摘要
p21活化激酶5(p21-activated kinase 5,PAK5)是一种丝氨酸/苏氨酸激酶,调节多种细胞进程,包括细胞骨架重构、细胞增殖、迁移和侵袭。研究表明,PAK5是调控乳腺癌进程的关键因子,但与衰老关系的研究尚未见报道。本研究利用CRISPR/Cas9慢病毒感染方法,构建敲低PAK5的人乳腺癌MDA-MB-231和BT474稳转细胞系。通过CCK-8和克隆形成实验检测敲低PAK5对乳腺癌细胞增殖的影响;通过流式细胞术检测敲低PAK5对细胞周期的影响;通过β-半乳糖苷酶染色观察敲低PAK5对细胞衰老的影响;Western印迹检测敲低PAK5的MDA-MB-231和BT474细胞衰老相关蛋白质p53、p21和p16的表达;使用CHX与MG132处理细胞,探究PAK5对p53蛋白质表达可能的调控机制。结果显示,敲低PAK5抑制细胞增殖(P<0.05),使细胞停滞在G0/G1期;而且,敲低PAK5通过上调细胞衰老相关蛋白质p53、p21及p16的表达(P<0.05)促进细胞衰老。进一步研究证明,PAK5可能泛素化降解p53。这些发现表明,敲低PAK5能够抑制乳腺癌细胞增殖,同时促进细胞衰老,为乳腺癌治疗提供新思路。
p21-activated kinase 5(PAK5),a kind of the PAK family of serine/threonine kinases,modulates various cellular processes,including cytoskeletal remodeling,cell proliferation,migration and metastasis.Although studies have suggested that PAK5 is a key regulator of breast cancer progression,the link of PAK5 to senescence has not been reported yet.In this study,the CRISPR/Cas9 lentivirus infection method was used to construct two PAK5 stably knockdown cell lines of human breast cancer(MDA-MB-231 and BT474).The effect of PAK5 knockdown on the proliferation of breast cancer cells was detected by CCK-8 and clone formation assays.The effect of PAK5 knockdown on cell cycle was detected by flow cytometry.The effect of PAK5 knockdown on cell senescence was observed byβ-galactosidase staining.Western blotting was performed to detect the expression of senescence-related proteins including p53,p21 and p16 in breast cancer cells.Cells were treated by CHX and MG132 to explore the possible mechanism of PAK5 regulating p53 protein expression.The results showed that knockdown of PAK5 inhibited cell proliferation and blocked cells in the G0/G1 phase(P<0.05);Moreover,knockdown of PAK5 promoted cell senescence by upregulating the expression of cellular senescence-related proteins p53,p21 and p16(P<0.05).Further studies have proved that PAK5 may degrade p53 by ubiquitination.These findings suggest that knockdown of PAK5 can inhibit breast cancer cell proliferation and promote cell senescence,providing potential targets for the treatment of breast cancer.
作者
胡冰涛
邢瑶
李洋
李丰
HU Bing-Tao;XING Yao;LI Yang;LI Feng(Department of Molecular Cell Biology,College of Life Sciences,China Medical University,Key Laboratory of Medical Cell Biology,Ministry of Education,Shenyang 110122,China)
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2021年第4期495-503,共9页
Chinese Journal of Biochemistry and Molecular Biology
基金
国家自然科学基金资助(No.31970741,31571457)
辽宁省科学技术计划项目(No.2019JH8/10300014)资助。
关键词
乳腺癌
p21活化激酶5
细胞增殖
细胞衰老
breast cancer
p21-activated kinase 5(PAK5)
cell proliferation
cell senescence
