Helicobacter pylori induces promoter hypermethylation and downregulates gene expression of IRX1 transcription factor on human gastric mucosa

Background and Aim: Gene silence of IRX1 tumor suppressor by promoter CpG methylation combined with loss of heterozygosity (LOH) has been identified in human gastric cancer. This study investigated the association between methylation of IRX1 and Helicobacter pylori infection in gastric mucosa tissues and cell line. Methods: IRX1 methylation was studied by methylation specific polymerase chain reaction (MSP) and bisulfate sequencing polymerase chain reaction (BSP) methods in gastric mucosa tissues from H. pylori‐positive chronic gastritis patients or H. pylori‐negative chronic gastritis patients. Promoter activity, methylation status and gene expressing level of IRX1 were evaluated by persistent infecting H. pylori on human gastric cells GES‐1 in vitro. Electron microscopy was used to observe the effect of H. pylori infection on GES‐1 gastric mucosa cells. Results: The methylation level of IRX1 promoter in H. pylori positive chronic gastritis and H. pylori negative chronic gastritis was 55.30% ± 13.17 versus 5.20% ± 6.31, respectively (P < 0.01). H. pylori infection stimulated increased microvillus, and mucous secretion on GES‐1 cells. Infection of H. pylori induced IRX1 promoter methylation and downregulation of the promoter activity as well as gene expression significantly. Conclusions: This study firstly demonstrated that H. pylori infection contributes to IRX1 promoter methylation on gastric mucosa.     

Expression of COX‐1, COX‐2 and inducible nitric oxide synthase in superficial gastritis,mucosal dysplasia and gastric carcinoma

OBJECTIVE : To investigate the significance of the expression of cyclooxygenase‐1 (COX‐1), cyclooxygenase‐2 (COX‐2) and inducible nitric oxide synthase (iNOS) in superficial gastritis, gastric mucosal dysplasia and gastric carcinoma, and to study the relationship between COX‐2, iNOS, gastric carcinoma and Helicobacter pylori infection. METHODS : Polyclonal antibodies to COX‐1, COX‐2 and iNOS were used detect their expression and the status of H. pylori infection in 92 specimens of paraffin‐embedded gastric tissue. Of the 92 patients, 33 had superficial gastritis, 30 had gastric mucosal dysplasia and 29 had gastric cancer. Helicobacter pylori was detected by toluidine blue staining. RESULTS : Expression of COX‐2 and iNOS in gastric cancer (65.5%, 62.1%) was significantly higher than that in gastritis (18.2%, 18.2%; P < 0.01). Expression of COX‐2 and iNOS in gastritis with H. pylori infection was higher than that in gastric mucosal dysplasia with H. pylori infection. The expression of COX‐2 and iNOS occurred concomitantly in gastritis, dysplasia and gastric cancer. CONCLUSION : Inflammation and H. pylori infection may be able to stimulate the expression of COX‐2 and iNOS, which might be involved in gastric carcinogenesis.     

Polymorphisms of DNA repair genes XRCC1 and XRCC3, interaction with environmental exposure and risk of chronic gastritis and gastric cancer

AIM: To evaluate the association between polymorphisms XRCC1 Arg194Trp and Arg399Gln and XRCC3 Thr241Met and the risk for chronic gastritis and gastric cancer, in a Southeastern Brazilian population. METHODS: Genotyping by PCR-RFLP was carried out on 202 patients with chronic gastritis (CG) and 160 patients with gastric cancer (GC), matched to 202 (CI) and 150 (C2) controls, respectively. RESULTS: No differences were observed among the studied groups with regard to the genotype distribution of XRCCl codons 194 and 399 and of XRCC3 codon 241. However, the combined analyses of the three variant alleles (194Trp, 399Gln and 241Met) showed an increased risk for chronic gastritis when compared to the GC group. Moreover, an interaction between the polymorphic alleles and demographic and environmental factors was observed in the CG and GC groups. XRCC1 194Trp was associated with smoking in the CG group, while the variant alleles XRCC1 399Gln and XRCC3 241Met were related with gender, smoking, drinking and H pylori infection in the CG and GC groups. CONCLUSION: Our results showed no evidence of a rela-tionship between the polymorphisms XRCCl Argl94Trp and Arg399Gln and XRCC3 Thr241Met and the risk of chronic gastritis and gastric cancer in the Brazilian population, but the combined effect of these variants may interact to increase the risk for chronic gastritis, considered a premalignant lesion. Our data also indicate a gene-environment interaction in the susceptibility to chronic gastritis and gastric cancer.     

Significance of calcium-sensing receptor expression in gastric cancer

Objective. The calcium-sensing receptor (CaSR) is known to have differential expression in various carcinomas and normal tissues. It has been shown to be involved in carcinogenesis or tumor suppression. However, its role in gastric cancer remains unknown. This study was performed to determine the CaSR expression level in gastric cancer and non-tumor gastric tissues and to examine the related clinicopathological factors. Materials and methods. Thirty-one pairs of gastric cancer tissues and matched non-tumor gastric tissues were obtained from surgical tissues after gastrectomy. Using real-time polymerase chain reaction, we measured CaSR mRNA expression. We evaluated the association between CaSR mRNA expression and clinicopathological variables based on the downregulation or upregulation of CaSR mRNA expression in gastric cancer tissues compared to those of matched non-tumor gastric tissues. By immunohistochemistry, we confirmed CaSR expression levels in gastric cancer tissues. Results. Downregulation of CaSR mRNA was observed in 77.4% of gastric cancer tissues compared to their matched normal tissues. Downregulated CaSR was associated with a tendency for deeper invasion into the proper muscle (p = 0.028) and more advanced stage (II–IV; p = 0.012). Conclusion. We conclude that downregulation of CaSR may contribute to the prevention or suppression of tumor outgrowth.     

Upregulation and CpG Island Hypomethylation of the TRF2 Gene in Human Gastric Cancer

The telomere-binding protein TRF2 regulates both telomere protection and telomere length. The fact that TRF2 is up-regulated in some tumors indicates that TRF2 plays a role in cancer. However, the role of TRF2 in gastric cancer has not been fully understood. The aim of this study is to evaluate the expression pattern of TRF2 in gastric cancer and examine the potential mechanism of TRF2 regulation. Our study revealed that the expression of TRF2 analyzed by immunohistochemistry was significantly higher in gastric cancers compared to noncancerous tissues. Moreover, TRF2 methylation was detected in six of 30 (20%) primary gastric cancers and 18 of 30 (60%) paired normal tissues, and the downregulation of TRF2 was strongly correlated with the methylation status (P < 0.001). Our results suggest that hypomethylation might contribute to the upregulation of TRF2 in gastric cancers and this overexpression may play a role in the pathogenesis of gastric cancers.     

MGMT and MLH1 methylation in Helicobacter pylori-infected children and adults

AIM:To evaluate the association between Helicobacter pylori(H.pylori) infection and MLH1 and MGMT methylation and its relationship with microsatellite instability(MSI).METHODS:The methylation status of the MLH1 and MGMT promoter region was analysed by methylation specific methylation-polymerase chain reaction(MSPPCR) in gastric biopsy samples from uninfected or H.pylori-infected children(n = 50),from adults with chronic gastritis(n = 97) and from adults with gastric cancer(n = 92).MLH1 and MGMT mRNA expression were measured by real-time PCR and normalised to a constitutive gene(β actin).MSI analysis was performed by screening MSI markers at 4 loci(Bat-25,Bat-26,D17S250 and D2S123) with PCR;PCR products were analysed by single strand conformation polymorphism followed by silver staining.Statistical analyses were performed with either the χ 2 test with Yates continuity correction or Fisher’s exact test,and statistical significance for expression analysis was assessed using an unpaired Student’s t-test.RESULTS:Methylation was not detected in the promoter regions of MLH1 and MGMT in gastric biopsy samples from children,regardless of H.pylori infection status.The MGMT promoter was methylated in 51% of chronic gastritis adult patients and was associated with H.pylori infection(P < 0.05);this region was methylated in 66% of gastric cancer patients,and the difference in the percentage of methylated samples between these patients and those from H.pylori-infected chronic gastritis patients was statistically significant(P < 0.05).MLH1 methylation frequencies among H.pylori-infected and non-infected chronic gastritis adult patients were 13% and 7%,respectively.We observed methylation of the MLH1 promoter(39%) and increased MSI levels(68%) in samples from gastric cancer patients in comparison to samples from H.pylori-infected adult chronic gastritis patients(P < 0.001 and P < 0.01,respectively).The frequency of promoter methylation for both genes was higher in gastric cancer samples than in H.pylori-positiv     

Potential involvement of leptin in carcinogenesis of hepatocellular carcinoma

AIM: To investigate the potential involvement of leptin in carcinogenesis of hepatocellular carcinoma (HCC) and to elucidate the etiology, carcinogenesis and progress of HCC. METHODS: Expressions of Ob gene product, leptin and its receptor, Ob-R were investigated in 36 cases of HCC specimens and corresponding adjacent non-tumorous liver tissues with immunohistochemical staining. The effect of leptin on proliferation of Chang liver cell line and liver cancer cell line SMMC-7721 was studied with cell proliferation assay (MTT). RESULTS: Leptin expression was detected in 36 cases of adjacent non-tumorous liver tissues (36/36, 100%) with moderate (++) to strong (+++) intensity; and in 72.22%(26/36) of HCC with weaker (+) intensity (P<0.05). Thirty of 36 (83.33%) cases of adjacent non-tumorous liver tissues were positive for Ob-R, with moderate (++) to strong (+++) intensity. In HCC, 11/36 (30.56%) cases were positive, with weak (+) intensity (P<0.05). In cell proliferation assay, leptin inhibited the proliferation of Chang liver cells. The cell survival rate was 10-13% lower than that of the untreated cells (P>0.05). Leptin had little effect on the proliferation of liver cancer cells (P>0.05). CONCLUSION: High level expression and decreased or absent expression of leptin and its receptor in adjacent non-tumorous liver cells and HCC cells, inhibitory effect of leptin on the proliferation of normal Chang liver cells and no effect of leptin on proliferation of liver cancer cells, may provide new insights into the carcinogenesis and progression of human HCC. It could be assumed that leptin acting as an inhibitor and/or promoter, is involved in the process of carcinogenesis and progress of human HCC.     

The expression level of TRAF1 in human gastric mucosa is related to virulence genotypes of Helicobacter pylori

Abstract

Objective. To investigate the expression level of tumor necrosis factor receptor-associated factor 1 (TRAF1) in gastric mucosa tissue in patients infected with Helicobacter pylori (H. pylori) and to analyze the relationship between TRAF1 expression and H. pylori virulence. Methods. Gastric tissue samples were collected from patients with gastritis, atrophic gastritis, intestinal metaplasia with atypical hyperplasia, and gastric cancer. The expression level of TRAF1 in each group was analyzed by real-time polymerase chain reaction (PCR) and Western blot analysis. Virulence genotypes of H. pylori were determined by PCR. Results. Significant differences in TRAF1 mRNA levels were observed between the gastritis and gastric cancer groups, and the atrophic gastritis and gastric cancer groups (p < 0.05). Moreover, significant differences in TRAF1 protein levels were observed between the gastritis and intestinal metaplasia with atypical hyperplasia groups, between the gastritis and gastric cancer groups, and between the atrophic gastritis and gastric cancer groups (all p < 0.05). The virulence genotypes of cytotoxin-associated gene A (cagA), vacAs1, and vacAm1 were more frequent in the TRAF1 high-level group than in the TRAF1 low-level group (p < 0.05). Conclusion. Higher TARF1 expression level is associated with infection by CagA⁺/vacAs1⁺/m1⁺ virulent H. pylori strains and may promote the proliferation of gastric mucosal cells and induce gastric cancer.     

错配修复基因 hMLH1 和hMSH2 在胃癌组织中的表达及意义

目的探讨错配修复基因hMLH1 和hMSH2 在胃癌发生中的作用.方法采用免疫组化SP法,检测散发性胃癌、癌旁黏膜和胃炎黏膜组织中hMLH1 和 hMSH2基因的表达情况.结果 hMSH2在胃癌组织中的阳性表达率(67.1%)显著高于癌旁黏膜(41.4%)和胃炎黏膜组织(41.2%)(P<0.05),而后两者无显著差异(P>0.05).hMSH2在低分化腺癌中的阳性率(80.6% )显著高于高中分化腺癌(57.7%)和黏液癌(53.8%)(P<0.05),而后两者无显著差异(P>0.05).hMLH1在胃炎黏膜组织的阳性表达率(38.2%)显著低于胃癌(77.1%)和癌旁黏膜组织(74.3%)(P<0.05),后两者无显著差异(P>0.05). hMLH1在黏液癌中的阳性率(38.5%)显著低于高中分化腺癌(80.8%)和低分化腺癌(90.3%)(P<0.05),后两者无显著差异(P>0.05).结论 hMSH2高表达可能是胃癌发生的标志之一;hMSH2表达与肿瘤分化程度及患者预后有关;hMLH1有可能作为胃癌预警组织学标记物.     

Decreased expression of gastrokine 1 in gastric mucosa of gastric cancer patients

AIM: To investigate the expression of gastrokine 1(GKN1) in normal gastric mucosa, precancerous lesions and gastric cancer tissues, and to analyse its correlations with tumour site and pathological pattern.METHODS: Thirty gastric cancer patients(12 cases of diffuse type and 18 cases of intestinal type), 13 atrophic gastritis patients and 15 healthy volunteers with almost normal gastric mucosa(superficial gastritis) were enrolled in this study. Helicobacter pylori(H. pylori) infection was examined in all subjects. All gastric mucosa biopsy specimens were obtained. Cancer-adjacent specimens were taken from corresponding gastric cancer patients. Immunohistochemistry and real-time PCR were performed to determine the expressions of the GKN1 protein and m RNA, respectively.RESULTS: H. pylori infection had no significant association with age, gender, tumour site or pathological pattern in all subjects. Compared with the superficial gastritis and atrophic gastritis groups, the expression of GKN1 protein(P = 0.011) and m RNA(P < 0.001) in gastric cancer was significantly decreased. The GKN1 m RNA level in diffuse type gastric cancer was significantly lower than in intestinal type gastric cancer(0.296 ± 0.076 vs 0.525 ± 0.164, P < 0.001).CONCLUSION: Compared with almost normal gastric mucosa, GKN1 expression in the gastric mucosa of gastric cancer patients is decreased; this is associated with progression and prognosis of gastric cancer.     

Overexpression of peroxisome proliferator‐activated receptor γ and retinoid X receptor α in gastric carcinomatous tissue

OBJECTIVE: To investigate the expression of peroxisome proliferator‐activated receptor γ (PPAR‐γ) and retinoid X receptor α (RXR‐α) in chronic gastritis, gastric mucosal dysplasia and gastric carcinoma and to identify any correlations between PPAR‐γ and RXR‐α expression in this progression sequence. METHODS: Immunohistochemical methods (avidin? biotin?peroxidase complex) were used to examine the expression of PPAR‐γ and RXR‐α in 53 patients with gastric carcinoma, 18 with gastric mucosal dysplasia and 30 with chronic atrophic gastritis. Thirty‐one patients with chronic non‐atrophic gastritis acted as controls. RESULTS: The positive rates of PPAR‐γ and RXR‐α in gastric carcinoma were 41.5 and 54.7%, 27.8 and 38.9% in gastric mucosal dysplasia, 10.0 and 20.0% in chronic atrophic gastritis, and 6.5 and 16.1% in chronic non‐atrophic gastritis, respectively. The expression of PPAR‐γ and RXR‐α increased during the progression from chronic non‐atrophic gastritis to chronic atrophic gastritis, then to gastric carcinoma. Compared with chronic gastritis, the expression of PPAR‐γ and RXR‐α in gastric mucosal dysplasia and gastric carcinoma was significantly increased (P < 0.05, P < 0.01). In gastric carcinoma, the expression of PPAR‐γ and RXR‐α was not associated with tumor cell differentiation or metastasis in the lymph nodes (P > 0.05). There was a positive correlation between the expression of PPAR‐γ and RXR‐α in gastric carcinoma (r= 0.54, P < 0.01). CONCLUSIONS: Overexpression of PPAR‐γ and RXR‐α protein is apparent in human gastric cancer. This might be an early event in carcinogenesis, and both PPAR‐γ and RXR‐α may play independent and/or synergistic roles in the progression of gastric carcinoma.     

DNA Methylation of NDRG2 in Gastric Cancer and Its Clinical Significance

Background

Gastric cancer is one of the most common digestive malignancies worldwide. N-myc downstream-regulated gene 2 (NDRG2) is a differentiation-related gene that is considered to be a metastasis suppressor gene. In this study, we examined the expression and DNA methylation of NDRG2 in gastric cancer cell lines and tissues, as well as its clinical significance.

Methods

Six gastric cancer cell lines and 42 paired normal and gastric cancer tissue samples were used to assess NDRG2 mRNA expression using RT-PCR. NDRG2 DNA methylation status was evaluated by methylation-specific PCR (MSP) in gastric cancer cell lines and tissues. The suppression of NDRG2 in BGC823 cells by siRNA transfection was utilized to detect the role of NDRG2 in gastric cancer progression.

Results

NDRG2 mRNA was down-regulated in gastric cancer cell lines and tissues, and its expression was just related to lymph node metastasis (p = 0.032). MSP showed methylation of NDRG2 in 54.0 % (47/87) of primary gastric cancer specimens and in 20.0 % (16/80) of corresponding nonmalignant gastric tissues. NDRG2 methylation was related to depth of tumor invasion, Borrmann classification and TNM stage (p < 0.05). Upon treatment with 5-aza-2′-deoxycytidine and trichostatin A, NDRG2 expression was upregulated in HGC27 cells, and demethylation of the highly metastatic cell line, MKN45, inhibited cell invasion. Furthermore, the suppression of NDRG2 by siRNA transfection enhanced BGC823 cells invasion.

Conclusions

Our results suggest that the aberrant methylation of NDRG2 may be mainly responsible for its downregulation in gastric cancer, and may play an important role in the metastasis of gastric cancer.     

胃癌RASSF1A基因启动子CpG岛甲基化与DNMT1表达的意义

目的探讨RAS相关区域家族1A（RASSF1A）基因启动子CpG岛甲基化和DNA甲基转移酶1（DN-MT1）的表达与胃癌发生的关系。方法运用甲基化特异性聚合酶链反应和免疫组织化学方法检测胃癌癌旁正常组织和癌组织RASSF1A基因启动子CpG岛甲基化发生率及DNMT1的表达情况。结果癌旁正常组织中RASSF1A基因启动子CpG岛甲基化发生率、DNMT1阳性表达率显著低于相应癌组织（P均〈0.01）。RASSF1A启动子CpG岛甲基化患者DNMT1阳性表达率与非甲基化患者比较无统计学差异（P〉0.05）。结论RASSF1A基因启动子区CpG岛甲基化和DNMT1的高表达可能与胃癌发生有一定关系。     

Hypermethylation of Syk gene in promoter region associated with oncogenesis and metastasis of gastric carcinoma

AIM: To investigate the relationship between methylation of Syk (spleen tyrosine kinase) gene in promoter region and oncogenesis, metastasis of gastric carcinoma. The relation between silencing of the Syk gene and methylation of Syk promoter region was also studied. METHODS: By using methylation-specific PCR (MSP) technique, the methylation of Syk promoter region in specimens from 61 gastric cancer patients (tumor tissues and adjacent normal tissues) was detected. Meanwhile, RT-PCR was used to analyse syk expression exclusively. RESULTS: The expression of the Syk gene was detected in all normal gastric tissues. Syk expression in gastric carcinoma was lower in 14 out of 61 gastric cancer samples than in adjacent normal tissues (chi(2)=72.3, P<0.05). No methylation of Syk promoter was found in adjacent normal tissues. hypermethylation of Syk gene in promoter was detected 21 cases in 61 gastric carcinoma patients. The rate of methylation of Syk promoter in gastric carcinoma was higher than that in adjacent normal tissues (chi(2)=25.1, P<0.05). In 31 patients with lymph node metastasis, 17 were found with Syk promoter methylation. A significant difference was noted between two groups (chi(2)=11.4,P<0.05). CONCLUSION: Hypermethylation leads to silencing of the Syk gene in human gastric carcinoma. Methylation of Syk promoter is correlated to oncogenesis and metastasis of gastric carcinoma. Syk is considered to be a potential tumor suppressor and anti-metastasis gene in human gastric cancer.     

p27蛋白与细胞周期蛋白D1在疣状胃炎和胃癌组织中的表达及其意义

背景：疣状胃炎是一种具有特殊形态的慢性胃炎,与胃癌可能存在一定关系,但其癌变的分子机制尚不清楚。p27蛋白与细胞周期蛋白D1（cyclin D1）的异常表达参与了胃癌的发生、发展。目的：探讨p27蛋白和cyclin D1在疣状胃炎和胃癌组织中的表达及其意义。方法：收集慢性非萎缩性胃炎、未成熟型疣状胃炎、成熟型疣状胃炎、胃癌组织标本各40例,采用免疫组化法检测p27蛋白、cyclin D1表达,并分析两者与疣状胃炎患者性别、年龄、Hp感染、肠化生、异型增生的关系。结果：未成熟型疣状胃炎组p27蛋白阳性表达率显著高于胃癌组（77．5％对45．0％,P〈0．05）,与慢性非萎缩性胃炎组相比无明显差异（P〉0．05）。成熟型疣状胃炎组p27蛋白阳性表达率显著低于慢性非萎缩性胃炎组和未成熟型疣状胃炎组（52．5％对85．0％、77．5％,P〈0．05）,与胃癌组相比无明显差异（P〉0．05）。未成熟型、成熟型疣状胃炎组cyclin D1阳性表达率均显著高于慢性非萎缩性胃炎组（40．0％、42．5％对12．5％,P〈0．05）,但均显著低于胃癌组（P〈0．05）。疣状胃炎患者p27蛋白表达与肠化生、异型增生有关（P〈0．05）,cyclin D1表达与肠化生有关（P〈0．05）;p27蛋白、cyclin D1表达与性别、年龄均无关。结论ip27蛋白、cyclin D1可能参与了疣状胃炎的发生和癌变过程。     

Altered Expression of a Li-Cadherin in Gastric Cancer and Intestinal Metaplasia

The aims of this study were to examine Li-cadherin expression in 74 gastric carcinoma tissues, 10 cases with normal gastric tissues, and 21 cases with intestinal metaplasia and to investigate the role of Li-cadherin in cell differentiation, cancer invasion, and metastasis. Expression of Li-cadherin was analyzed by immunohistochemistry and semiquantitative polymerase chain reactio and correlated with clinicopathological parameters. Immunohistochemistry showed that Li-cadherin was mainly present on the cell membrane and there was no staining for liver–intestine cadherin in normal tissues. The reduction of Li-cadherin mRNA expression was inversely correlated with the grade of differentiation (P < 0.05). Significant differences in the expression of liver–intestine cadherin were found in lymphatic metastasis of the tumors (P < 0.05), but the expression of liver–intestine cadherin was not associated with gender (P=0.748), serosal invasion (P=0.136), TNM stage (P=0.172), Helicobacter pylori infection (P=0.572), liver metastasis (P=0.374), or peritoneal metastasis (P=0.621). Multivariate analysis revealed that the expression of Li-cadherin is an important predictor of lymph node metastasis. We conclude that there is a significant correlation between Li-cadherin expression and the differentiation of gastric carcinoma, and Li-cadherin can be a good marker to detect gastric cancer at early stages. Increased Li-cadherin expression may contribute to gastric cancer invasion to lymph nodes.