| Objective: Breast cancer is the most common malignancy of women worldwide. 90% of the patients died in clinical practice are due to the recurrence and metastasis of breast cancer. Therefore, the mechanisms, involved in the genesis, development, invasion and metastasis of breast cancer, have become a research focus. Recently, TET1 has been discovered capable of mediating the sequential oxidation of 5-methylcytosine(5m C) to 5-hydroxymethylcytosine(5hm C), leading to eventual DNA demethylation. The amount of 5hm C and TET mutation have been found to be closely associated with the genesis,development and prognosis of different tumors, but their mechanisms still remain controversial.The expression of TET1 in breast cancer tissue and benign breast lesion was detected to investigate the relation between TET1 level and the clinicopathological features of breast cancer. Then expression of TET1 was determined in different breast cancer cell lines. At last, to probe into the effects of TET1 on the proliferation, cell cycle distribution, migration of MCF-7 cells and its possible mechanisms, plasmid vector containing TET1 was transfected into MCF-7 cells to construct stable transfection cell line.Methods:1 62 patients with breast cancer and 10 patients with benign breast lesion were recruited from January 2012 to June 2014 at breast cancer center of the Fourth Hospital of Hebei Medical University. The expression of TET1 in breast cancer tissue and benign cancer lesion was detected by Max Vision TM One Step immunohistochemical stain after surgery. Then TET1 level was compared with the clinicopathological features of breast cancer, such as age, tumor size, lymph node status, pathological stage, histological grade, vessel tumor embolus, molecular subtype and the level of ER, PR, HER-2, P53 and Ki-67, according to the reports provided by the pathology department.2 The expression of TET1 in four different breast cancer cell lines, such as SK-BR-3, MCF-7, MDA-MB-231 and MDA-MB-453, was detected by PCR and Western blot. The non-tumorderived renal epithelial cells, HEK293, and umbilical cord derived mesenchymal stem cells(hmsc) were set as control groups.3 The plasmid carrying TET1 and e GFP was respectively transfected into MCF-7 cells to construct stable transfection cell lines, MCF-7-TET1 and MCF-7-vector. TET1 expression level was detected by SYBRGreen quantitive real-time PCR.4 Cell morphology was observed by light microscope. Cell proliferation was detected by CCK-8 and the flow cytometry was adopted to determine cell cycle distribution. The migration ability was detected by wound healing essay and the expression level of EMT-related genes, such as CDH1, VIM, TGFB1, and CTNNB1, was measured by quantitive real-time PCR.5 Statistical analyses were performed by SPSS 19.0 software. Results of immunohistochemical stain were analyzed using Chi-square test and Spearman order correlated examination. In the cell experiments, differences of paired groups were assessed by paired-sample t test and differences among multiple groups were evaluated by one-way ANOVA method with multiple comparisons made by Tukey test. Results were expressed as mean±SD. For each protocol, at least three independent experiments were performed. All statistical tests were considered significant at P<0.05.Results:1 In the 62 cases of primary breast cancer specimens, 33 cases displayed low expression of TET1, which accounted for 53.23%, while 29 cases displayed high expression of TET1. In the 10 cases of benign breast lesion specimens, no cases displayed low expression, which meant that the low expression rate of TET1 was 0% in patients with benign breast lesions. The expression difference of TET1 between primary breast cancer and benign breast lesion was of statistical significance, with χ2=7.799 and P=0.005.2 The cases with primary breast cancer were divided into 2 groups according to tumor size, ≤2cm group with 32 cases and >2cm group with 30 cases. The expression difference of TET1 between above two groups was of statistical significance, with χ2=3.218 and P=0.040.3 The cases with primary breast cancer were divided into 2 groups according to the status of lymph node metastasis, N0 group with 29 cases and N1~N3 group with 33 cases. The expression difference of TET1 between above two groups was of statistical significance, with χ2=5.120 and P=0.024.4 The cases with primary breast cancer were divided into 3 groups according to pathological stage, stage 0~Ⅰ group with 19 cases, stage Ⅱ group with 23 cases and stage Ⅲ group with 20 cases. The expression difference of TET1 in above three groups was of statistical significance, with χ2=11.480 and P=0.003. Then we adopted Spearman order correlated examination to analyze the statistics and found that TET1 expression was negatively correlated with the pathological stage of primary breast cancer, with r=-0343 and P=0.006.5 The cases with primary breast cancer were divided into 2 groups according to histological grade, grade Ⅱ group with 36 cases and grade Ⅲ group with 20 cases. 6 other cases could not be identified. The expression difference of TET1 between above two groups was of statistical significance, with χ2=4.134 and P=0.042.6 The cases with primary breast cancer were divided into 2 groups according to the status of vessel tumor embolus, tumor embolus group with 35 cases and non-tumor embolus group with 26 cases. Another case could not be identified. The expression difference of TET1 between above two groups was of statistical significance, with χ2=9.932 and P=0.002.7 The expression of TET1 was not associated with the age of patient, different subtypes of breast cancer and the expression of ER, PR, HER-2, P53 and Ki-67, with P>0.05.8 RT-PCR technique was used to detect the relative expression of TET1 m RNA in six different cell lines, such as HEK-293, SK-BR-3, MCF-7, MDA-MB-231, MDA-MB-453 and hmsc, results of which were 0.399±0.007, 0.073±0.005, 0.103±0.002, 0.088±0.003, 0.111±0.003 and 0.025±0.001, respectively. The relative expression of TET1 m RNA in above six different cell lines detected by q PCR was 1.070±0.250, 0.260±0.150,0.160±0.100,0.060±0.020,0.190±0.220 and 0.080±0.050, respectively. The expression difference of TET1 between HEK-293 cell line and other 5 cell lines was of statistical significance(P<0.05).The relative expression of TET1 protein in above six different cell lines detected by Western blot was 1.252±0.016,0.773±0.008,0.789±0.029,0.860±0.061,0.672±0.002 and 1.711±0.093, respectively. Then the expression difference of TET1 between HEK-293 and hmsc cell lines, and other 4 breast cancer cell lines respectively, was of statistical significance(P<0.05).9 A stable overexpression of TET1 in MCF-7 cells was successfully obtained. MCF-7 and MCF-7-vector cells were collected to detect fluorescent cell number(%) and fluorescence intensity using flow cytometry. The fluorescent cell number(%) in MCF-7 and MCF-7-vector cells was 3.067±0.006 and 97.557±0.090 respectively and the fluorescence intensity was 11.873±0.111 and 3320.353±41.787 respectively. The increases of fluorescent cell number and fluorescence intensity were of statistical significance(P<0.05). TET1 m RNA level, detected by CYBRGreen q PCR method, was 203.75±25.67 fold increased in MCF-7-TET1 compared to MCF-7-vector(P<0.0001).10 Cell proliferation was detected by CCK-8 method. The result showed that there was no significant difference(P>0.05) between blank control group(MCF-7) and negative control group(MCF-7-vector) but cell proliferation was significantly decreased in MCF-7-TET1(P<0.01), compared with control groups.11 Flow cytometry results revealed that there was no significant difference(P>0.05) in cell cycle distribution between MCF-7 and MCF-7-vector after culturing for 72 h, but G0/G1 phase cell proportion in MCF-7-TET1(57.96±0.74%) was significantly higher than that in control groups(45.57±2.23% in MCF-7 and 45.29±0.71% in MCF-7-vector), while S phase and G2/M phase became lower and sub-G0 phase remained unchanged. The difference was of of statistical significance(P<0.05).12 Wound healing assay indicated that the migration ability of cells in MCF-7-TET1 declined(P<0.01), compared with that in blank control group(MCF-7) and negative control group(MCF-7-vector).13 The detection of expression of EMT-related genes by q PCR showed that the level of CDH1 and CTNNB1 increased(P<0.01) while the differences of VIM and TGFB1 level were of no significance(P>0.05).Conclusions:1 The expression of TET1 decreases in breast cancer tissue as compared with benign breast lesion tissue. Our results demonstrate that a decrease in TET1 expression in breast cancer is associated with tumor size above 2cm, lymph node metastasis, late pathological stage, high histological grade and vessel tumor embolus.2 Compared with non-tumorderived epithelial cell line, TET1 expression in breast cancer cell lines decreases significantly.3 A model of MCF-7 cells stably overexpressing TET1 is successfully constructed.4 Overexpression of TET1 suppresses cell proliferation through inducing G0/G1 arrest.5 Overexpression of TET1 suppresses cell migration by inducing EMT through regulating Wnt/β-catenin pathway. |
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