Salsolinol as an RNA m~6A methylation inducer mediates dopaminergic neuronal death by regulating YAP1 and autophagy

Salsolinol(1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline,Sal)is a catechol isoquinoline that causes neurotoxicity and shares structural similarity with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine,an environmental toxin that causes Parkinson's disease.However,the mechanism by which Sal mediates dopaminergic neuronal death remains unclear.In this study,we found that Sal significantly enhanced the global level of N~6-methyladenosine(m~6A)RNA methylation in PC12 cells,mainly by inducing the downregulation of the expression of m~6A demethylases fat mass and obesity-associated protein(FTO)and alk B homolog 5(ALKBH5).RNA sequencing analysis showed that Sal downregulated the Hippo signaling pathway.The m~6A reader YTH domain-containing family protein 2(YTHDF2)promoted the degradation of m~6A-containing Yes-associated protein 1(YAP1)mRNA,which is a downstream key effector in the Hippo signaling pathway.Additionally,downregulation of YAP1 promoted autophagy,indicating that the mutual regulation between YAP1 and autophagy can lead to neurotoxicity.These findings reveal the role of Sal on m~6A RNA methylation and suggest that Sal may act as an RNA methylation inducer mediating dopaminergic neuronal death through YAP1 and autophagy.Our results provide greater insights into the neurotoxic effects of catechol isoquinolines compared with other studies and may be a reference for assessing the involvement of RNA methylation in the pathogenesis of Parkinson's disease.

DNA Methylation of KLRC1 and KLRC3 in Autoimmune Thyroiditis:Perspective of Different Water Iodine Exposure

Objective This study aimed to identify differentially methylated genes(DMGs) associated with natural killer cells in patients with autoimmune thyroiditis(AIT), focusing on the influence of varying water iodine exposure levels.Methods Participants were divided into categories based on median water iodine(MWI)concentrations: iodine-fortified areas(IFA, MWI < 10 μg/L), iodine-adequate areas(IAA, 40 ≤ MWI ≤ 100μg/L), and iodine-excessive areas(IEA, MWI > 300 μg/L). A total of 176 matched AIT cases and controls were recruited and divided into 89, 40, and 47 pairs for IFA, IAA, and IEA, respectively. DMGs were identified using 850K Bead Chip analysis for 10/10 paired samples. Validation of DNA methylation and m RNA expression levels of the DMGs was conducted using Methyl Target^(TM) and QRT-PCR for 176/176paired samples.Results KLRC1, KLRC3, and SH2D1B were identified as significant DMGs. Validation revealed that KLRC1 was hypomethylated and highly expressed, whereas KLRC3 was hypermethylated and highly expressed in individuals with AIT. Furthermore, KLRC1 was hypomethylated and highly expressed in both IFA and IEA.Conclusion The DNA methylation status of KLRC1 and KLRC3 may play crucial roles in AIT pathogenesis. Additionally, DNA methylation of KLRC1 seems to be influenced by different iodine concentrations in water.

TCERG1L hypermethylation is a risk factor of diabetic retinopathy in Chinese children with type 1 diabetes

●AIM:To identify the differential methylation sites(DMS)and their according genes associated with diabetic retinopathy(DR)development in type 1 diabetes(T1DM)children.●METHODS:This study consists of two surveys.A total of 40 T1DM children was included in the first survey.Because no participant has DR,retina thinning was used as a surrogate indicator for DR.The lowest 25%participants with the thinnest macular retinal thickness were included into the case group,and the others were controls.The DNA methylation status was assessed by the Illumina methylation 850K array BeadChip assay,and compared between the case and control groups.Four DMS with a potential role in diabetes were identified.The second survey included 27 T1DM children,among which four had DR.The methylation patterns of the four DMS identified by 850K were compared between participants with and without DR by pyrosequencing.●RESULTS:In the first survey,the 850K array revealed 751 sites significantly and differentially methylated in the case group comparing with the controls(|Δβ|>0.1 and Adj.P<0.05),and 328 of these were identified with a significance of Adj.P<0.01.Among these,319 CpG sites were hypermethylated and 432 were hypomethylated in the case group relative to the controls.Pyrosequencing revealed that the transcription elongation regulator 1 like(TCERG1L,cg07684215)gene was hypermethylated in the four T1DM children with DR(P=0.018),which was consistent with the result from the first survey.The methylation status of the other three DMS(cg26389052,cg25192647,and cg05413694)showed no difference(all P>0.05)between participants with and without DR.●CONCLUSION:The hypermethylation of the TCERG1L gene is a risk factor for DR development in Chinese children with T1DM.

Pancreatic agenesis and altered m6A methylation in the pancreas of PDX1-mutant cynomolgus macaques

As an essential transcriptional activator,PDX1 plays a crucial role in pancreatic development andβ-cell function.Mutations in the PDX1 gene may lead to type 4 maturityonset diabetes of the young(MODY4)and neonatal diabetes mellitus.However,the precise mechanisms underlying MODY4 remain elusive due to the paucity of clinical samples and pronounced differences in pancreatic architecture and genomic composition between humans and existing animal models.In this study,three PDX1-mutant cynomolgus macaques were generated using CRISPR/Cas9 technology,all of which succumbed shortly postpartum,exhibiting pancreatic agenesis.Notably,one tri-allelic PDX1-mutant cynomolgus macaque(designated as M4)developed a pancreas,whereas the two monoallelic PDX1-mutant cynomolgus macaques displayed no anatomical evidence of pancreatic formation.RNA sequencing of the M4 pancreas revealed substantial molecular changes in both endocrine and exocrine functions,indicating developmental delay and PDX1haploinsufficiency.A marked change in m6A methylation was identified in the M4 pancreas,confirmed through cultured PDX1-mutantisletorganoids.Notably,overexpression of the m6A modulator METTL3 restored function in heterozygous PDX1-mutant islet organoids.This study highlights a novel role of m6A methylation modification in the progression of MODY4 and provides valuable molecular insights for preclinical research.

MLH1 promoter germline-methylation in selected probands of Chinese hereditary non-polyposis colorectal cancer families

AIM： To detect the MLH1 gene promoter germline- methylation in probands of Chinese hereditary non- polyposis colorectal cancer （HNPCC）, and to evaluate the role of methylation in MLH1 gene promoter and molecular genetics in screening for HNPCC.METHODS： The promoter germline methylation of MLH1 gene was detected by methylation-specific PCR （MSP） in 18 probands from unrelated HNPCC families with high microsatellite-instability （MSI-H） phenotype but without germline mutations in MSH2, MLH1 and MSH6 genes. At the same time, 6 kindreds were col- lected with microsatellite-stability （MSS） phenotype but without germline mutations in MSH2, MIH1 and MSH6 genes as controls. The results of MSP were confirmed by clone sequencing. To ensure the reliability of the results, family H65 with nonsense germline mutation at c.2228C 〉 A in MSH2 gene was used as the negative control and the cell line sw48 was used as the known positive control along with water as the blank control. Immunochemical staining of MIH1 protein was performed with Envision two-step method in those patients with aberrant methylation to judge whether the status of MLH1 gene methylation affects the expression of MLH1 protein.RESULTS： Five probands with MIH1 gene promoter methylation were detected in 18 Chinese HNPCC families with MSI-H phenotype but without germline mutations in MSH2, MLH1 and MSH6 genes. Two of the five probands from families H10 and H29 displayed exhaustive-methylation, fulfilling the Japanese criteria （JC） and the Amsterdam criteria （AC）, respectively. The other 3 probands presented part-methylation fulfilling the AC. Of the 13 probands with unmethylation phenotype, 8 fulfilled the JC and the Bethesda guidelines （BG）, 5 fulfilled the AC. The rate of aberrant methylation in MLH1 gene in the AC group （22.2%, 4/18） was higher than that in the JC/BG groups （5.6%, 1/18） in all HNPCC families with MSI-H phenotype but without germline mutations in PISH2, PIIH1 and MSH6 genes. However, no proband with methylation in MLH1 gene was found in the families with MSS phenotype and without germline mutations in MSH2, MLH1 and MSH6 genes. No expression of MLH1 protein was found in tumor tissues from two patients with exhaustive-methylation phenotype, whereas positive expression of MLH1 protein was observed in tumor tissues from patients with partial methylation phenotype （excluding family H42 without tumor tissue）, indicating that exhaustive-methylation of MLH1 gene can cause defective expression of MLH1 protein.CONCLUSION： Methylation phenotype of MLH1 gene is correlated with microsatellite phenotype of MMR genes, especially with MSI-H. Exhaustive-methylation of MLH1 gene can silence the expression of MLH1 protein. MLH1 promoter methylation analysis is a promising tool for molecular genetics screening for HNPCC.

低压低氧对肠黏膜屏障及LINE-1核酸内切酶变异体GCRG213表达的影响

背景长散在核元件-1(long interspersed nuclear element-1,LINE-1或L1)是目前基因组中唯一活跃的自主反转录转座元件,低压低氧环境可以改变L1的甲基化程度。目的探究低压低氧环境对肠黏膜中L1核酸内切酶(L1 endonuclease,L1-EN)变异体GCRG213表达水平及以闭合蛋白(Occludin)和紧密连接蛋白-1(Claudin-1)表达水平为指标的肠黏膜机械屏障功能的影响。方法利用免疫组织化学染色(immunohistochemical staining,IHC)检测人正常小肠、结肠组织及小鼠正常结肠组织的GCRG213表达,观察GCRG213蛋白在正常肠道中的分布规律。将雄性C57BL/6小鼠随机分为实验组和对照组。实验组模拟海拔6000 m环境,饲养7 d构建低压低氧小鼠模型,对照组常压常氧饲养。将人正常结肠上皮细胞NCM-460随机分为常氧组以及低氧24 h、48 h组,常氧组于常氧环境正常培养,低氧组分别于0.3%O2浓度下培养24 h、48 h构建低氧细胞模型。使用qPCR、Western blot技术检测GCRG213、Occludin和Claudin-1在小鼠结肠组织及人NCM-460细胞中表达水平的变化。结果人正常小肠、结肠及小鼠结肠组织IHC结果一致显示,GCRG213表达于肠黏膜柱状上皮细胞胞质中,而在杯状细胞中未见表达。在动物实验中,与常氧组相比,低压低氧组小鼠结肠组织GCRG213在mRNA水平(P<0.05)和蛋白水平(P<0.01)均表达上调;同时,Occludin在蛋白水平表达下调(P<0.05)。在细胞实验中,低氧暴露后NCM-460细胞中GCRG213在mRNA(P<0.001)和蛋白水平(P<0.01)均表达上调,其中低氧24 h组GCRG213表达水平低于低氧48 h组,Occludin、Claudin-1在mRNA水平(P<0.01;P<0.001)表达下调,其中低氧24 h组表达水平均低于低氧48 h组,Occludin蛋白水平表达在低氧48 h组出现下调(P<0.05)。结论L1-EN变异体GCRG213在人和小鼠正常结肠组织黏膜柱状上皮细胞胞质内存在表达;在体内外模型中,低压低氧暴露下结肠黏膜上皮细胞GCRG213表达升高,同时观察到低压低氧对结肠黏膜紧密连接完整性产生影响,表现为Occludin、Claudin-1蛋白表达水平的降低。

N^(6)-腺苷甲基化修饰及其对LINE-1的调控机制

长散布元件-1(long interspersed elements-1,LINE-1)是现今在人类基因组中唯一具有自主转座能力的转座子,其转座会引起细胞基因组结构和功能的改变,是导致多种严重疾病的重要因素。在转座过程中,LINE-1 mRNA是转座中间体的核心,宿主细胞对其进行相关修饰直接影响转座。N^(6)-腺苷甲基化修饰(m^(6)A)是真核细胞RNA上最丰富且动态可逆的表观遗传修饰。目前发现m^(6)A修饰也存在于LINE-1 mRNA上,参与LINE-1整个生命周期的调控,影响其转座和基因组中LINE-1相邻基因的表达,进而影响基因组稳定性、细胞自我更新与分化潜能,在人类发育和疾病中具有重要作用。本文介绍了LINE-1 m^(6)A修饰的位置、功能以及相关机制,并总结了LINE-1的m^(6)A修饰对其转座调控的研究进展,以期为相关疾病发生发展的机制研究和治疗提供新的思路。

Unveiling DNA methylation in Alzheimer’s disease:a review of array-based human brain studies

The intricacies of Alzheimer’s disease pathogenesis are being increasingly illuminated by the exploration of epigenetic mechanisms,particularly DNA methylation.This review comprehensively surveys recent human-centered studies that investigate whole genome DNA methylation in Alzheimer’s disease neuropathology.The examination of various brain regions reveals distinctive DNA methylation patterns that associate with the Braak stage and Alzheimer’s disease progression.The entorhinal cortex emerges as a focal point due to its early histological alterations and subsequent impact on downstream regions like the hippocampus.Notably,ANK1 hypermethylation,a protein implicated in neurofibrillary tangle formation,was recurrently identified in the entorhinal cortex.Further,the middle temporal gyrus and prefrontal cortex were shown to exhibit significant hypermethylation of genes like HOXA3,RHBDF2,and MCF2L,potentially influencing neuroinflammatory processes.The complex role of BIN1 in late-onset Alzheimer’s disease is underscored by its association with altered methylation patterns.Despite the disparities across studies,these findings highlight the intricate interplay between epigenetic modifications and Alzheimer’s disease pathology.Future research efforts should address methodological variations,incorporate diverse cohorts,and consider environmental factors to unravel the nuanced epigenetic landscape underlying Alzheimer’s disease progression.

Effect of insulin and metformin on methylation and glycolipid metabolism of peroxisome proliferator-activated receptor γcoactivator-1A of rat offspring with gestational diabetes mellitus

Objective:To discuss the effect of insulin and metformin on amethylation and glycolipid metabolism of peroxisome proliferator-activated receptor γ coactivator-1A(PPARGC1A) of rat offspring with gestational diabetes mellitus(GDM).Methods:A total of 45 pregnant rats received the intraperitoneal injection of streptozotocin to establish the pregnant rat model of GDM.A total of 21 pregnant rats with GDM were randomly divided into three groups,with 7ruts in each group,namely the insulin group,metformin group and control group.Rats in the insulin group received the abdominal subcutaneous injection of 1 mL/kg recombinant insulin glargine at 18:00 every day.Rats in the metformin group received the intragastric infusion of metformin hydrochloride at 18:00 every day,with the first dose of 300 mg/kg.The doses of two groups were adjusted every 3 d to maintain the blood glucose level at 2.65-7.62 mmol/L.Rats in the control group received the intragastric infusion of 1 mL normal saline at 18:00 every day.After the natural delivery of pregnant rats.10 offspring rats were randomly selected from each group.At birth,4 wk and 8 wk after the birth of offspring rats,the weight of offspring rats was measured.The blood glucose level of offspring rats was measured at 4wk and 8 wk,while the level of serum insulin,triglyceride and leptin was measured at 8 wk.Results:The weight of offspring rats at birth in the insulin group and metformin group was significantly lower than the one in the control group(P<0.05),and there was no significant difference at 4 wk and 8 wk among three groups(P>0.05).The fasting blood glucose and random blood glucose in the insulin group and metformin group at 4 wk and 8 wk were all significantly lower than ones in the control group(P<0.05);there was no significant difference between the insulin group and metformin group(P>0.05).The expression of PPARGC1 A mRNA in the insulin group and metformin group was significantly higher and the methylation level of PPARGC1 A was significantly lower than the one in the control group(P<0.05),but there was no significant difference between the insulin group and metformin group(P>0.05).Insulin and leptin at 8 wk in the insulin group and metformin group were significantly higher,while triglyceride was significantly lower than the one in the control group(P<0.05);triglyceride level of rats in the insulin group was significantly higher than the one in the metformin group(P<0.05).There was no significant difference in insulin and leptin level of offspring rats between the insulin group and metformin group(P>0.05).Conclusions:GDM can induce the methylation of PPARGC1 A of offspring rats to reduce the expression of PPARGC1 A mRNA and then cause the disorder of glycolipid metabolism when the offspring rats grow up;the insulin or metformin in the treatment of pregnant rats with GDM can reduce the methylation level of PPARGC1 A and thus improve the abnormal glycolipid metabolism of offspring rats.

Promoter methylation and mRNA expression of DKK-3 and WIF-1 in hepatocellular carcinoma

AIM:To investigate the promoter methylation status and mRNA expression of DKK-3 and WIF-1 gene in hepatocellular carcinoma(HCC).METHODS:DKK-3 and WIF-1 acted as Wnt-antagonists and tumor suppressors,but hypermethylation of the gene promoter and low mRNA expression activated Wnt signaling aberrantly and induced the development of HCC.Methylation status of the DKK-3 and WIF-1 gene promoter was investigated using methylation specific polymerase chain reaction(PCR) in tumor and adjacent non-cancerous tissues from 33 HCC patients and 20 normal liver tissues served as control.The expression of DKK-3 and WIF-1 mRNA was also determined by real-time quantitative reverse transcriptase PCR.The relationship between methylation,mRNA expression,and clinical data,as well as methylation and mRNA expression of the two genes were analyzed.RESULTS:The methylation of DKK-3 and WIF-1 genes in HCC increased significantly compared with adjacent non-cancerous tissues and normal control tissues(χ2 =7.79,P < 0.05;χ2 = 4.89,P < 0.05),and no significant difference in methylation between adjacent non-cancerous tissues and normal control tissues was observed.In HCC tissues,significant differences in the DKK-3 promoter methylation were observed in age and cirrhosis,and significant differences of the WIF-1 promoter methylation were observed in HBsAg and cirrhosis.The average expression of DKK-3 mRNA in HCC and adjacent non-cancerous tissues was increased significantly compared with normal control tissues.The average expression of WIF-1 mRNA showed no significant difference among the three tissues.The mRNA expression of DKK-3 gene in HCC was decreased as the pathological grade increased.CONCLUSION:The aberrant promoter methylation and decreased expression of DKK-3 and WIF-1 may be an important mechanism in HCC,and may be a far-reaching significance in early diagnosis and therapy of HCC.

Promoter methylation status of hMLH1,MGMT,and CDKN2A/p16 in colorectal adenomas

AIM:To investigate aberrant DNA methylation of CpG islands and subsequent low-or high-level DNA microsatellite instability(MSI)which is assumed to drive colon carcinogenesis. METHODS:DNA of healthy individuals,adenoma(tu-bular or villous/tubulovillous)patients,and colorectal carcinoma patients who underwent colonoscopy was used for assessing the prevalence of aberrant DNA methylation of human DNA mismatch repair gene mutator L homologue 1(hMLH1),Cyclin-dependent kinase inhibitor 2A(CDKN2A/p16),and O-6-methylguanine DNA methyltransferase(MGMT),as well as their rela- tion to MSI. RESULTS:The frequency of promoter methylation for each locus increased in the sequence healthy tissue/adenoma/carcinoma.MGMT showed the highest frequency in each group.MGMT and CDKN2A/p16 presented a statistically significant increase in promoter methylation between the less and more tumorigenic forms of colorectal adenomas(tubular vs tubullovillous and villous adenomas).All patients with tubulovillous/villous adenomas,as well as all colorectal cancer patients,showed promoter methylation in at least one of the examined loci.These findings suggest a potentially crucial role for methylation in the polyp/adenoma to cancer progres- sion in colorectal carcinogenesis.MSI and methylation seem to be interdependent,as simultaneous hMLH1, CDKN2A/p16,and MGMT promoter methylation was present in 8/9 colorectal cancer patients showing the MSI phenotype. CONCLUSION:Methylation analysis of hMLH1,CD- KN2A/p16,and MGMT revealed specific methylation profiles for tubular adenomas,tubulovillous/villous adenomas,and colorectal cancers,supporting the use of these alterations in assessment of colorectal tumorigenesis.

Microsatellite instability and MLH1 promoter hypermethylation in colorectal cancer

Colorectal cancer （CRC） is caused by a series of genetic or epigenetic changes, and in the last decade there has been an increased awareness that there are multiple forms of colorectal cancer that develop through different pathways. Microsatellite instability is involved in the genesis of about 15% of sporadic colorectal cancers and most of hereditary nonpolyposis cancers. Tumors with a high frequency of microsatellite instability tend to be diploid, to possess a mucinous histology, and to have a surrounding lymphoid reaction. They are more prevalent in the proximal colon and have a fast pass from polyp to cancer. Nevertheless, they are associated with longer survival than stage-matched tumors with microsateUite stability. Resistance of colorectal cancers with a high frequency of microsatellite instability to 5-fluorouracilbased chemotherapy is well established. Silencing the MLH1 gene expression by its promoter methylation stops the formation of MLH1 protein, and prevents the normal activation of the DNA repair gene. This is an important cause for genomic instability and cell proliferation to the point of colorectal cancer formation. Better knowledge of this process will have a huge impact on colorectal cancer management, prevention, treatment and prognosis.

Promoter Hypermethylation of KiSS-1 Gene in Gastric Cancer

Objective： To investigate the association between KiSS-1 methylation and clinicopathological characteristics of gastric cancer and evaluate the role of peritoneal lavage fluid in detecting peritoneal metastases. Methods： The methylation status of KiSS-1 gene in 40 gastric cancer specimens, the corresponding adjacent normal mucosa, lymph nodes and peritoneal lavage fluid was investigated by methylation-spcific polymerase chain reaction（MS-PCR）. Results： Aberrant methylation of KiSS-1 gene was detected in 55%（22/40） of the adjacent normal mucosa, 82.5% （33/40）of gastric cancer specimens, 80.95%（17/21） of the lymph nodes, and 42.5%（17/40） of peritoneal lavage fluid. Methylation in gastric carcinoma and lymphonode was more frequent than in non-neoplastic gastiric mucosa. Presence of KiSS-1 methylation in peritoneal lavage fluid was significantly correlated with tumor invasion （P=0.043）. The accuracy of KiSS-1 methylation in peritoneal lavage fluid for diagnosing peritoneal metastasis was 70%, with a sensitivity of 77.8% and a specificity of 67.7%. Conclusion： Aberrant methylation of KiSS-1 gene is a common event in the occurrence and progression of gastric carcinoma, which may provide useful information for the early diagnosis of peritoneal metastases and a new therapy for gastric cancer.

KISS1 methylation and expression as predictors of disease progression in colorectal cancer patients

AIM: To examine the effect of aberrant methylation of the KISS1 promoter on the development of colorectal cancer (CRC) and to investigate reversing aberrant methylation of the KISS1 promoter as a potential therapeutic target.

MT1M and MT1G promoter methylation as biomarkers for hepatocellular carcinoma

AIM: To investigate the potential of promoter methylation of two tumor suppressor genes (TSGs) as biomarkers for hepatocellular carcinoma (HCC).

Hypermethylation of TGF-β1 gene promoter in gastric cancer

AIM:To examine transforming growth factor-β1(TGF-β1)promoter methylation in gastric cancer and to determine if Helicobacter pylori(H.pylori)or interleukin(IL)-1β could induce TGF-β1 hypermethylation in vitro.METHODS:We examined the frequency and extent of TGF-β1 promoter methylation using methylationspecific PCR in the gastric tissues from 47 gastric cancer patients and 39 non-gastric cancer subjects.H.pylori infection was confirmed by a positive result from either a serological test,histological analysis or C13urea breath test.GES-1 and MKN-45 cells co-cultured with H.pylori or treated with IL-1β for 12,24 and 48 h in vitro tested the effects of H.pylori or IL-1β on TGF-1β.RESULTS:Twenty-four/forty-seven(51%)cases of gastric cancer(GC)tissues showed TGF-β1 promoter methylation,15/47(31.9%)cases of matched noncancerous gastric mucosa tissues from the GC patients,and 11/39(28%)case of the normal gastric mucosa tissues from non-GC subjects showed TGF-β1 promoter methylation(51%vs 28%,P<0.05).Significantly higher levels of methylation of TGF-β1 were found in the tumor tissues than in non-tumor tissues from GC patients(0.24±0.06 vs 0.17±0.04,P<0.05)and normal gastric tissues from non-GC subjects(0.24±0.06 vs 0.15±0.03,P<0.05).TGF-β1 methylation was found in 48.3% of H.pylori-positive gastric mucosal tissues whereas only 23.1% of H.pylori-negative gastric mucosal tissues showed TGF-β1 methylation(48.3%vs 23.1%,P<0.05).IL-1β appeared to induce a dose-dependent methylation of TGF-β1 and the strongest methylation was observed in GES-1 cells treated with 2.5 ng/mL of IL-1β for 48 h.Further studies showed that pre-treatment of GES-1 cells with 20ng/mL IL-1RA for 1 h could partially abolish the effect of IL-1β on TGF-β1 methylation.Infection of GES-1cells by H.pylori was not found to induce significant TGF-β1 promoter methylation.CONCLUSION:Our data revealed that TGF-1 promoter is methylated in GC patients.IL-1β may be an important mediator for H.pylori induced gene methylation during GC development.

BRCA1 affects global DNA methylation through regulation of DNMT1

Global DNA hypomethylation at CpG islands coupled with local hypermethylation is a hallmark for breast cancer, yet the mechanism underlying this change remains elusive. In this study, we showed that DNMT1, which encodes a methylation maintenance enzyme, is a transcriptional target of BRCA1. BRCA1 binds to the promoter of the DNMT1 gene through a potential OCT1 site and the binding is required for maintaining a transcriptional active configuration of the promoter in both mouse and human cells. We further demonstrated that impaired function of BRCA1 leads to global DNA hypomethylation, loss of genomic imprinting, and an open chromatin configuration in several types of tissues examined in a BRCA1 mutant mouse model at premaligant stages. BRCA1 deficiency is also associated with significantly increased expression levels of several protooncogenes, including c-Fos, Ha-Ras, and c-Myc, with a higher expression in tumors, while premalignant mammary epithelial cells displayed an intermediate state between tumors and controls. In human clinical samples, reduced expression of BRCA1 correlates with decreased levels of DNMT1, and reduced methylation of CpG islands. Thus, BRCA1 prevents global DNA hypomethylation through positively regulating DNMT1 expression, and this provides one of mechanisms for BRCAl-associated breast cancer formation.

Methylation of PTCH1a gene in a subset of gastric cancers

AIM： To establish if PTCHla transcriptional regulation region （TRR） is methylated in gastric cancer and its influence in gastric tumorigenesis.METHODS： The CpG islands in PTCHla TRR were analyzed by Methyl Primer Express v1.0 software. The region from -643 to -355 bp （the transcription initiation site of PTCHla was designated as 0） that contained 19 CpG sites was chosen for bisulfitesequencing PCR （BSP） and methylation-specific PCR （MSP） detection. The gastric cancer cell line AGS was treated with 5-aza-2′-deoxycytidine （5-Aza-dC; 1 μmol/L） for 3 d. Alterations in PTCHla TRR methylation in treated AGS cells was measured through BSP clone sequences, and their PTCH1 expression was measured by quantitative RT-PCR. The cell cycle and apoptosis were observed with flow cytometry through propidium iodide （PI） staining or annexin V/PI double staining. The prevalence of PTCHla TRR methylation was investigated in 170 gastric cancer tissue samples and the adjacent normal tissues by MSP. The correlation of PTCH1a TRR methylation with PTCH1 expression or with patients＇ clinical features was analyzed.RESULTS： Methylation of PTCHla TRR was observed in AGS ceils and a subset of gastric cancer tissues （32%, 55/170）, while no methylation amplification products were observed in any normal tissues by MSP. The methylation of PTCH1α TRR was correlated negatively with PTCH1 expression （Spearman＇s r = -0.380, P = 0.000）. However, methylation of PTCHla TRR was not related to the gastric cancer patients＇ clinical features, such as sex, age of onset, clinical stage, lymph node metastasis or histological grade. The methylation of PTCHla TRR in AGS cells was almost converted to non-methylation after 5-Aza-dC treatment, which increased PTCH1 expression （5.3 ± 2.5 times; n = 3） and apoptosis rate （3.0 ± 0.26 times; P 〈 0.05; n = 3）.CONCLUSION： Methylation of PTCH1α TRR is present in a subset of gastric cancers and correlated negatively with PTCH1 expression. This may be an early event in gastric tumorigenesis and a new treatment target.

Methylation of RAR-β2, RASSF1A, and CDKN2A Genes Induced by Nickel Subsulfide and Nickel-carcinogenesis in Rats

Objective To investigate the expression variation of RAR‐β2, RASSF1A, and CDKN2A gene in the process of nickel‐induced carcinogenesis. Methods Nickel subsulfide （Ni 3 S 2 ） at dose of 10 mg was given to Wistar rats by intramuscular injection. The mRNA expression of the three genes in induced tumors and their lung metastasis were examined by Real‐time PCR. The methylation status of the 5’ region of these genes were detected by Quantitative Real‐time methylation specific PCR. Results The mRNA expressions of the three genes both in muscle and lung tumor were decreased distinctly in comparison with normal tissue. But hypermethylation was found only in muscle tumor. Conclusion These findings suggest that loss of function or decrease of RAR‐β2, RASSF1A, and CDKN2A, as well as the hypermethylation of 5’ region of these genes, are related with nickel exposure.

Methylation of DAPK and THBS1 genes in esophageal gastric-type columnar metaplasia

AIM: To explore methylation of DAPK, THBS1, CDH-1, and p14 genes, and Helicobacter pylori(H. pylori) status in individuals harboring esophageal columnar metaplasia.METHODS: Distal esophageal mucosal samples obtained by endoscopy and histologically diagnosed as gastric-type(non-specialized) columnar metaplasia, were studied thoroughly. DNA was extracted from paraffin blocks, and methylation status of deathassociated protein kinase(DAPK), thrombospondin-1(THBS1), cadherin-1(CDH1), and p14 genes, was examined using a methyl-sensitive polymerase chain reaction(MS-PCR) and sodium bisulfite modification protocol. H. pylori cag A status was determined by PCR.RESULTS: In total, 68 subjects(33 females and 35 males), with a mean age of 52 years, were included. H. pylori cag A positive was present in the esophageal gastric-type metaplastic mucosa of 18 individuals. DAPK, THSB1, CDH1, and p14 gene promoters were methylated by MS-PCR in 40(58.8%), 33(48.5%), 46(67.6%), and 23(33.8%) cases of the 68 esophageal samples. H. pyloristatus was associated with methylation of DAPK(P = 0.003) and THBS1(P = 0.019).CONCLUSION: DNA methylation occurs in cases of gastric-type(non-specialized) columnar metaplasia of the esophagus, and this modification is associated with H. pylori cag A positive infection.