Association Study Of Wnt Antagonists Methylation With Signal Pathway Activation And Prognosis In Patients With Gastric Cancer

Gastric cancer(GC)is the second largest cancer in China,its incidence and mortality are only behind to lung cancer.The patient's 5-year survival rate is only about 30%.The development of gastric cancer is closely related to the abnormal activation and inactivation of multiple signaling pathways.Among them,Wnt signaling pathway play an important role in the gastric carcinogenesis.In recent years,it was found that Wnt inhibitor genes methylation are an important mechanism for the pathway activation.And studies have reported that abnormal changes in epigenetics is an important factor for gastric cancer.Based on this,we focus on several key inhibitor genes of the Wnt signaling pathway.In the first part,we selected six Wnt antagonist genes(SFRP2,SFRP4,SFRP5,DKK1,DKK2,APC).CpG islands were analyzed by methPrimer online tool.Amplification and sequencing primers were designed.Pyrosequencing and Real-time PCR were used to detect the methylation level and mRNA expression level of these genes.The association of these molecular data and clinical information of gastric cancer were analyzed.We found aberrant promoter methylation of SFRP2,SFRP4,DKK1,and DKK2 observed in GC tissues was significantly higher than that in the control.The mRNA expression levels of SFRP2 and DKK2 genes were significantly decreased in GC.Co-methylation of Wnt antagonists played an important role in tumorigenesis.SFRP2 and DKK2 were concurrently hypermethylated and the hypermethylation of these two genes cause activation of WNT signaling pathway.Survival analysis showed that hypermethylation of SFRP2 and DKK2 genes was closely related to the poor overall survival of patients,indicating that the two genes methylation may be a potential prognostic marker.In the second part,we constructed DKK2 gene lentiviral expression vector.The function of the gene was studied by CCK8 cell proliferation,clonal formation,apoptosis,cell migration and cell invasion.The results showed that overexpression of DKK2 gene could significantly decrease the growth rate and clonal ability of gastric cancer cell,and DKK2 could also induce cell apoptosis.In addition,overexpression of DKK2 gene can significantly reduce the migration and invasion of gastric cancer cell,indicating that DKK2 as a tumor suppressor gene is very important in the development of gastric cancer.In the third part,we used TCGA data to analyze all Wnt signaling genes(including canonical and non-canonical signaling pathways)from gene mutations,copy number mutations,mRNA expression,and methylation levels with clinical and molecular information.It was found that the mutations of Wnt signaling pathway(mainly gene amplification and gene deletion)occurred in high frequency in GC.The abnormal frequency of mRNA expression levels of 13 genes were higher than 10%in gastric cancer.SMAD4 gene mutation had marginally significant poor overall survival.The high expression of SENP2 gene had marginally significant good overall survival rate of GC.In the methylation cluster analysis,gastric cancer samples were clustered into three distinct groups.And the group is associated with molecular classification,CIMP status,tumor level of GC.there was a significant increase in ?-catenin activity in the hypermethylated group,indicating that the abnormal increase of methylation was an important factor for the activation of WNT signaling pathway.