摘要
该文旨在探讨次大风子素(hydnocarpin,HC)对三阴性乳腺癌(triple negative breast cancer,TNBC)的抑制作用及分子机制。采用CCK-8、实时无标记细胞分析技术(RTCA)、克隆形成实验检测HC处理后MDA-MB-231、MDA-MB-4362种TNBC细胞系增殖能力的变化;通过高内涵成像分析技术、划痕愈合实验、Transwell侵袭实验检测HC对TNBC细胞迁移侵袭能力的影响;Western blot检测HC处理后上皮-间质转化(epithelial-mesenchymal transition,EMT)及侵袭迁移相关蛋白E-cadhe-rin、vimentin、Snail、MMP-2、MMP-9的表达变化;Western blot、RT-qPCR检测Yes相关蛋白(Yes-associated protein,YAP)的表达及下游靶基因CTGF、Cyr61 mRNA及蛋白水平的表达变化;TNBC细胞瞬时转染Flag-YAP,过表达YAP,通过CCK-8实验、Transwell侵袭实验及划痕愈合实验检测YAP作为HC抑制TNBC恶性进展关键靶分子的作用;蛋白酶体抑制剂与HC共处理、泛素化免疫共沉淀实验检测HC诱导YAP降解的途径;基于靶点稳定性的药物亲和反应实验和微量热泳动技术检测HC与YAP及E3泛素连接酶Ccr4-not transcription complex subunit 4(CNOT4)之间的结合作用。结果表明HC显著抑制TNBC细胞增殖、克隆形成、侵袭及EMT;HC下调CTGF、Cyr61的mRNA及蛋白表达;HC下调YAP总蛋白水平,但对其基因水平无影响;过表达YAP能够拮抗HC对TNBC细胞增殖、迁移及侵袭的抑制作用;HC促进YAP通过蛋白酶体途径降解并上调YAP的泛素化水平;微量热泳动技术、基于靶点稳定性的药物亲和反应实验结果显示,HC、YAP、CNOT4分子之间存在直接结合作用。以上结果说明HC通过CNOT4介导的YAP泛素化及蛋白酶体降解抑制三阴性乳腺癌的恶性进展。
This study aims to investigate the effect and mechanism of hydnocarpin(HC)in treating triple negative breast cancer(TNBC).Cell counting kit-8(CCK-8),xCELLigence real-time cellular analysis(RTCA),and colony formation assay were employed to determine the effects of HC on the proliferation of two TNBC cell lines:MDA-MB-231 and MDA-MB-436.The effects of HC on the migration and invasion of TNBC cells were detected by high-content analysis,wound-healing assay,and Transwell assay.The changes in the epithelial-mesenchymal transition(EMT)and the expression of invasion-and migration-associated proteins[E-cadherin,vimentin,Snail,matrix metalloproteinase-2(MMP-2),and MMP-9]were detected by Western blot.Western blot and RT-qPCR were employed to determine the protein and mRNA levels of Yes-associated protein(YAP)and downstream targets(CTGF and Cyr61).TNBC cells were transfected with Flag-YAP for the overexpression of YAP,and the role of YAP as a key target for HC to inhibit TNBC malignant progression was examined by CCK-8 assay,Transwell assay,and wound-healing assay.The pathway of HC-induced YAP degradation was detected by the co-treatment of proteasome inhibitor with HC and ubiquitination assay.The binding of HC to YAP and the E3 ubiquitin ligase Ccr4-not transcription complex subunit 4(CNOT4)was detected by microscale thermophoresis(MST)assay and drug affinity responsive target stability(DARTS)assay.The results showed that HC significantly inhibited the proliferation,colony formation,invasion,and EMT of TNBC cells.HC down-regulated the protein and mRNA levels of CTGF and Cyr61.HC downregulated the total protein level of YAP,while it had no effect on the mRNA level of YAP.The overexpression of YAP antagonized the inhibitory effects of HC on the proliferation,migration,and invasion of TNBC cells.HC promoted the degradation of YAP through the proteasome pathway and up-regulated the ubiquitination level of YAP.The results of MST and DARTS demonstrated direct binding between HC,YAP,and CNOT4.The above results indicated that HC inhibited the malignant progression of TNBC via CNOT4-mediated degradation and ubiquitination of YAP.
作者
欧虹灵
吴慧
任宇亮
司渊
段钟琪
刘雪文
OU Hong-ling;WU Hui;REN Yu-liang;SI Yuan;DUAN Zhong-qi;LIU Xue-wen(School of Basic Medical Sciences,Hubei University of Medicine,Shiyan 442000,China;Hubei Key Laboratory of Wudang Local Chinese Medicine Research,Hubei University of Medicine,Shiyan 442000,China;Hubei Key Laboratory of Embryonic Stem Cell Research,Hubei University of Medicine,Shiyan 442000,China)
出处
《中国中药杂志》
CAS
CSCD
北大核心
2023年第16期4483-4492,共10页
China Journal of Chinese Materia Medica
基金
国家自然科学基金项目(82203409)
湖北省自然科学基金面上项目(2022CFB548,2023AFB851)
湖北省教育科学规划课题(2021GB101)
湖北医药学院研究生科技创新项目(YC2022031)
2022年大学生创新创业训练计划项目(202210929009)。
