冬凌草甲素上调PLK1对Jurkat细胞细胞周期的影响

Effect of oridonin up-regulating PLK1 on the cell cycle of Jurkat cells

目的·探索冬凌草甲素对Jurkat细胞增殖的抑制作用及其机制。方法·采用CCK-8法、流式细胞术、瑞士吉姆萨染色、蛋白质印迹法,检测冬凌草甲素处理后Jurkat细胞的增殖情况、细胞周期、凋亡情况及有丝分裂相关蛋白激酶表达情况。蛋白质印迹法检测冬凌草甲素处理后,Jurkat细胞中Polo样蛋白激酶1(Polo-like kinase 1,PLK1)下游蛋白的磷酸化水平。通过计算机模拟、免疫沉淀、蛋白质印迹法、细胞热力学迁移实验方法研究冬凌草甲素与PLK1的结合方式及结合位点。组间比较采用t检验,P<0.05为差异具有统计学意义。结果·冬凌草甲素通过上调PLK1蛋白含量并促进其激酶活性,从而诱导Jurkat细胞发生G2/M期阻滞;冬凌草甲素可与PLK1蛋白直接结合抑制其通过泛素蛋白酶体途径降解;如PLK1蛋白的Cys67或Cys133氨基酸残基发生突变则抑制冬凌草甲素与其直接结合,且抑制冬凌草甲素对PLK1蛋白的稳定作用。结论·冬凌草甲素通过直接结合PLK1蛋白抑制其泛素化修饰及降解,促进其活性,诱导Jurkat细胞发生G2/M期阻滞;PLK1蛋白的Cys67和Cys133位点是蛋白与冬凌草甲素结合的关键位点。

Objective·To explore the inhibitory effect of oridonin on the proliferation of Jurkat cells and its mechanism.Methods·CCK-8 method,flow cytometry,Swiss Giemsa staining,and Western blotting were used to detect the proliferation,cell cycle and apoptosis of Jurkat cells and the expression of mitosis-related protein kinases after oridonin treatment.Western blotting was used to detect the phosphorylation level of PLK1 downstream proteins in Jurkat cells after oridonin treatment.Computer simulation,immunoprecipitation,Western blotting and Celluar Thermal Shift Assay were used to study the binding mode and binding site of oridonin and PLK1.Student's-t test was performed for comparison between each two groups.Statistical significance was accepted at a value of P<0.05.Results·Oridonin could induce G2/M phase arrest in Jurkat cells by up-regulating the content of PLK1 protein and promoting its kinase activity.Oridonin could directly bind to PLK1 protein to inhibit its degradation through the ubiquitin proteasome pathway.The mutation of Cys67 or Cys133 amino acid residues of PLK1 protein inhibited its direct binding to oridonin.And the mutation inhibited the stabilizing effect of oridonin on PLK1 protein.Conclusion·Oridonin inhibits PLK1 protein ubiquitination modification and degradation by directly binding to its protein.Oridonin can also promote PLK1 protein kinase activity and induce G2/M phase arrest in Jurkat cells.The Cys67 and Cys133 sites of the PLK1 protein are the key sites for the protein to bind to oridonin.