| Objective: To examine the expressions of DNA methyltransferase 3a(DNMT3a)in colorectal cancer tissue and normal mucosa using Taqman real time PCR and study the relationship between the expressions and the clinicopathological characteristics of patients.And to observe the effects of DNMT3 a on proliferation and apoptosis of colon cell line HCT116 by Cell Counting Kit-8(CCK-8),FCM and Transwell experiment.To evaluate diagnosis value of DNMT3 a for colorectal cancer as tumour marker,elucidate the biological function of DNMT3 a in the development of tumor,provide theoretical and experimental basis for early diagnosis of colorectal cancer.Methods:1 25 paired fresh colorectal tissues and their matched normal mucosa tissues were collected from Hebei Medical University Fourth Hospital and Hebei Medical University First Hospital between May,2012 and March,2015.2 Examination the expressions of DNMT3 a in colorectal cancer tissue and normal mucosa using by Taqman real time PCR,and study the relationship between the expressions and the clinicopathological characteristics of patients.3 Cell culture was performed for colon cell line HCT116.The small interfering RNA(siRNA)of DNMT3 a was transfected into HCT116 cells by Lipofectamine 2000,and the control group(transfected with negative control siRNA)was set up.Examination the expressions of DNMT3 a after transfection 24 h in the two groups.4 After transfection of DNMT3 a siRNA,the OD value was measured by CCK-8 method at 1,2,3,4 and 5 day,and the cell growth curve was drawn to observe the cell proliferation.5 Flow Cytometry were performed in negative control and experimental group to analyze the effect of DNMT3 a on apoptosis of HCT116 cell.6 Transwell technique was used to detect the changes of tumor cell migration ability before and after transfection.7 All experimental data were analyzed by SPSS 13.0 statistical software.The results were measured by(meansstandard±deviations)or median(quartile).Wilcoxon-test or t-test was used to analyze comparison of paired sample,Mann-Whitney-test to analyze independent sample,two-Way ANOVA to analyze repeated measurement data.P<0.05 was regarded as statistical significance.Results:1 DNMT3 a in 25 cases of colorectal cancer tissue and corresponding normal mucosa the expression level of cutting edge were 2.332(1.3685,4.653)and 0.9073(0.2708,1.5185),the expression of colorectal cancer tissues than in normal mucosa tissues increased significantly(P < 0.0001).2 The expression level of DNMT3 a and the pathological type of the patients with colorectal cancer is closely related to T stage 3,4 patients was significantly higher than that in patients with T1,T2(P =0.0239),5cm,tumor patients was significantly higher than 5cm patients(P =0.0043),and gender,age,pathological type,differentiation degree and have no lymph node metastasis(P >0.05).3 After transfection with 24 h,the expression of DNMT3 a in the experimental group was significantly lower than that in the negative control group,the difference was statistically significant.4 Transfection of DNMT3 a siRNA in the experimental group after transfection of 1d,2d,3d,4d,5d,5 times OD values are lower than that of the negative control group,the difference was statistically significant(each P <0.05).5 In HCT116 cells,the apoptosis rate of the experimental group(3.35± 0.451)% was significantly higher than that of the control group(2.733±0.745)%,the difference was not statistically significant(P=0.4016)6 24 hours after transfection assays of Transwell cells,the cells in the experimental group the number of migration(130+ 12),negative control group,the number of cell migration(231 + 14),migration ability in experimental group was significantly lower than the negative control group(P=0.0395).Conclusions:1 DNMT3 a was highly expressed in colorectal cancer and was significantly higher in tumors with tumor> 5cm,T stage 3 and 4 than in patients with tumor ?5cm and T stage earlier.The results showed that DNMT3 a was found in colorectal The occurrence and development of cancer may play the role of oncogene,and may through a mechanism to promote the occurrence of rectal cancer and tumor malignancy progress.2 Down-regulation of DNMT3 a expression,can inhibit the proliferation of colon cancer cells and reduce their ability to migrate,and can promote colon cancer cell apoptosis process.3 In-depth study of DNMT3 a cancer can provide a theoretical basis for the pathogenesis of colon cancer.And provide new ideas and targets for early diagnosis,prognosis and targeted therapy of colon cancer patients. |
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