli BL21 (DE3), among which T7 tag and S tag were obtained by two rounds of purification steps and TEV cleavage, and maintained their intrinsic immunogenicity. Furthermore, Cecropin A and Melittin, two different types of widely explored anti-microbial peptides, were produced likewise and verified to possess anti-microbial/anti-tumor bioactivities. No significant bacterial growth inhibition was observed during the fusion protein expression, indicating that the fusion form not only mediated the secretion but also decreased the toxicity of anti-microbial peptides (AMPs) to the host bacteria. To the best of our knowledge, this is the first report to achieve the secretory expression of these two AMPs in E. coli with considerable potential for manufacturing and industrialization purposes. CONCLUSIONS: The results demonstrate that the HlyA based novel production platform of E. coli allowed the efficient secretory production and purification of peptides, thus suggesting a promising strategy for the industrialized production of peptide pharmaceuticals or reagents.

COX7A1 suppresses the viability of human non-small cell lung cancer cells via regulating autophagy.

COX7A1 is a subunit of cytochrome c oxidase, and plays an important role in the super-assembly that integrates peripherally into multi-unit heteromeric complexes in the mitochondrial respiratory chain. In recent years, some researchers have identified that COX7A1 is implicated in human cancer cell metabolism and therapy. In this study, we mainly explored the effect of COX7A1 on the cell viability of lung cancer cells. COX7A1 overexpression was induced by vector transfection in NCI-H838 cells. Cell proliferation, colony formation and cell apoptosis were evaluated in different groups. In addition, autophagy was analyzed by detecting the expression level of p62 and LC3, as well as the tandem mRFP-GFP-LC3 reporter assay respectively. Our results indicated that the overexpression of COX7A1 suppressed cell proliferation and colony formation ability, and promoted cell apoptosis in human non-small cell lung cancer cells. Besides, the overexpression of COX7A1 blocked autophagic flux and resulted in the accumulation of autophagosome via downregulation of PGC-1α and upregulation of NOX2. Further analysis showed that the effect of COX7A1 overexpression on cell viability was partly dependent of the inhibition of autophagy. Herein, we identified that COX7A1 holds a key position in regulating the development and progression of lung cancer by affecting autophagy. Although the crosstalk among COX7A1, PGC-1α and NOX2 needs further investigation, our study provides a novel insight into the therapeutic action of COX7A1 against human non-small cell lung cancer.

Paracrine action of human placental trophoblast cells attenuates cisplatin-induced acute kidney injury.

AIMS: The action of cell-based therapy against acute kidney injury (AKI) has been demonstrated by different groups for years. However, which kind of cells hold best therapeutic effect remains unclear. In this study, we mainly explored whether human placental trophoblast cells hold the potential to be applied in AKI therapy. MAIN METHODS: To study the renoprotective effect, the trophoblast cells were isolated from human placenta and characterized by flow cytometry first. The AKI model was induced using cisplatin in NOD-SCID mice. The therapeutic effect of human placental trophoblast cells on renal function, apoptosis and inflammation were analyzed respectively. KEY FINDINGS: The administration of trophoblast cells isolated from human placenta improved the pathological changes of kidney tissues and renal dysfunction induced by cisplatin. In addition, the placental trophoblast cell-based treatment also showed anti-apoptotic effect and decreased the level of apoptotic genes (Bax and Caspase 3) expression in damaged kidney tissues obviously. All of the inflammatory components (MCP-1, IL-10 and RANTES) in kidney tissues were down-regulated with the therapy of placental trophoblast cells. Further analysis indicated that the paracrine effects of human placental trophoblast cells may hold a key position in the AKI therapy process. SIGNIFICANCE: In this study, we mainly developed a novel therapeutic strategy to treat cisplatin-induced AKI with human placental trophoblast cells. Even though the detailed mechanism and the optimizations of this cell-based therapy still need further investigation, the application of placental trophoblast cell holds special potential in the treatment of patients with AKI.

5­Azacytidine treatment results in nuclear exclusion of DNA methyltransferase­1, as well as reduced proliferation and invasion in human cytomegalovirus­infected glioblastoma cells.

Glioblastoma (GBM) is the most aggressive form of brain tumor in adults, with a devastating outcome. Emerging evidence shows that human cytomegalovirus (HCMV) proteins and nucleic acids are present in GBM tissues. DNA methylation is important for the initiation and progression of cancer and is an established host response against invading nucleic acids. The expression and localization of DNA methyltransferase 1 (DNMT­1) was assessed, and the effects of DNA methylation inhibitor 5­azacytidine (5AZA) were analyzed in the context of the viral replication, proliferation and invasion capacities of HCMV­infected GBM U343MG cells. In addition, the expression of various HCMV proteins and DNMT­1 was examined in GBM tissue specimens obtained from five patients. DNMT­1 was localized in the nucleus of cells expressing HCMV­immediate early, whereas in cells expressing HCMV­glycoprotein gB (gB), extranuclear/cytoplasmic localization was observed. This was also observed in vitro in U343MG cells. In addition, DNMT­1 was localized to the extranuclear/cytoplasmic space of cells lining blood vessel walls within the GBM tumors. Treatment of infected U343MG cells with 5AZA did not affect viral replication, but reduced cell invasion and proliferation (P=0.05 and P<0.0001, respectively). However, 5AZA treatment of uninfected cells did not affect cell invasion (P=0.09), but proliferation was significantly reduced (P<0.0001). These findings may be of importance in further investigations aimed at using DNA methylation and viral inhibitors in GBM therapy.

A genome-wide scan suggests a susceptibility locus on 5p 13 for nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) occurs with high frequency in Southeast Asian populations. The high prevalence and familial clustering of NPC in these populations suggest that genetic factors may contribute to the increased cancer risk by affecting susceptibility. The aim of the present study was to map chromosomal loci linked to susceptibility genes predisposing for NPC. We carried out a genome-wide scan by multipoint affected-only allele-sharing methods in 15 Chinese NPC families with two to six affected members per family. The families were from the Guangdong province in the south of China, where the highest risk of NPC is documented. These samples were genotyped using 800 microsatellite markers covering all autosomal chromosomes with an average marker distance of 5 cM. Using multipoint linkage analysis, four loci (2q, 5p, 12p, and 18p) showed LOD scores above 1.5. After genotyping additional markers in these four regions, only one locus on 5p13 showed an increased LOD of 2.1. In further haplotype analysis, affected individuals in six families shared three marker haplotypes between D5S674 and D5S418. In conclusion, a region on 5p13 may harbor a susceptibility gene for NPC.

A major EBNA1 variant from Asian EBV isolates shows enhanced transcriptional activity compared to prototype B95.8.

Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) has an instrumental role in maintaining EBV latent infection by controlling EBV episome replication and regulating viral transcription. It is a ubiquitously expressed protein during latent viral infection and in EBV-associated tumors. The EBNA1 C-terminus interacts functionally with the Qp and Cp that control viral gene expression in latency I/II and III, respectively. EBNA1 has been classified into five subtypes due to sequence variation in the DNA-interacting C-terminus. By DNA sequence analysis of its C-terminus, we detected a main sub-variant (V-val-v1) of EBNA1 with valine located in both positions 487 and 528 from matched samples including NPC biopsies and peripheral blood taken from Vietnamese (9), Chinese (12) NPC patients and healthy donors (5). In the FR-region of oriP from nine NPC biopsies from Vietnam we also frequently found substitutions, deletions and variable numbers of repeats. Using a luciferase reporter system, EBNA1 and FR both derived from Asian isolates induced higher transcriptional activity than those from B95-8 virus.

Intermolecular disulfide bonds between unpaired cysteines retard the C-terminal trans-cleavage of Npu DnaE.

Npu DnaE is a naturally occurred split intein possessing robust trans-splicing activity and could be engineered to perform rapid C-terminal cleavage module by a single mutation D118G. Unfortunately, however, for this modified selfcleaving module, reducing agents were needed to trigger the rapid cleavage, which prevents the utilization in purification of disulfide bonds containing recombinant proteins. In this study, we demonstrated that the unpaired cysteine residues in Npu DnaE tend to form disulfide bonds, and contributed to the reduction of the cleavage under non-reducing conditions. This redox trap can be disrupted by site-directed mutation of these unpaired cysteines. The results further indicated that the position 28 and 59 may play certain roles in the correct folding of the active conformation.

Aberrant methylation of CDH13 gene in nasopharyngeal carcinoma could serve as a potential diagnostic biomarker.

CDH13 encodes a cell adhesion molecule, H-cadherin. In this study, we examined CDH13 methylation in nasopharyngeal carcinoma (NPC). Methylation specific PCR results showed that CDH13 was methylated in 20% (1/5) NPC cell lines, 100% (2/2) NPC xenografts and 89.7% (52/58) of the NPC primary tumors, while only methylated in 10% (1/10) normal nasopharyngeal epithelia (P<0.05). CDH13 expression in NPC cell lines and NPC xenografts analyzed by RT-PCR showed that expressions of CDH13 were reversely correlated with their methylation status. In CDH13-silenced cell line, demethylating agent 5-aza-deoxycytidine could dramatically restore CDH13 expression. Taken together, CDH13 promoter is aberrantly methylated in NPC both in vitro and in vivo, and promoter methylation plays a pivotal role in the silencing of H-cadherin expression. Furthermore, the high sensitivity (81%) and specificity (0% false positives) of detecting CDH13 methylation from nasopharyngeal swabs suggest it could be utilized as a tool for early diagnosis.

Polymorphisms of XRCC1 genes and risk of nasopharyngeal carcinoma in the Cantonese population.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is one of the most common cancers in southern China. In addition to environmental factors such as Epstein-Barr virus infection and diet, genetic susceptibility has been reported to play a key role in the development of this disease. The x-ray repair cross-complementing group 1 (XRCC1) gene is important in DNA base excision repair. We hypothesized that two common single nucleotide polymorphisms of XRCC1 (codons 194 Arg-->Trp and 399 Arg-->Gln) are related to the risk of NPC and interact with tobacco smoking. METHODS: We sought to determine whether these genetic variants of the XRCC1 gene were associated with the risk of NPC among the Cantonese population in a hospital-based case control study using polymerase chain reaction-restriction fragment length polymorphism analysis. We conducted this study in 462 NPC patients and 511 healthy controls. RESULTS: After adjustment for sex and age, we found a reduced risk of developing NPC in individuals with the Trp194Trp genotype (OR = 0.48; 95% CI, 0.27-0.86) and the Arg194Trp genotype (OR = 0.79; 95% CI, 0.60-1.05) compared with those with the Arg194Arg genotype. Compared with those with the Arg399Arg genotype, the risk for NPC was not significantly different in individuals with the Arg399Gln genotype (OR = 0.82; 95% CI, 0.62-1.08) and the Gln399Gln genotype (OR = 1.20; 95% CI, 0.69-2.06). Further analyses stratified by gender and smoking status revealed a significantly reduced risk of NPC among males (OR = 0.32; 95% CI, 0.14-0.70) and smokers (OR = 0.34; 95% CI, 0.14-0.82) carrying the XRCC1 194Trp/Trp genotype compared with those carrying the Arg/Arg genotype. No association was observed between Arg399Gln variant genotypes and the risk of NPC combined with smoking and gender. CONCLUSION: Our findings suggest that the XRCC1 Trp194Trp variant genotype is associated with a reduced risk of developing NPC in Cantonese population, particularly in males and smokers. Larger studies are needed to confirm our findings and unravel the underlying mechanisms.

Detection of nasopharyngeal carcinoma in Morocco (North Africa) using a multiplex methylation-specific PCR biomarker assay.

BACKGROUND: Silencing of tumor suppressor genes (TSGs) or activation of oncogenes by, e.g., aberrant promoter methylation, may be early events during carcinogenesis. The methylation status of such genes can be used for early detection of cancer. We are pursuing this approach in our efforts to develop markers for early detection and follow-up of nasopharyngeal carcinoma (NPC). We set out to develop this approach to allow identification of NPC from Morocco and then also compared with NPC samples from different geographical locations and different ethnicity with different NPC incidences, Epstein-Barr virus (EBV) prevalence, and environments. RESULTS: By multiplex methylation-specific PCR (MMSP), multiple relevant genes can be detected simultaneously, to achieve high sensitivity and specificity. The strong association of EBV with NPC is also very useful in such an approach. We have initially screened for 12 potential marker genes including EBV genes coding for EBV nuclear antigen 1 (EBNA1) and latent membrane protein-1 (LMP1) and ten potential TSGs obtained from previously published data. The resulting assay included EBNA1, LMP1, and three cellular TSGs: ITGA9, RASSF1A, and P16. We evaluated this assay on 64 NPC patient biopsies from Morocco, Italy, and China compared to deoxyribonucleic acid (DNA) from 20 nasopharyngeal control tissues. In the Moroccan NPC cohort (n = 44), prevalence of the EBNA1 gene showed the highest sensitivity (36/44; 82 %) with 94 % specificity. Out of eight (18 %) EBNA1 negative Moroccan samples, only three were positive for at least one methylated cellular gene. By detection of cellular marker genes, the sensitivity increased from 82 to 89 % (39/44). In the whole material of 64 biopsies from three geographical locations, at least any one marker (viral or cellular) could be detected in 91 % of biopsies with 90 % specificity. In a pilot evaluating assay performance on serum DNA from NPC and controls including samples from Italy (n = 11) and China (n = 5), at least any one marker from the MMSP assay could be detected in 88 %, but the specificity was only 50 %. CONCLUSIONS: An MMSP assay has the potential for detection of NPC by screening in high-risk populations. Serum-derived DNA seems not as good as earlier published NPC swab DNA for screening purpose.

Integrin α9 gene promoter is hypermethylated and downregulated in nasopharyngeal carcinoma.

Epigenetic silencing of tumor suppressor genes (TSGs) by promoter methylation can be an early event in the multi-step process of carcinogenesis. Human chromosome 3 contains clusters of TSGs involved in many cancer types including nasopharyngeal carcinoma (NPC), the most common cancer in Southern China. Among ten candidate TSGs identified in chromosome 3 using NotI microarray, ITGA9 and WNT7A could be validated. 5'-aza-2' deoxycytidine treatment restored the expression of ITGA9 and WNT7A in two NPC cell lines. Immunostaining showed strong expression of these genes in the membrane and cytoplasm of adjacent control nasopharyngeal epithelium cells, while they were weakly expressed in NPC tumor cells. The ITGA9 promoter showed marked differentially methylation between tumor and control tissue, whereas no differentially methylation could be detected for the WNT7A promoter. The expression level of ITGA9 in NPC tumors was downregulated 4.9-fold, compared to the expression in control. ITGA9 methylation was detected by methylation specific PCR (MSP) in 56% of EBV positive NPC-cases with 100% specificity. Taken together, this suggests that ITGA9 might be a TSG in NPC that is involved in tumor cell biology. The possibility of using ITGA9 methylation as a marker for early detection of NPC should further be explored.