Cinobufotalin powerfully reversed EBV-miR-BART22-induced cisplatin resistance via stimulating MAP2K4 to antagonize non-muscle myosin heavy chain IIA/glycogen synthase 3β/β-catenin signaling pathway
Cinobufotalin powerfully reversed EBV-miR-BART22-induced cisplatin resistance via stimulating MAP2K4 to antagonize non-muscle myosin heavy chain IIA/glycogen synthase 3β/β-catenin signaling pathway
复制标题
Cinobufotalin 通过刺激 MAP2K4 拮抗非肌肉肌球蛋白重链 IIA/糖原合酶 3β/β-连环蛋白信号通路，强力逆转 EBV-miR-BART22 诱导的顺铂耐药性
DOI：
10.1016/j.ebiom.2019.08.040
复制
发表时间：
2019-10-01
期刊：
影响因子：
11.1
通讯作者：
Fang, Weiyi
中科院分区：
文献类型：
--
作者：
Liu, Yiyi;Jiang, Qingping;Fang, Weiyi
背景：鼻咽癌是一种与EB病毒(EBV)相关的肿瘤。EBV编码的miR-BART22在鼻咽癌中的作用尚不清楚。本研究旨在明确EBV-miR-BART22作为促癌因子的具体作用机制，并评价华蟾素对EBV-miR-BART22高表达鼻咽癌细胞的作用。方法：采用实时荧光定量聚合酶链式反应、Western blotting、免疫组织化学和原位杂交等方法，检测鼻咽癌患者鼻咽癌组织和细胞中miR-BART22和MAP2K4的表达，并评价其临床意义。通过球体形成实验、侧群分析、Transwell、Boyden、体内移植瘤模型等方法检测miR-BART22对细胞转移、干细胞分化和顺铂化疗耐药的影响。Western blotting、免疫荧光染色、荧光素酶报告实验、芯片、EMSA和Co-IP分析等。以探讨EBV-miR-BART22在鼻咽癌中的详细分子机制。最后，我们在体内外实验中评价了华蟾素对EBV-miR-BART22高表达鼻咽癌细胞的作用及其分子基础。结果：EBV-miR-BART22不仅促进了肿瘤的干化和转移，而且在体内外增强了对顺铂(DDP)的耐药性。机制分析表明，EBV-miR-BART22通过PI3K/AKT/c-jun诱导转录，直接靶向MAP2K4，上调非肌肉肌球蛋白重链IIA(Myh9)的表达。此外，Myh9与糖原合成酶3β(GSK3β)蛋白相互作用，通过激活PI3K/AKT/c-jun诱导的泛素转录，后者与TRAF6 E3连接酶结合，进而与GSK3β蛋白结合，从而诱导其泛素降解。因此，GSK3β蛋白的减少促进了β-连环蛋白的表达和核移位，从而诱导了肿瘤干细胞和上皮向间充质转化(EMT)信号。此外，我们观察到一种新的化学合成化合物华蟾素通过上调MAP2K4抑制Myh9/GSK3β/β-catenin及其下游的肿瘤干细胞和EMT信号，显著抑制EBV-miR-BART22诱导的DDP化疗耐药。临床资料显示miR-BART22表达增加和MAP2K4表达降低导致鼻咽癌患者预后不良。解释：我们的研究为华蟾素逆转EBV-miR-BART22诱导的顺铂耐药和鼻咽癌EMT提供了新的机制。(C)2019年提交人。爱思唯尔出版公司(Elsevier B.V.)
Background: Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV)-related tumor. The role of EBV-encoding miR-BART22 is still unclear in NPC. This study aimed to identify the detailed mechanisms by which EBV-miR-BART22 functions as a tumor-promoting factor and evaluate the action of cinobufotalin in treating EBV-miR-BART22-overexpressing NPC cells.Methods: Using real-time PCR, western blotting, immunohistochemistry, and In situ hybridization, we detected the expression of miR-BART22 and MAP2K4 in tissues and cells, as well as evaluated their clinical relevance in NPC patients. The effects of miR-BART22 on cell metastasis, stemness and DDP chemoresistance were examined by sphere formation assay, side population analysis, transwell, boyden, in vivo xenograft tumor mouse model et al. Western blotting, immunofluorescence staining, luciferase reporter assay, ChIP, EMSA and Co-IP assay et al. were performed to explore the detailed molecular mechanism of EBV-miR-BART22 in NPC. Finally, we estimated the effects and molecular basis of Cinobufotalin on EBV-miR-BART22-overexpressing NPC cells in vitro and in vivo assays.Findings: We observed that EBV-miR-BART22 not only promoted tumor stemness and metastasis, but also enhanced the resistance to Cisplatin (DDP) in vitro and in vivo. Mechanistic analysis indicated that EBV-miR-BART22 directly targeted the MAP2K4 and upregulated non-muscle myosin heavy chain IIA (MYH9) expression by PI3K/AKT/c-Jun-induced transcription. Further, MYH9 interacted with glycogen synthase 3 beta(GSK3 beta) protein and induced its ubiquitin degradation by activating PI3K/AKT/c-Jun-induced ubiquitin transcription and the latter combined with increased TRAF6 E3 ligase, which further bound to GSK3 beta protein. Reductions in the GSK3 beta protein thus promoted beta-catenin expression and nuclear translocation, which induced tumor stemness and the epithelial-to-mesenchymal transition (EMT) signals. Furthermore, we observed that cinobufotalin, a new chemically synthesized compound, significantly suppressed EBV-miR-BART22-induced DDP chemoresistance by upregulating MAP2K4 to suppress MYH9/GSK3 beta/beta-catenin and its downstream tumor stemness and EMT signals in NPC. Finally, clinical data revealed that increased miR-BART22 and reduced MAP2K4 expression caused the poor prognoses of NPC patients.Interpretation: Our study provides a novel mechanism that cinobufotalin reversed the DDP chemoresistance and EMT induced by EBV-miR-BART22 in NPC. (C) 2019 The Authors. Published by Elsevier B.V.