从DNA甲基化的视角探讨骨肉瘤化疗耐药机制及其在临床预期中转化医学研究

Osteosarcoma is the most common primary bone malignancy in China, notorious for high malignancy 、morbidity and mortality. In addition to the surgery, the neo- adjuvant chemotherapy and postoperative adjuvant chemotherapy are the primary means of treatment. However, chemoresistance is a major source of failure of chemotherapy, threating lives of patients due to lack of effective prediction of chemoresistance. To avoid abuse of the unwarranted chemotherapy, we intend to perform the comparative genomic analyses on levels of both DNA methylation and RNA expression of the chemoresistant versus sensitive osteosarcoma cell lines to discover a group of key chemoresistant genes, expression of which is negatively regulated by the DNA methylation. A systematic study ranging from molecule up to the organism level will be followed for mechanistic insights of how chemoresistance of osteosarcoma develops. Meanwhile, we will perform DNA methylation analysis of 2-5 candidate genes in peripheral plasma of osteosarcoma patients who benefit from the chemotherapy versus who do not, to determine the predictive value of this DNA methylation analysis for chemotherapy tolerance / sensitivity of osteosarcoma in clinic

骨肉瘤是我国最常见的原发恶性骨肿瘤，以恶性度高和致残及病死率高为特征。除手术治疗以外, 术前新辅助化疗和术后辅助化疗是治疗骨肉瘤的主要手段。但是，骨肉瘤细胞的耐药性严重制约骨肉瘤化疗疗效，威胁骨肉瘤患者生命。为避免临床用药盲目性，我们将通过对化疗药物耐药/敏感骨肉瘤细胞株的DNA甲基化谱、mRNA表达谱的系统分析，寻找一批转录受到DNA甲基化状态负调的，影响骨肉瘤化疗耐药性的关键侯选基因。继而系统地阐明这些候选基因在决定骨肉瘤化疗耐药/敏感性的作用机制。同时，将通过对临床收集的患者外周血DNA中候选基因甲基化状态的分析，以期发现能作为临床化疗前预测耐药性的标志物。

骨肉瘤是我国最常见的原发恶性骨肿瘤，以恶性度高和致残及病死率高为特征。除手术治疗以外,术前新辅助化疗和术后辅助化疗是治疗骨肉瘤的主要手段。但是，骨肉瘤细胞的耐药性严重制约骨肉瘤化疗疗效，威胁骨肉瘤患者生命。为避免临床用药盲目性，我们将通过对化疗药物耐药/敏感骨肉瘤细胞株的DNA甲基化谱、mRNA表达谱的系统分析，寻找一批转录受到DNA甲基化状态负调的，影响骨肉瘤化疗耐药性的关键侯选基因。继而系统地阐明这些候选基因在决定骨肉瘤化疗耐药/敏感性的作用机制。我们对9株骨肉瘤细胞系对5种化疗药物耐药性评估，筛选出药物耐受细胞株SJSA-1和MNNG/HOS和药物敏感细胞株G-292和143B。继而使用综合亲和柱层析富集高度甲基化组分和对其深度测序的DNA methylome 技术平台对化疗耐药/敏感细胞系的DNA甲基化组和基因表达组学分析，筛选化疗耐药密切相关的一批候选基因包括miR-34a-5p、miR-20a-5p、miR-34b-3p、miR-193a-3p、miR-193a-5p、CD117、AGTR1、DLL1、KIF26B、SRR和Rab27B等。并通过qPCR、western、基因干扰、细胞周期、细胞凋亡、体外裸鼠实验等方法对特异目的基因表达水平与细胞系化疗敏感性的关系进行验证。相关结果发表SCI论文6篇，培养硕士研究生2名。这对早期发现对化疗耐药的患者，及早调整化疗方案，寻找能预测骨肉瘤化疗耐药性的标志物，用于指导临床用药，具有非常重要的意义。