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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 investigate whether SMYD3 regulates the proliferation of MCF-7 breast cancer cells by transfecting it to inhibit or enhance the expression of SMYD3 gene. Methods: MTT assay and soft agar colony formation assay were used to study the effect of SMYD3 gene expression on cell proliferation. RT-PCR and Western blotting were used to detect the expression of SMYD3, ERK / MAPK Pathway-related proteins and their phosphorylation products, as well as the changes of apoptosis and cell cycle related protein expression. Results: After SMYD3 gene was transfected into MCF-7 cells, the expression of SMYD3 mRNA and protein was down-regulated and the growth of MCF-7 cells was inhibited. The expression of ERK1 / 2, CyclinD1 and CDK4 were down-regulated. The formation of experiments showed that enhanced SMYD3 gene expression can significantly promote MCF-7 cell clone formation. Conclusion: SMYD3 can enhance the proliferation of tumor cells by activating the ERK / MAPK signal transduction pathway and up-regulating the expression of cyclinD1 and CDK4.