Abstract：
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 down-regulated 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.
摘要：
本研究旨在探讨hydnocarpin（HC）治疗三阴性乳腺癌（TNBC）的作用及机制。细胞计数试剂盒-8(CCK-8),xCELLigence实时细胞分析(RTCA)，和集落形成试验用于确定HC对两种TNBC细胞系MDA-MB-231和MDA-MB-436的增殖的影响。通过高含量分析检测HC对TNBC细胞迁移和侵袭的影响,伤口愈合试验，和Transwell分析。上皮-间质转化（EMT）的变化以及侵袭和迁移相关蛋白的表达[E-cadherin，波形蛋白,蜗牛,基质金属蛋白酶-2(MMP-2),Westernblot检测MMP-9]。采用Western印迹和RT-qPCR测定Yes相关蛋白(YAP)和下游靶标(CTGF和Cyr61)的蛋白和mRNA水平。用Flag-YAP转染TNBC细胞用于过表达YAP,CCK-8检测YAP作为HC抑制TNBC恶性进展的关键靶标的作用，Transwell分析，和伤口愈合试验。通过蛋白酶体抑制剂与HC和泛素化分析的共同处理来检测HC诱导的YAP降解途径。HC与YAP和E3泛素连接酶Ccr4-not转录复合物亚基4（CNOT4）的结合通过微尺度热电泳（MST）测定和药物亲和力响应靶标稳定性（DARTS）测定来检测。结果表明，HC显著抑制细胞增殖，菌落形成,入侵,和TNBC细胞的EMT。HC下调CTGF和Cyr61的蛋白质和mRNA水平。HC下调YAP的总蛋白水平，而对YAP的mRNA水平没有影响。YAP的过表达拮抗HC对细胞增殖的抑制作用,迁移,和TNBC细胞的侵袭。HC通过蛋白酶体途径促进YAP的降解，并上调YAP的泛素化水平。MST和DARTS的结果表明HC之间的直接结合，YAP,CNOT4以上结果表明HC通过CNOT4介导的YAP降解和泛素化抑制了TNBC的恶性进展。
