PAK5与p53竞争结合DDX5促进乳腺癌细胞的增殖  

PAK5 and p53 competitively combine with DDX5 to promote the proliferation of breast cancer cells

作　　者：蔡伊航 王乐严 赵鑫[1] 李洋[1] CAI Yihang;WANG Leyan;ZHAO Xin;LI Yang(Molecular Cell Biology,College of Life Sciences,China Medical University,Liaoning Shenyang 110122,China)

机构地区：[1]中国医科大学生命科学学院分子细胞生物学教研室,辽宁沈阳110122

出　　处：《现代肿瘤医学》2025年第5期749-755,共7页Journal of Modern Oncology

基　　金：辽宁省教育厅青年科学家项目(编号:LJ212410159117);辽宁省沈阳市科技局2023年助力中国医科大学高质量发展专项(编号:23-506-3-01-34)。

摘　　要：目的:探讨p21活化激酶-5(p21-activated kinase 5,PAK5)通过磷酸化修饰对DDX5与p53结合的影响和可能机制。方法:为了探究PAK5对DDX5与p53结合的影响,在过表达PAK5的乳腺癌细胞中,采用Co-IP验证DDX5与p53结合能力的变化,并在细胞外利用GST pulldown实验验证PAK5对DDX5与p53直接结合能力的影响。构建DDX5截短质粒,GST pulldown实验验证PAK5和p53与DDX5的结合结构域。构建DDX5磷酸化位点突变质粒,纯化不同磷酸化状态的DDX5蛋白,GST pulldown实验验证DDX5和p53的结合状态。Real-Time PCR实验证实PAK5抑制成熟的miR-143和miR-145的表达。最后,通过CCK-8实验检测过表达不同磷酸化状态的DDX5对乳腺癌细胞增殖的影响。结果:过表达PAK5减弱了DDX5与p53的结合,促进乳腺癌细胞的增殖。结论:PAK5可能通过磷酸化DDX5,与p53竞争性结合DDX5,抑制成熟的miR-143和miR-145的表达,从而促进乳腺肿瘤的生长。Objective:To explore the effects and possible mechanisms of phosphorylation modification of p21-activated kinase 5(PAK5)on the binding of DDX5 to p53.Methods:In order to explore the effect of PAK5 on the binding of DDX5 and p53,in breast cancer cells overexpressing PAK5,Co-IP was used to verify the change of the binding ability of DDX5 and p53,and GST pulldown experiment was used to verify the effect of PAK5 on the direct binding ability of DDX5 and p53.Construct a DDX5 truncated plasmid and validate the binding domains of PAK5 and p53 with DDX5 through GST pulldown experiment.Construct a DDX5 phosphorylation site mutant plasmid,purify DDX5 proteins with different phosphorylation states,and validate the binding status of DDX5 and p53 through GST pulldown experiment.The Real-Time PCR experiment confirmed that PAK5 inhibits the expression of mature miR-143 and miR-145.Finally,CCK8 experiment was used to detect the effect of overexpression of DDX5 with different phosphorylation states on the proliferation of breast cancer cells.Results:Overexpression of PAK5 weakened the binding of DDX5 to p53,and promoted the proliferation of breast cancer cells.Conclusion:PAK5 may promote the growth of breast tumors by phosphorylating DDX5 and competitively binding to DDX5 with p53,and thereby inhibiting the expression of mature miR-143 and miR-145.

关 键 词：磷酸化 竞争性结合 p21活化蛋白5(PAK5) DEAD box RNA解旋酶5(DDX5) 

分 类 号：R737.9[医药卫生—肿瘤]