TET1蛋白对人乳腺癌MDA-MB-231细胞株增殖和侵袭能力的影响及其相关机制

Effects of TET1 on Proliferation and Invasion of Human Breast Cancer MDA-MB-231 Cells and Related Mechanism

目的探讨TET1对乳腺癌MDA-MB-231细胞增殖、转移、侵袭等生物学行为的影响,并初步探究其相关分子机制。方法应用慢病毒载体建立TET1过表达稳转细胞株(MDA-MB-231-TET1)及阴性对照组稳转细胞株(MDA-MB-231-NC),Real-time PCR及Western blot法检测TET1的表达情况。CCK-8法、流式细胞术检测细胞的增殖及细胞周期分布,细胞划痕实验及Transwell小室检测细胞迁移及侵袭能力;Western blot及免疫荧光实验检测EMT相关蛋白(E-cadherin、N-cadherin、Vimentin、β-catenin)分子的表达。结果成功构建过表达TET1细胞株(P=0.03)。相较于阴性对照组和空白对照组细胞,MDA-MB-231-TET1组细胞增殖、迁移及侵袭明显受到抑制(P<0.001),G2/M期细胞周期阻滞(P=0.002);同时,伴随E-cadherin表达升高(P<0.001),N-cadherin(P=0.003)、Vimentin(P=0.041)表达降低,β-catenin(P<0.001)表达降低,并且TET1可抑制β-catenin向细胞核内转移,进而抑制EMT的发生。结论 TET1可抑制MDA-MB-231细胞增殖,并能抑制其迁移及侵袭,其机制可能与通过Wnt/β-catenin通路抑制EMT的发生相关。

Objective To investigate the effects of ten-eleven translocation （TET1） on the proliferation, metastasis and invasion of breast cancer MDA-MB-231 cells and possible mechanisms. Methods Breast cancer cell line MDA-MB-231 were stably transfected with recombinant lentiviral expression vector of TET1 and enhanced green fluorescent protein （eGFP）. The mRNA and protein expressions of TET 1 were detected by Real-time PCR and Western blot. Cell proliferation was detected by CCK-8 assay and flow cytometry was adopted to determine cell cycle distribution. The migration and invasion abilities of cells were detected by wound healing assay and transwell assay. The expression levels of EMT-related protein, such as E-cadherin, N-cadherin, Vimentin and [3-catenin, were measured by Western blot and immunofluorescence. Results A stable overexpression of TET1 cell line was successfully obtained （P=0.030）. Cell proliferation, migration and invasion abilities were significantly decreased in MDA-MB-231-TETl cells（P〈0.001）, compared with control group. The overexpression of TETI suppressed cell proliferation through inducing G2/M arrest （P=0.002）. At the same time, the level of E-cadherin was significantly increased（P〈0.001） and N-cadherin, Vimentin, and β-catenin were decreased（P=-0.003, P=0.041, P〈0.001） in MDA-MB-231-TET1 cells. TET1 inhibited the nuclear translocation of β-cantenin and then repressed EMT. Conclusion The overexpression of TET1 suppresses the proliferation, migration and invasion abilities of MDA-MB-231 cells by inducing EMT through regulating Wnt/β-catenin pathway in vitro.