The Study Of Genome-wide DNA Methylation Involved In The Development And Prognosis Of Gastric Cancer

Gastric cancer is one of the most common malignancies that seriously threaten the health of people in our country.Because of the lack of early,specific and sensitive screening biomarkers,it has brought great difficulties to the early diagnosis and treatment of gastric caner.Therefore,it is of great significance to investigate the etiology,pathogenesis and prognosis of gastric cancer,which can provide help to screen reliable and predictable indicators of the risk and prognosis of gastric cancer.It is significant to the prevention,intervention,treatment and predicting prognosis of gastric cancer.The development and progression of gastric cancer is a complex multi-stage processes involving in genetic and epigenetic changes of cell regulation and aberrant growth,which is the result of the interaction of environmental and genetic factors.Epidemiological investigation showed that infection of Helicobacter pylori(Hp),dietary factors(high salt,N-nitroso compounds,lack of fresh vegetables and fruit intake),smoking,obesity and family history,were closely related to gastric cancer risk.Various environmental risk factors in different stages exert on different genes can cause epigenetic changes,and then lead to related gene changes of expression levels,leading to the development of gastric cancer.Among them the methylation of the regulatory regions of genes is one of the key regulators of the body environmental responses.Methylation of CpG islands in the promoter region plays an important role in the regulation of gene expression.However,the mechanism of DNA methylation at the whole genome level in the development and progression of gastric carcinoma is still scarce.And the molecular mechanism of methylated abnormality of gene regulatory region in the development of gastric cancer has not been fully elucidated.In addition,whether the genetic variation in the CpG islands is associated with the genetic effect and the occurrence and development of gastric cancer need to be further studied.The aim of this study was to explore the mechanism of epigenetics and the interaction between genetic and epigenetic effects in the development and progression of gastric cancer.Part 1 KCNMA1 cooperating with PTK2 is a novel tumor suppressor in gastric cancer and is associated with disease deveopmentBackgroundThe methylation of gene promoter exert on the important effect on the gene expression.Inactivation of tumor suppressor genes by promoter hypermethylation plays a key role in the tumorigenesis.It is necessary to uncover the detailed pattern of whole genome-wide abnormal DNA methylation during the development of gastric cancer(GC).MethodsWe performed a genome-wide methylation detection using 12 paired of the GC tissues and their corresponding normal tissues by Illumina Human methyaltion 450K chip.Methylation-specific PCR(MSP)and bisulphite sequencing(BSP)were used to measure methylation status of specific CpG site.Based on the bioinformatic analysis,the cell phenotypes and mouse model experiments were constructed to detect effect of the target gene.Using the Kaplan-Meier survival curve,the clinical impact of KCNMA1 was assessed in GC patients.ResultsThe CpG site cg24113782 located at the promoter of KCNMA1 showed the most significant difference,contributing to the commonly silenced KCNMA1in gastric cancer cells and primary GC tissues.The promoter methylation of KCNMA1 was detected in 68,7%(77/112)of tumor tissues,compared with 16.2(18/112)of normal tissues(P<0.001).The survival curve indicated that KCNMA1 hypermethylation was significantly associated with the shortened survival in GC patients(P = 0.036).KCNMA1 significantly inhibited biological malignant behavior of gastric cancer cell by inducing cell apoptosis in vitro,and suppressed xenograft tumor growth in subcutaneous mouse models(both P<0.001).Furthermore,the anti-tumorigenic effect of KCNMA1 was mediated through depressing the expression of PTK2,ConclusionKCNMA1 is a critical tumor suppressor in gastric carcinogenesis by regulating the expression of PTK2 and its hypermethylation is an independent prognostic factor in patients with gastric cancer.Part 2 The mechanism study of whole genome SNPs associated with DNA methylation(CpG-SNPs)involved in the gastric cancer riskBackgroundA large number of studies have confirmed that SNPs in genome can influence the methylation status of CpG sites in themself or nearby CpG islands,leading to individual differences in the methylation level of CpG islands.SNPs may alter their CpG islands methylation status,thus disturb transcription factor binding,which can affect the downstream gene transcription regulation and lead to tumorigenesis.Therefore,it is of great significance to explore the interaction between genetic and epigenetic at the genome-wide level to reveal the biological function of the risk locus involving in gastric cancer development.MethodsIn this study,we combine the GC GWAS and Encyclopedia of DNA Elements(ENCODE)to genotype all the variations in the genomic CpG islands by Illumina Human Omni Zhong Hua-8 Bead Chip.Moreover,Molecular biology technologies were used to explore the molecular mechanism of significant SNPs,and uncover the function of the host gene.ResultsWe found the SNP rs2990245 located on the promoter of GBAP1,a pseudogene,showed the most significant related with GC risk after bonferroni correction.In the dominant model,compared with individuals carrying T genotype,the GC risk of people with C genotype was reduced by 20%(OR = 0.80,95%CI = 0.68-0.94,P =0.0076).In addition,in the recessive model,we found the individuals with CC genotype have significantly low GC risk compared with TT and CT genotype(OR=0.40,95%CI = 0.24-0.65,P = 0.0002).Furthermore,we identified that rs2990245 can regulate the methylation of GBAP1 promoter,thus effect the expression of GBAP1.The dual-luciferase reporter assay showed the transcriptional activity of rs2990245T genotype was higher compared with C genotype.In addition,compared with adjacent gastric mucosa,the expression of GBAP1 was significantly higher in the gastric cancer tissues,and the expression of GBAP1 is obviously lower in the cell nucleus than that in the cytoplasm.Moreover,we found the GBAP1 can act as competing endogenous RNAs(ceRNA)to competitively adsorb miR-212 with homologous gene GBA.Significantly positive correlation between GBAP1 and GBA was observed in TCGA and GEO database and validated in our database.ConclusionSNP rs2990245 was significantly associated with the gastric cancer risk.It can influence the expression of GBAP1 by regulating the methylation of its promoter.GBAP1 completely combined with miR-212-3p and released the expression of GBA to participate in the gastric cancer.