ceRNA network construction and comparison of gastric cancer with or without Helicobacter pylori infection

Gastric cancer (GC) is a lethal disease, and among its variety of etiological factors, Helicobacter pylori (H. pylori) infection is the strongest risk factor. However, the genetic and molecular mechanisms underlying H. pylori-related GC need further elucidation. We investigated the competing endogenous RNA (ceRNA) network differences between H. pylori (+) and H. pylori (−) GC. The long noncoding RNA (lncRNA), microRNA (miRNA), and messenger RNA (mRNA) expression data from 32 adjacent noncancerous samples and 18 H. pylori (+) and 141 H. pylori (−) stomach adenocarcinoma samples were downloaded from the TCGA database. After construction of lncRNA–miRNA–mRNA ceRNA networks of H. pylori (+) and H. pylori (−) GC, Panther and Kobas databases were used to analyze the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Finally, survival analysis was used to discover the key genes. In H. pylori (+) GC, we identified a total of 1,419 lncRNAs, 82 miRNAs, and 2,501 mRNAs with differentially expressed profiles. In H. pylori (−) GC, 2,225 lncRNAs, 130 miRNAs, and 3,146 mRNAs were differentially expressed. Furthermore, three unique pathways (cytokine–cytokine receptor interaction, HIF-1 signaling pathway, and Wnt signaling pathway) were enriched in H. pylori (+) GC. According to the overall survival analysis, three lncRNAs (AP002478.1, LINC00111, and LINC00313) and two mRNAs (MYB and COL1A1) functioned as prognostic biomarkers for patients with H. pylori (+) GC. In conclusion, our study has identified the differences in ceRNA regulatory networks between H. pylori (+) and H. pylori (−) GC and provides a rich candidate reservoir for future studies.     

Epigenetic alterations of CYLD promoter modulate its expression in gastric adenocarcinoma: A footprint of infections

Gastric cancer (GC) is one of the most common causes of cancer-related death in the world, with multiple genetic and epigenetic alterations involved in disease development. CYLD tumor suppressor gene encodes a multifunctional deubiquitinase which negatively regulates various signaling pathways. Deregulation of this gene has been found in different types of cancer. This study aimed to evaluate for the first time the CpG island methylation pattern of CYLD gene promoter, and its expression level in gastric adenocarcinoma. CYLD messenger RNA expression and promoter methylation in 53 tumoral and their non-neoplastic counterpart tissues were assessed using quantitative polymerase chain reaction and bisulfite sequencing. Also, we investigated the impacts of the infectious agents including Helicobacter pylori (H. pylori), EBV, and CMV on CYLD expression and promoter methylation in GC. Results showed that the expression level of CYLD was downregulated in GC, and was significantly associated with gender (female), patient’s age (<60), high grade, and no lymph-node metastasis (p = 0.001, 0.002, 0.03, and 0.003, respectively). Among the 31 analyzed CpG sites located in about 600 bp region within the promoter, two CpG sites were hypermethylated in GC tissues. We also found a significant inverse association between DNA promoter methylation and CYLD expression (p = 0.02). Furthermore, a direct association between H. pylori, EBV, and CMV infections with hypermethylation and reduced CYLD expression was observed (p = 0.04, 0.03, and 0.03, respectively). Our findings indicate that CYLD is downregulated in GC. Infectious agents may influence CYLD expression.     

Epigenetic silencing of DACH1 induces the invasion and metastasis of gastric cancer by activating TGF‐β signalling

Gastric cancer (GC) is the fourth most common malignancy in males and the fifth most common malignancy in females worldwide. DACH1 is frequently methylated in hepatic and colorectal cancer. To further understand the regulation and mechanism of DACH1 in GC, eight GC cell lines, eight cases of normal gastric mucosa, 98 cases of primary GC and 50 cases of adjacent non‐tumour tissues were examined. Methylation‐specific PCR, western blot, transwell assay and xenograft mice were used in this study. Loss of DACH1 expression correlated with promoter region methylation in GC cells, and re‐expression was induced by 5‐Aza‐2′‐deoxyazacytidine. DACH1 is methylated in 63.3% (62/98) of primary GC and 38% (19/50) of adjacent non‐tumour tissues, while no methylation was found in normal gastric mucosa. Methylation of DACH1 correlated with reduced expression of DACH1 (P < 0.01), late tumour stage (stage III/IV) (P < 0.01) and lymph node metastasis (P < 0.05). DACH1 expression inhibited epithelial–mesenchymal transition and metastasis by inhibiting transforming growth factor (TGF)‐β signalling and suppressed GC cell proliferation through inducing G2/M phase arrest. The tumour size is smaller in DACH1‐expressed BGC823 cell xenograft mice than in unexpressed group (P < 0.01). Restoration of DACH1 expression also sensitized GC cells to docetaxel. These studies suggest that DACH1 is frequently methylated in human GC and expression of DACH1 was controlled by promoter region methylation. DACH1 suppresses GC proliferation, invasion and metastasis by inhibiting TGF‐β signalling pathways both in vitro and in vivo. Epigenetic silencing DACH1 may induce GC cells' resistance to docetaxel.     

In vitro DNA cytosine methylation of cis-regulatory elements modulates c-Ha-ras promoter activity in vivo.

The effect of DNA cytosine methylation on promoter activity was assessed using a transient expression system employing pHrasCAT. This 551 bp Ha-ras-1 gene promoter region is enriched with 84 CpG dinucleotides, six functional GC boxes, and is prototypic of many genes possessing CpG islands in their promoter regions. Bacterial modification enzymes HhaI methyl transferase (MTase) and HpaII MTase, alone or in combination with a human placental DNA methyltransferase (HP MTase) that methylates CpG sites in a generalized manner, including asymmetric elements such as GC box CpG's, were used to methylate at different types of sites in the promoter. Methylation of HhaI and HpaII sites reduced CAT expression by approximately 70%-80%, whereas methylation at generalized CpG sites with HP MTase inactivated the promoter by greater than 95%. The inhibition of H-ras promoter activity was not attributable to methylation-induced differences in DNA uptake or stability in the cell, topological form of the plasmid, or methylation effects in non-promoter regions.     

Transmembrane protein 106A is silenced by promoter region hypermethylation and suppresses gastric cancer growth by inducing apoptosis

Inactivation of tumour suppressor genes by promoter methylation plays an important role in the initiation and progression of gastric cancer (GC). Transmembrane 106A gene (TMEM106A) encodes a novel protein of previously unknown function. This study analysed the biological functions, epigenetic changes and the clinical significance of TMEM106A in GC. Data from experiments indicate that TMEM106A is a type II membrane protein, which is localized to mitochondria and the plasma membrane. TMEM106A was down‐regulated or silenced by promoter region hypermethylation in GC cell lines, but expressed in normal gastric tissues. Overexpression of TMEM106A suppressed cell growth and induced apoptosis in GC cell lines, and retarded the growth of xenografts in nude mice. These effects were associated with the activation of caspase‐2, caspase‐9, and caspase‐3, cleavage of BID and inactivation of poly (ADP‐ribose) polymerase (PARP). In primary GC samples, loss or reduction of TMEM106A expression was associated with promoter region hypermethylation. TMEM106A was methylated in 88.6% (93/105) of primary GC and 18.1% (2/11) in cancer adjacent normal tissue samples. Further analysis suggested that TMEM106A methylation in primary GCs was significantly correlated with smoking and tumour metastasis. In conclusion, TMEM106A is frequently methylated in human GC. The expression of TMEM106A is regulated by promoter hypermethylation. TMEM106A is a novel functional tumour suppressor in gastric carcinogenesis.     

Development of a novel gene signature in patients without Helicobacter pylori infection gastric cancer

Gastric cancer (GC) is one of the most fatal common cancers in worldwide. Helicobacter pylori (H. pylori) infection is closely related to the development of GC, although the mechanism is still unclear. In our study, we aim to develop a robust messenger RNA (mRNA) signature associated with H. pylori (-) GC that can sensitively and efficiently predict the prognostic. The RNA-seq expression profile and corresponding clinical data of 598 gastric cancer samples and 63 normal samples obtained from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus database. Using gene set enrichment analysis H. pylori (+) GC and H. pylori (-) GC patients and normal samples to select certain genes for further analysis. Using univariate and multivariate Cox regression model to establish a gene signature for predicting the overall survival (OS). Finally, we identified G2/M related seven-mRNA signature (TGFB1, EGF, MKI67, ILF3, INCENP, TNPO2, and CHAF1A) closely related to the prognosis of patients with H. pylori (-) GC. The seven-mRNA signature was identified to act as an independent prognostic biomarker by stratified analysis and multivariate Cox regression analysis. It was also validated on two test groups from TCGA and GSE15460 and shown that patients with high-risk scores based on the expression of the seven mRNAs had significantly shorter survival times compared to patients with low-risk scores (P < .0001). In this study, we developed a seven-mRNA signature related to G2/M checkpoint from H. pylori (-) GCs that as an independent biomarker potentially with a good performance in predicting OS and might be valuable for the clinical management for patients with GC.     

The Correlation of MGMT Promoter Methylation and Clinicopathological Features in Gastric Cancer: A Systematic Review and Meta-Analysis

The silencing of the tumor suppressor gene O-6-methylguanine-DNA methyltransferase (MGMT) by promoter methylation commonly occurs in human cancers. The relationship between MGMT promoter methylation and gastric cancer (GC) remains inconsistent. This study aimed to evaluate the potential value of MGMT promoter methylation in GC patients. Electronic databases were searched to identify eligible studies. The pooled odds ratio (OR) and the corresponding 95% confidence interval (95% CI) were used to evaluate the effects of MGMT methylation on GC risk and clinicopathological characteristics. In total, 31 eligible studies including 2988 GC patients and 2189 nonmalignant controls were involved in meta-analysis. In the pooled analysis, MGMT promoter methylation was significantly associated with GC risk (OR = 3.34, P < 0.001) and substantial heterogeneity (P < 0.001). Meta-regression and subgroup analyses based on the testing method, sample material and ethnicity failed to explain the sources of heterogeneity. Interestingly, MGMT methylation showed a trend associated with gender, and methylation is lower in males compared with females (OR = 0.76, 95% CI = 0.56–1.03). We did not find a significant association in relation to tumor types, clinical stage, age status or H. pylori status in cancer (all P > 0.1). MGMT promoter methylation may be correlated with the prognosis of GCs in disease free survival (DFS) or overall survival (OS) for univariate analysis. MGMT promoter methylation may play a crucial role in the carcinogenesis and prognosis of GC. MGMT methylation was not correlated with tumor types, clinical stage, age status, H. pylori status. However, the result of the association of MGMT methylation and gender should be considered with caution.     

Metformin sensitizes insulin signaling through AMPK-mediated PTEN down-regulation in preadipocyte 3T3-L1 cells

Insulin resistance is the primary cause responsible for type 2 diabetes. Phosphatase and tensin homolog (PTEN) plays a negative role in insulin signaling and its inhibition improves insulin sensitivity. Metformin is a widely used insulin-sensitizing drug; however, the mechanism by which metformin acts is poorly understood. To gain insight into the role of PTEN, we examined the effect of metformin on PTEN expression. Metformin suppressed the expression of PTEN in an AMP-activated protein kinase (AMPK)-dependent manner in preadipocyte 3T3-L1 cells. Knock-down of PTEN potentiated the increase in insulin-mediated phosphorylation of Akt/ERK. Metformin also increased the phosphorylation of c-Jun N-terminal kinase (JNK)-c-Jun and mammalian target of rapamycin (mTOR)-p70S6 kinase pathways. Both pharmacologic inhibition and knock-down of AMPK blocked metformin-induced phosphorylation of JNK and mTOR. Knock-down of AMPK recovered the metformin-induced PTEN down-regulation, suggesting the involvement of AMPK in PTEN regulation. PTEN promoter activity was suppressed by metformin and inhibition of mTOR and JNK by pharmacologic inhibitors blocked metformin-induced PTEN promoter activity suppression. These findings provide evidence for a novel role of AMPK on PTEN expression and thus suggest a possible mechanism by which metformin may contribute to its beneficial effects on insulin signaling.     

Important role of the LKB1-AMPK pathway in suppressing tumorigenesis in PTEN-deficient mice

The LKB1 tumour suppressor phosphorylates and activates AMPK (AMP-activated protein kinase) when cellular energy levels are low, thereby suppressing growth through multiple pathways, including inhibiting the mTORC1 (mammalian target of rapamycin complex 1) kinase that is activated in the majority of human cancers. Blood glucose-lowering Type 2 diabetes drugs also induce LKB1 to activate AMPK, indicating that these compounds could be used to suppress growth of tumour cells. In the present study, we investigated the importance of the LKB1-AMPK pathway in regulating tumorigenesis in mice resulting from deficiency of the PTEN (phosphatase and tensin homologue deleted on chromosome 10) tumour suppressor, which drives cell growth through overactivation of the Akt and mTOR (mammalian target of rapamycin) kinases. We demonstrate that inhibition of AMPK resulting from a hypomorphic mutation that decreases LKB1 expression does not lead to tumorigenesis on its own, but markedly accelerates tumour development in PTEN(+/-) mice. In contrast, activating the AMPK pathway by administration of metformin, phenformin or A-769662 to PTEN(+/-) mice significantly delayed tumour onset. We demonstrate that LKB1 is required for activators of AMPK to inhibit mTORC1 signalling as well as cell growth in PTEN-deficient cells. Our findings highlight, using an animal model relevant to understanding human cancer, the vital role that the LKB1-AMPK pathway plays in suppressing tumorigenesis resulting from loss of the PTEN tumour suppressor. They also suggest that pharmacological inhibition of LKB1 and/or AMPK would be undesirable, at least for the treatment of cancers in which the mTORC1 pathway is activated. Most importantly, our results demonstrate the potential of AMPK activators, such as clinically approved metformin, as anticancer agents, which will suppress tumour development by triggering a physiological signalling pathway that potently inhibits cell growth.     

Myeloperoxidase, xanthine oxidase and superoxide dismutase in the gastric mucosa of helicobacter pylori positive and negative pediatric patients

The aim of this study was determination and comparison of the levels of myeloperoxidase (MPO), xanthine oxidase (XO), and superoxide dismutase (SOD) in gastric mucosa of children who were infected and noninfected with Helicobacter pylori (HP). The MPO, and XO enzyme activities were detected via kinetic measurement, and the MPO, XO and SOD enzyme protein levels were detected via Western blot, in antral mucosa specimens of 43 patients who underwent upper gastrointestinal endoscopy with various indications. The diagnosis of HP infection was made with a positive rapid urease test and histopathologic detection. MPO activity and enzyme protein levels were measured in 14 [8 HP (+) and 6 HP (−)], and in 9 [5 HP (+) and 4 HP (−)] while XO activity and enzyme protein levels were measured in 16 [10 HP (+) and 6 HP (−)] and in 9 [5 HP (+) and 4 HP (−)] patients, respectively. SOD protein level was detected in 13 [7 HP (+) and 6 HP (−)] patients. Of 43 patients 25 were HP (+) and 18 were HP (−). MPO activities were 75.6 ± 40.5 and 98.8 ± 44.1 U/g. protein (p = 0.302) while XO activities were 0.5 ± 0.3 and 0.4 ± 0.2 U/g. protein in HP (+) and HP (−) patients, respectively (p = 0.625). Measured enzyme protein levels of MPO, XO and SOD were found statistically indifferent in HP (+) and HP (−) patients (p = 0.327, p = 0.086, and p = 0.775, respectively). The results of this study revealed that, MPO, XO and SOD conditions in gastric mucosa alone were not affected from HP presence. That's why MPO, XO, and SOD may not have important roles in the pathogenesis of HP related gastric disease in children.     

Tumour suppressor PTEN enhanced enzyme activity of GPx,SOD and catalase by suppression of PI3K/AKT pathway in non-small cell lung cancer cell lines

Phosphates and tensin homologue deleted on chromosome 10 (PTEN) is a tumour suppressor gene which dephosphorilates phosphoinositol 3,4,5 triphosphates. Therefore PTEN can regulate PI3K/AKT pathway in cells. Because of promoter methylation or gene deletion, PTEN expression is commonly decreased or lost in non-small cell lung cancer (NSCLC) cell lines. Therefore, we hypothesized that PTEN could regulate the activity of superoxide dismutase (CuZnSOD), glutathione peroxidase (GPx) and catalase. We first recreated PTENwt, G129R and G129E expressions in lung cell lines, in which endogenous PTEN expression was not detected. Then, we showed that PTEN could suppress AKT activity by its lipid phosphatase domain. We then examined the effect of recreated PTEN expressions in NSCLC cells. While PTENwt expression caused enhanced activity of SOD, GPx and catalase in transfected cells lines, neither G129R nor G129E expression effected enzyme activities. These results suggest that PTEN can up-regulate SOD, GPx and catalase activity by inhibition of PI3K/AKT pathway in NSCLC cell lines.     

腹部脂肪组织APN基因DNA甲基化及mRNA表达与维吾尔族T2DM的相关性

为探讨APN基因启动子区DNA甲基化及mRNA表达与新疆维吾尔族T2DM发生、发展的相关性,文章选择新疆维吾尔族正常个体50例、肥胖个体48例、肥胖伴T2DM个体26例,收集腹部网膜脂肪组织,利用变性高效液相色谱技术检测APN基因启动子区DNA甲基化情况,应用Real-time PCR方法检测APN 基因mRNA表达情况。结果显示,APN基因启动子区DNA甲基化阳性率在正常对照(34%)、肥胖(47.9%)及T2DM组(65.4%)逐渐增加,差异具有统计学意义(P<0.05)。Real-time PCR结果显示,正常对照组APN mRNA相对拷贝数(0.7162)显著高于肥胖(0.4244)及T2DM组(0.4093),差异具有统计学意义(P<0.05)。非T2DM个体相关性分析提示,APN mRNA相对拷贝数与空腹血清葡萄糖(Fasting plasma glucose, FPG)、糖化血红蛋白(Glycosylated hemoglobin, HbA₁c)、甘油三酯(Triglyceride, TG)水平显著负相关(P<0.05)。APN基因启动子区DNA甲基化与其mRNA表达负相关,甲基化阳性组相对拷贝数(0.2700)显著低于阴性组(0.7870),差异具有统计学意义(P<0.01)。以上结果提示,APN基因启动子区DNA甲基化通过抑制其 mRNA表达导致糖脂代谢紊乱,可能参与了新疆维吾尔族肥胖及T2DM的发生、发展过程。     

Circulating cell-free DNA of methylated insulin-like growth factor-binding protein 7 predicts a poor prognosis in hepatitis B virus-associated hepatocellular carcinoma after hepatectomy

The initiation and progression of hepatocellular carcinoma (HCC) is a multistage process involving a variety of changes at the gene level. Methylation of insulin-like growth factor-binding protein 7 (IGFBP7) plays a crucial role in HCC development. The main purpose of this study was to investigate the relationship between oxidative stress, DNA methyltransferases (DNMTs) expression, and IGFBP7 methylation, and to evaluate the prognostic value of serum IGFBP7 methylation status in patients with HCC after hepatectomy. We enrolled 155 patients with HCC undergoing surgical resection. The IGFBP7 methylation status, DNMTs mRNA levels and malondialdehyde (MDA), xanthine oxidase (XOD), reduced glutathione hormone (GSH), and glutathione-S-transferases (GST) levels were detected. MDA and XOD levels were significantly higher in IGFBP7 methylated group than unmethylated group, while GSH level was lower in methylated group than unmethylated group. The DNMT1 and DNMT3a mRNA levels were higher in IGFBP7 methylated group than unmethylated group. Kaplan–Meier curve analysis revealed that IGFBP7 promoter methylation was significantly correlated with overall survival (OS) (p?IGFBP7 methylation was an independent prognostic predictor for OS (p?=?.000) and early tumour recurrence (ETR) (p?=?.008) in HCC after hepatectomy. Our results indicated that IGFBP7 promoter methylation was associated with oxidative stress and DNMTs expression. Meanwhile, IGFBP7 promoter methylation was associated with OS and ETR, indicating that it might serve as a potentially independent prognostic factor in patients with HCC after hepatectomy.     

Epigenetic inactivation of PCDH10 in human prostate cancer cell lines

PCDH10 (protocadherin-10), a novel tumour suppressor gene, is down-regulated in several human cancers due to hypermethylation of promoter CGIs (CpG islands). Here, we investigated the expression of PCDH10 in different normal adult tissues and in a panel of prostate cancer cell lines. PCDH10 was widely expressed in normal tissues with higher levels in the prostate. The expression of PCDH10 was markedly reduced or silenced in prostate cancer cell lines compared with normal adult prostate tissue. Decreased PCDH10 expression was correlated with the methylation status of the PCDH10 promoter. Furthermore, the DNA demethylating agent 5'-azacytidin restored PCDH10 expression by suppressing PCDH10 promoter methylation in prostate cancer cell lines. Treatment with Trichostatin A alone had no significant effect on the expression of PCDH10 but enhanced the effect of 5'-azacytidin. In conclusion, we found that the decreased PCDH10 expression in prostate cancer cells was associated with the aberrant methylation of PCDH10 promoter CGI. Our results may contribute to the understanding of the role of PCDH10 inactivation in the progression of prostate cancers.     

Genetic polymorphisms potentially associated with response to metformin in postmenopausal diabetics suffering and not suffering with cancer

Metformin is a well-known antidiabetic medication, which, besides diabetes, may be involved into modulation of other age-related pathologies, including cancer. The study concerns 12 gene polymorphisms divided into 2 groups consisting of 6 genes each. The first group was composed from so-called “standard” (S) polymorphisms, for which the connection with metabolic response to metformin is already established. The second group included polymorphisms of genes encoding proteins possibly connected with diabetes mellitus type 2 (DM2), impaired glucose tolerance or cancer and entitled here as “associated” (A). A total of 156 postmenopausal women (average age 60.7 ± 0.7) were included, 37 of them healthy, 64 with type DM2 and concurrent treatment-naïve cancer (mostly breast, endometrial or colorectal cancer), 32 with DM2 without cancer, and 23 with treatment-naïve cancer and normal glucose tolerance. The leading metformin response S-marker in combined group of DM2 patients was the CC variant of OCT1-R61C polymorphism of organic cation transporter protein 1 gene. In cancer patients without DM2, this position belonged to AC and AA genotypes of OCT1_rs622342 polymorphism. Among the A-polymorphisms, GA variant of sex hormone-binding globulin gene SHBG_D356N was less frequently observed in DM2 patients with or without cancer. Besides, in diabetics, the same polymorphic variant of SHBG as well as GC genotype of oxidized lipoprotein receptor OLR1_G501C and GG genotype of locus rs11065987 near BRAP gene were carried rather often in combination with “metformin-positive” variant of OCT1_R61C. In addition, carriers of OCT1_R61C and OCT1_rs622342 polymorphisms with potentially positive reaction to metformin had higher insulin resistance score (HOMA-IR) values. Received data lead to the conclusion that postmenopausal diabetics, both with and without cancer, differ in genetic stigmata of potential response to metformin less than they differ from cancer patients without DM2. As genetic polymorphisms associated with metabolic and anticancer metformin (and, possibly, phenformin) effects may be different, this subject requires further investigation.