TET1 Induces DNA Hydroxymethylation Of Gastric Cancer Related Genes PTEN And EpCAM

Objective The epigenetic regulatory effect on tumor related genes has been one of important fields of tumor research.The most classical epigenetic modification is DNA methylation and demethylation.DNA hydroxymethylation is a novel epigenetic modification.DNA hydroxymethylation is mainly induced by TET family which includes TET1,TET2 and TET3.Hydroxymethylation in most tissues is completed by TET1.It has been reported that TET1 level down regulated in many kinds of tumors.Consequently,the genome 5-hm C content was also down regulated.These phenomenons indicate important role of 5-hm C produced by TET1 in cell malignant transformation.In this study we explored TET1 function on gastric cancer development and progesssion.Methods TET1 and 5-hm C was detected by Immunohistochemistry(IHC),Western blot(WB)and Dot blot content in gastric cancer(GC)tissues and gastric cancer cells.Chi-square test was used to analyze the correlationship between TET1 expression and clinicopathologic characteristics.sh RNA and TET1 over-expression plasmid were used to knock down(KD)or over express TET1 in GC cell lines.Then CCK-8 assay,would healing assay and transwell assay were used to observe the influence of TET1 on GC cells.Quantitative PCR(q PCR)was also used to detect the expression of TET1 target genes.And we used Bisulfite sequencing PCR(BSP)to detect methylation status in the promoters of these potential target genes.Furthermore,Glu MS-PCR was used to detect the 5-hmC content in the promoters of these genes.In this research,we also used WB to detect AKT and FAK pathway after TET1 was KD or over-expressed in order to clarify how TET1 regulated biological function of GC cells.At last,we injected GC cells subcutaneously and intraperitneally on nude mice to demonstrate that TET1 KD could promote tumor growth and metastasis.Results(1)Compared with adjacent non-tumor tissues,TET1 expression was significantly down-regulated in GC tissues(Wilcoxon test,p=0.036)and 5-hm C content in genomic DNA was also down-regulated.(2)Kaplan-meier survival analysis showed that GC patients with higher TET1 level had better prognosis than those with lower TET1 expression(p<0.01).(3)When TET1 was knocked down in NCI-N87 cell,obvious cell growth could be observed in CCK-8 assay(p<0.05)and the potential of migration and invasion also increased(p<0.01).While TET1 over-expression in SGC7901 inhibited cell growth(p<0.05)and cell metastasis also be suppressed(p<0.01).(4)TET1 could alter the 5-m C and 5-hm C content in the promoter of GC related genes such as PTEN.When TET1 was knocked down in NCI-N87,the percentage of methylation in PTEN promoter was up-regulated from 1.5% to 12%.While TET1 knocked down in SGC7901 down regulated the percentage of methylation in PTEN promoter from 3% to 0.9%.Through Ch IP assay,we confirmed the direct binding of TET1 to PTEN promoter.(5)In animal experiments,we observed bigger and heavier tumor in TET1 KD group as compared with NC group(p<0.05).And TET1 knocked down induced more tumor nodes in abdominal cavity as compared with NC group(p<0.01).Conclusion We found that TET1 content was down-regulated in GC tissues as compared with its adjacent non-tumor tissues.TET1 down-regulated TET1 induced hypermethylation in PTEN promoter,inhibited PTEN expression,activated AKT and FAK pathway.These made GC cells possessed increasing potential of metastasis.So we clarified the mechanism of cell malignant transformation induced by TET1 low expression in GC cells.