SMYD3调控人乳腺癌MCF-7细胞增殖机制的研究

Study on Regulation Mechanisms of SMYD3 on Proliferation of MCF-7 Human Breast Cancer Cells

目的:通过转染技术抑制或增强SMYD3基因的表达,以探讨SMYD3对乳腺癌MCF-7细胞增殖的调控机制机制。方法:以MTT法和软琼脂克隆形成实验研究抑制或增强SMYD3基因表达对细胞增殖的影响,以RT-PCR法和Western印迹法检测MCF-7细胞经转染后,胞内SMYD3、ERK/MAPK通路相关蛋白及其磷酸化产物,以及凋亡和细胞周期调控相关蛋白表达水平的变化。结果:用针对SMYD3基因的shRNAs表达质粒转染MCF-7细胞后,其SMYD3基因mRNA和蛋白表达水平下调,细胞生长受到抑制,细胞中ERK1/2、CyclinD1和CDK4蛋白水平明显下调;软琼脂克隆形成实验显示,增强SMYD3基因表达能明显促进MCF-7细胞克隆形成。结论:SMYD3可通过激活ERK/MAPK信号转导通路和上调细胞周期相关蛋白CyclinD1和CDK4的水平提高肿瘤细胞的增殖能力。

Objective： To study the function of SMYD3 in the proliferation regulation of human breast cancer MCF-7 cells by inhibiting or activating the expression of SMYD3 gene by transfection technique. Methods： The MTT assay and soft agar cloning assay were used to explore the influence of SMYD3 gene inhibition and activation on MCF-7 cell proliferation. RT-PCR and Western blot assays were conducted to investigate the intracellular expression levels of SMYD3, ERK/MAPK pathway related proteins, as well as apoptosis and cell circle regulation related proteins, after gene transfection. Results： After SMYD3 shRNA expression vector was transfected into MCF-7 cells, the SMYD3 gene transcription level and the SMYD3 expression level were down-regulated greatly, and the growth of the cells was inhibited. Besides, down-regulation of the expression levels of ERK1/2, CyclinDl and CDK4 was also observed in MCF-7 cells, in which the SMYD3 gene expression was inhibited. The soft agar cloning assay demonstrated that the clone number of MCF-7 cells rised obviously after the activation of SMYD3 gene. Conclusion： SMYD3 could promote the proliferation of tumor cells via activating the MAPK/ERK pathway and up-regulating the expression levels of CyclinD1 and CDK4.