基于DNA甲基化测序技术研究青蒿琥酯干预脑胶质瘤的分子机制  

Molecular mechanism of artesunate on intervening glioblastoma based on DNA methylation sequencing technology

作　　者：闫向英 毛霞[1] 李涛[1] Koji Mizuno Katsuki Okuyama 林娜[1] 张彦琼 Takashi Sato YAN Xiang-ying;MAO Xia;LI Tao;Koji Mizuno;Katsuki Okuyama;LIN Na;ZHANG Yan-qiong;Takashi Sato(Institute of Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China;Department of Biochemistry,Tokyo University of Pharmacy and Life Sciences,Tokyo 192-0392,Japan)

机构地区：[1]中国中医科学院中药研究所,北京100700 [2]东京药科大学生物化学系,东京192-0392

出　　处：《中草药》2023年第6期1814-1824,共11页Chinese Traditional and Herbal Drugs

基　　金：国家自然科学基金青年项目(8210143224);中国中医科学院优秀青年科技人才(创新类)(ZZ15-YQ-029)。

摘　　要：目的探究青蒿琥酯干预胶质母细胞瘤(glioblastoma,GBM)的药效及其潜在分子机制。方法采用Cell TiterBlue检测青蒿琥酯对U87和U2512种人源胶质母细胞瘤细胞活性和增殖的影响;流式细胞术检测青蒿琥酯对2种细胞周期的调节作用;采用q RT-PCR和Western blotting检测青蒿琥酯对2种细胞自噬水平的影响。采用DNA甲基化联合网络药理学从表观遗传学层面研究青蒿琥酯干预GBM的靶标基因,并建立蛋白质-蛋白质相互作用网络,通过计算网络中各节点的网络拓扑特征值,筛选出青蒿琥酯干预GBM的核心靶点并进行生物功能富集分析;采用AutoDock v4.2.6和Auto Dock Vina v1.2.0软件开展分子对接虚拟计算,考察青蒿琥酯与其候选关键靶点的结合能力,并采用Western blotting验证青蒿琥酯对2种脑胶质瘤细胞中关键靶点表达的影响。结果青蒿琥酯可显著抑制U87细胞的活性和增殖(P<0.05、0.01、0.001),而对U251细胞的药效不显著;高剂量青蒿琥酯可影响2种细胞周期分布;高剂量青蒿琥酯可显著提高U87细胞中3种自噬标志分子[自噬关键分子酵母Atg6同系物(ATG6 autophagy related 6 homolog,Beclin1)、泛素结合蛋白(ubiquitin-binding protein,p62)、微管相关蛋白1轻链3(microtubule-associated protein 1 light chain 3,LC3)]的m RNA表达水平(P<0.001),而仅高剂量青蒿琥酯升高U251细胞中p62的m RNA表达水平(P<0.05);低、中、高剂量青蒿琥酯可显著提高U87细胞中Beclin1、p62、LC3B蛋白表达水平(P<0.05、0.01),仅中、高剂量青蒿琥酯可以影响U251细胞中Beclin1、p62、LC3B蛋白表达。利用给药前后的U87与U251细胞进行DNA甲基化测序与网络药理学整合分析,在获得青蒿琥酯敏感型和耐药型脑胶质瘤细胞的差异基因集,以及青蒿琥酯干预2种细胞效应基因集的基础上,发现其干预2类脑胶质瘤细胞的关键网络靶标共同参与GBM恶性进展密切相关的血小板衍生生长因子受体α(platelet Objective To explore the efficacy and mechanism of artesunate in intervening glioblastoma.Methods Cell Titer-Blue was used to detect the effect of artesunate on viability and proliferation of U87 and U251 human glioblastoma cells;Flow cytometry was used to detect the regulation effect of artesunate on two cell cycles;qRT-PCR and Western blotting were used to detect the effect of artesunate on autophagy level of two kinds of cells.Further,target genes of artesunate intervention in glioblastoma were studied from the epigenetic level by using the analysis strategy of DNA methylation combined with network pharmacology,and a protein-protein interaction network was established.By calculating the network topological eigenvalues of each node in network,core targets of artesunate intervention in glioblastoma were screened out and biological function enrichment analysis was carried out.Finally,AutoDock v4.2.6 and AutoDock Vina v1.2.0 software were used to carry out molecular docking virtual calculation,to investigate the binding ability of artesunate and its candidate key targets,and verify the influence of artesunate on the expressions of key targets in two types of glioma cells by Western blotting.Results Artesunate significantly inhibited the activity and proliferation of U87 cells(P<0.05,0.01,0.001),but had no significant effect on U251 cells.High-dose artesunate could affect the distribution of two cell cycles.High-dose artesunate significantly increased the m RNA expression levels of three autophagy markers[ATG6 autophagy related 6 homolog(Beclin1),ubiquitin-binding protein(p62),microtubule-associated protein 1 light chain 3(LC3)]in U87 cells(P<0.001),while only high-dose artesunate increased the mRNA expression level of p62 in U251 cells(P<0.05).Low-,medium-and high-dose of artesunate could significantly improve the expression levels of Beclin1,p62 and LC3B proteins in U87 cells(P<0.05,0.01),only medium-and highdose of artesunate could affect Beclin1,p62 and LC3B protein expressions in U251 cells.Furthermore,DNA met

关 键 词：青蒿琥酯 胶质母细胞瘤 DNA甲基化分析 耐药 PDGFRA-RAS-BRAF-MEK-ERK信号通路 

分 类 号：R285.5[医药卫生—中药学]