Research Of TETs Gene Expression Characteristic And Active Demethylation By 5-aza-dC In Gastric Cancer Cells

Background: Gastric cancer is the second leading cause-related death worldwide owing to its high prevalence,its occurrence and development process of gastric cancer involves a variety of genes,it is a multi-stage and complex ways.Not only associate with genetic changes(such as mutation,gene deletions),but also inextricably linked with epigenetics.Epigenetics which contains DNA methylation refers to the nucleotide sequence does not change,but the expression level of genes changes,thus has an influence on the life activities.DNA methylation play an important role in the development oftumor.Hypermethylation of tumor suppressor gene promoter causes gene inactivation.DNA methylation refers to the cytosines in CpG dinucleotide were catalyticed into 5mC by DNMTs,SAM as a methyldonor.CpG hypermethylation would silence many genes associated with tumor.DNA methylation is a dynamic and reversible process,5mC can occur demethylation,which include passive demethylation and active methylation.Passive demethylation refer to the activity of DNMTs is inhibited by DNMTs inhibitor,such as 5-aza-dC,and the methylation level of new DNA duplexes reduce.Active methylation catalyticed by TET protein family,TET protein is α-ketoglutarate and Fe2+ dependent dioxygenase,not only mediate 5-mC oxidize to 5-hmC,but also 5-fC and 5caC,subsequently recognized and excised by thymine DNA glycosylase(TDG)in vivo and in vitro,and then converted to Cthrough the base excision repair(BER)pathway.TET protein family not only regulate the growth and development of mammalian,but also play an important role in tumogenesis.In previous work,we used DNA methyltransferase inhibitor 5-aza-dC treated gastric cancer cell lines.Found that,some genes which hypermethylation low expression,such as TIMP3,expression increased.This result show that the activity of DNMTs was inhibited,the methylation level of hypermethylation induced,the development of tumor was blocked.Intrestingly,we also observed the change of TET protein.After 5-aza-dC treated,the expression of TET1 gene enhanced in gastric cancer cell lines.We speculate that,as a DNA methyltransferase inhibitor,5-aza-dC may induce active demethylation occur by upregulating TET1 gene when it inhibited DNMTs.Objective: 1)Investigate the expression of TET1,TET2,5mC,5hmCof gastric cancer tissues and relatively normal tissue adjacent to carcinoma.2)Detect the expression of the factors in MGC803 cell line and investigate whether 5-aza-dC have influence on active demethylation.Methods: 1)In gastric cancer and adjacent normal tissues specimens,using RT-qPCRand IHC to detect the expression of TET1,TET2 gene and the level of 5mC,5hmC.2)In gastric cancer cells,using RT-PCR,RT-qPCR,ICCto detect the expression of DNMTs,TETs,TIMP3 and RASSF1 Agene,the level of 5mC,5hmC.Results:1)TET1 gene expression in gastric cancer was lower than the relatively normal tissue adjacent to carcinoma,the difference was statistically significant(p<0.05);TET2 gene expression in gastric cancer was not statistically significant with the relatively normal tissue adjacent to carcinoma.2)The level of 5mC in gastric cancer was higher than the relatively normal tissue adjacent to carcinoma,the difference was statistically significant(p<0.05);The level of 5hmC in gastric cancer was lower than the relatively normal tissue adjacent to carcinoma,the difference was statistically significant(p < 0.05).3)After using demethylating agent 5-aza-dC in MGC803,DNMTsgene expression was decreased gradually;TET1 gene expression was significantly increased(p < 0.05);TET2gene expression has no significantlychange;TIMP3gene expression was increased gradually;RASSF1Agene expression was increased gradually;4)After using demethylating agent 5-aza-dC in MGC803,the level of 5mC decreased gradually;the level of 5hmC increased gradually after TET1 protein enter cell nucleus.Conclusion: 1)Because of TET1 gene expression decreased,the active demethylation mediated by TET1 protein is inhibited in the development of gastric cancer,this is an important reason for gastric cancer cell genomic methylation feature.2)The proportion of 5mC/5hmC in gastric cancer genome is significantly higher than pre-cancerous and normal tissues,suggest that dramatic of 5mC and 5hmC is closely related with the development of gastric cancer.3)5-aza-dC not only mediates passive demethylation through inhibiting the activity of DNMTs,but also participates in active demethylation by increasing TET1 gene expression,changes the methylation states of gastric cancer cell genomic by two mechanisms,make sure some silenced tumor suppressor genes expression again.4)Detection and analysis of TET1,5mC,5hmC can be a mean of gastric cancer early diagnosis,and also can be an important biomarker in clinic treatment and prognosis on tumou.