The Effects Of TET1 On The Biological Behavior Of MDA-MB-231 Cells

Objective:Breast cancer is one of the most common malignancies of women. Rescently, the incidence and mortality of breast cancer is increasing in our country. Even though early detection methods and treatment options greatly improved, the metastasis and invasion are still the main reasons of the death among the breast cancer patients. Therefore, the mechanisms, involved in the invasion and metastasis of breast cancer, have become a research focus. Increasing evidence suggests that TET1 catalyzesthe oxidation of 5-methylcytosine(5mC) to 5-hydroxymethylcytosine(5hmC), which might represent an intermediate form of an active DNA demethylation process and regulate gene expression. The TET1 mutation has been found to be closely associated with the genesis,development and prognosis of different tumors. Hence, exploring the mechanism of invasion would improve the clinical outcome for breast cancer patients.To investigate the effects of TET1 on proliferation, metastasis and invasion of MDA-MB-231 cells and its possible mechanisms, lentiviral vector containing TET1 precusor was used to get the ectopic overexpression TET1 in the MDA-MB-231 cells.Methods:1 The Lentiviral carrying TET1 was packaged with the method of transfecting 293 T cell lines in vitro. Then, MDA-MB-231 cells were infected with the fresh virus according to kit instruction. Then, MDA-MB-231 cell line that stably overexpressed TET1 was selected. TET1 expression levels were detected by SYBR Green quantitative real-time PCR and Western blot.2 The 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.3 The growth and migration abilities of cells were detected by wound healing essay and TranswellTM. The expression levels of EMT-related protein, such as E-cadherin, N-cadherin, Vimentin and β-catenin, were measured by Western blot and immunofluorescence.4 Tumor growth, tumor volume and quality were observed after inoculation with blank control group(MDA-MB-231), negative control group(MDA-MB-231-NC) and MDA-MB-231-TET1 in vivo.5 Statistical analyses were performed by SPSS 13.0 software. Differences of paired groups were assessed by paired-sample t test and differences among multiple groups were evaluated by one-way ANOVA method. 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 A stable overexpression of TET1(MDA-MB-231-TET1) cell line was successfully obtained. TET1 mRNA level, detected by SYBR Green qPCR method, was 3.11±4.61 fold increase in MDA-MB-231-TET1 compared to MDA-MB-231-NC(P<0.01). TET1 protein level, detected by Western blot, was 2.67±1.55 fold increase in MDA-MB-231-TET1 compared to MDA-MB-231-NC(P< 0.05).2 Cell proliferation was detected by CCK-8 method. The result showed that there was no significant difference(P>0.05) between blank control group(MDA-MB-231) and negative control group(MDA-MB-231-NC), but cell proliferation was significantly decreased in MDA-MB-231-TET1(P<0.01), compared with control groups.3 Flow cytometry results revealed that there was no significant difference(P>0.05) in cell cycle distribution between MDA-MB-231 and MDA-MB-231-NC, while in MDA-MB-231-TET1, the G2/M phase cell proportion(13.24±3.64%) was significantly higher than that in control groups(4.30±1.36%)(P< 0.01).4 Wound healing assay indicated that the migration ability of cells in MDA-MB-231-TET1 declined(P<0.001), compared with that in blank control group(MDA-MB-231) and negative control group(MDA-MB-231-NC).5 TranswellTM assay indicated that the invasion ability of cells in MDA-MB-231-TET1 declined(P<0.001), compared with that in blank control group(MDA-MB-231) and negative control group(MDA-MB-231-NC).6 The Cell morphology was observed under light microscope, the morphology of MDA-MB-231-TET1 cells did not change significantly. The detection of expression of EMT-related protein by Western blot and immunofluorescence indicated that the level of E-cadherin(P<0.001) was significantly increased and N-cadherin, Vimentin(P<0.05) were decreased in MDA-MB-231-TET1 compared with control groups. β-catenin was expressed in cytoplasm and nucleus among three groups. However, the expression of β-catenin was significantly increased in the nucleus, suggesting that β-catenin gather to the nucleus in blank control group(MDA-MB-231) and negative control group(MDA-MB-231-NC)(P<0.05).7 Observing the tumor size of nude mice, there was no significant difference among the three groups(P > 0.05).Conclusions:1 A model that stably overexpression TET1 in MDA-MB-231 cells is successfully obtained.2 Overexpression of TET1 supresses cell proliferation through inducing G2/M arrest.3 Overexpression of TET1 suppresses cell proliferation, migration and invasion abilities by inducing EMT through regulating Wnt/β-catenin pathway in vitro.