Impact Of DNMT1 On The Proliferation And Invasion Of Gastric Carcinoma Cells And Its Mechanism

DNA methyltransferase 1(DNMT1)is the most abundant DNA methylation transferase in mammalian cells.It is significantly up-regulated in a variety of tumor tissues and it often preceded abnormal methylation status.Thus it is the early molecular change that is the characteristic of tumor cells.DNMT1 mainly mediated methylation pattern of the establishment and maintenance.The high expression of DNMT1 can cause abnormal methylation patterns,tumor suppressor gene silencing and oncogene activation,which may result in tumorigenesis.Gastric cancer is one of the most common malignant tumors in our country.The pathogenesis of gastric cancer is still not clear.Due to the lack of effective biomarkers and early diagnosis,most patients have a poor prognosis in the progressive stage.Studying the relationship between DNMT1 and gastric cancer is helpful to clarify the mechanism of the occurrence and progression of gastric cancer,and to provide a new method for the diagnosis and treatment of gastric cancer.The aim of this study is to explore the effect and mechanism of DNMT1 on cell proliferation in gastric cancer.This study will detect the expression of DNMT1 in gastric cancer tissue and observe the effect of DNMT1 on the proliferation and invasion of gastric cancer cells and the expression of related molecules.And we will investigate the relationship between DNMT1 and the development of gastric cancer and its mechanism in order to provide theoretical basis for further enriching the diagnosis and treatment of gastric cancer.Method:1.We evaluated DNMT1 levels in 60 gastric cancer tissues and their corresponding non-tumorous tissues by Real-time PCR.2.We established slow virus vector and plasmid vector of DNMT1-shRNA,tested the packaging and virus titer of the slow virus,evaluated the interference effect of interference vector on target gene,transfected MKN45?SGC7901?MKN28 gastric cancer cells and built stable strains of DNMT1-shRNA.At the same time,we established a slow virus negative control(NC-shRNA)transfected target cells and non transfected cell lines as control group.3.Cell proliferation assay analyzed the proliferation effect of MKN45?SGC7901 and MKN28 gastric cancer cells which were transfected by DNMT1-shRNA with control group.4.The migration ability of MKN45?SGC7901?MKN28 cell lines which was transfected by DNMT1-shRNA,and control cell lines were analyzed by transwell migration assay.5.The expression of CDC25 B in MKN45?SGC7901?MKN28 cell lines and control group was detected by Western-blotting and Real-time PCR assays.Result:1.Real-time PCR showed the DNMT1 levels were increased in gastric cancer tissues(P<0.05).The expression level of DNMT1 in 52 cases of gastric carcinoma in 60 cases(86.7%,52/60)was significantly higher than that in the corresponding adjacent tissues,and 17 cases of gastric carcinoma in 60 cases(28.3%,17/60)was up-regulated more than 2 times.The expression of DNMT1 was significantly higher in patients,who suffer from gastric cancer with lymph node metastasis,tumor adjacent vascular invasion and III to IV stage of TNM,higher than in other patients who suffer from gastric cancer without lymph node metastasis,without cancer adjacent vascular invasion and I to II stage of TNM.2.Cell proliferation assay:the proliferation ability of gastric cancer cell line,which was transfected by DNMT1-shRNA,was significantly down-regulated compared with that of the control group(P<0.05).It suggested that DNMT1 could promote the proliferation of gastric cancer cells.3.Cell migration test: interfering DNMT1 could significant inhibit the migration of gastric cancer cells(P<0.05).4.Western-blotting:interfering the expression of CDC25 B protein could reduce the protein expression levels in MKN45?SGC7901?MKN28 cell lines.The results of Real-time PCR also showed that interfering the expression of DNMT1 could make mRNA expression level of CDC25B down-regulate in gastric cancer cells.It might indicate that CDC25 B was the downstream target gene of DNMT1,and DNMT1 might affect the occurrence and progression of gastric carcinoma by regulating CDC25 B.Conclusion:DNMT1 is up regulated in gastric cancer,and it could promote proliferation and distant metastasis of gastric cancer cells.DNMT1 expression may be one of the mechanisms that affect the occurrence and progression of gastric cancer by regulating the expression of CDC25B.