【作者】 杨杰；

【导师】 查振刚；

【作者基本信息】 暨南大学 ， 外科学（骨外科）， 2020， 博士

【摘要】 软骨肉瘤是是第二大常见的原发性骨恶性肿瘤,其治疗主要为手术切除。软骨肉瘤固有地对常规化疗和放疗具有抗性,所以,临床缺乏对晚期软骨肉瘤的有效治疗。因此,研究软骨肉瘤耐药机制对于发现新的分子靶点至关重要。有氧糖酵解(Warburg效应)被认为是肿瘤的特征,新的研究发现Warburg效应可以影响抗癌药物疗效。同时,硫氧还蛋白互作蛋白(TXNIP)在氧化应激,细胞增殖与凋亡以及代谢调控中发挥着重要作用。最近的研究表明,TXNIP在许多人类肿瘤如乳腺癌、肝癌、肺癌、膀胱癌以及胃癌中低表达,同时,在恶性肿瘤糖酵解过程中发挥着关键作用。在此过程中,TXNIP常常通过调节葡萄糖转运蛋白GLUT1介导糖酵解的调控。首先,我们通过高浓度JQ1处理软骨肉瘤细胞的方法,建立JQ1耐药软骨肉瘤细胞模型;接着,在获得稳定耐药软骨肉瘤细胞系后,我们利用CCK-8、流式周期术、Western blot、细胞计数法以及克隆形成实验,发现JQ1耐药软骨肉瘤细胞系对JQ1以及顺铂耐药,证实此细胞系具有潜在的多重耐药性。为了进一步研究JQ1软骨肉瘤耐药潜在机制,本研究采用i TRAQ蛋白质组学在JQ1耐药软骨肉瘤细胞中发现379个差异蛋白,其中211个位上调差异蛋白;通过对差异蛋白GO功能注释以及KEGG通路注释等信息学分析,发现JQ1耐药软骨肉瘤细胞可能存在恶性程度能力增强以及糖酵解增加。本研究利用划痕实验、Transwell实验以及Western blot证实JQ1耐药软骨肉瘤细胞迁移能力增加;接着,我们利用Seahorse XF96糖酵解能力测定实验、葡萄糖剥夺实验联合细胞计数实验以及克隆形成实验以及q PCR检测证实JQ1耐药软骨肉瘤细胞糖酵解能力增强;同时,我们通过q PCR以及Western blot发现TXNIP在JQ1耐药软骨肉瘤细胞中低表达;利用过表达TXNIP实验联合Seahorse XF96糖酵解能力测定实验以及葡萄糖剥夺实验,发现TXNIP过表达的JQ1耐药软骨肉瘤细胞对葡糖糖剥夺产生耐受,同时减弱耐药细胞糖酵解的能力;通过q PCR以及Western blot检测GLUT1的表达以及定位,发现GLUT1在JQ1耐药软骨肉瘤细胞中高表达,主要在细胞膜与细胞器表达,同时伴随着TXNIP表达的降低。综上所述,首先,我们建立了JQ1耐药软骨肉瘤细胞系,并证实了JQ1耐药软骨肉瘤细胞具有JQ1以及顺铂耐药性。其次,发现JQ1耐药软骨肉瘤细胞迁移能力以及糖酵解能力增强。最后阐明TXNIP/GLUT1信号通路介导软骨肉瘤细胞JQ1耐药的糖酵解增加。这为耐药软骨肉瘤治疗提供理论与实验依据,为新抗癌药的研发奠定基础。更多还原

【Abstract】 Chondrosarcoma is the second-most frequent primary malignant tumor of bone.Surgical resection is the most acceptable treatment approach for chondrosarcoma.Meanwhile,there is no effective treatment for advanced chondrosarcoma in the clinic,because chondrosarcoma is inherently resistant to conventional chemotherapy and radiotherapy.Therefore,That understanding the chemotherapy resistance mechanism of chondrosarcoma is very important for developing new molecularly targeted therapies.Aerobic glycolysis(Warburg effect)has been considered to be a characteristic of tumors.Recent research has found that the Warburg effect plays a vital role in the efficacy of chemotherapy.Thioredoxin-interacting protein(TXNIP)plays a vital role in oxidative stress,cell proliferation and apoptosis,and metabolic regulation.Recent studies have shown TXNIP is low expressed in a serious of human cancer,and it plays an indispensable role in the glycolysis process of malignant tumors.In this process,GLUT1 was usually needed for the TXNIP function.First,we established a JQ1-resistant chondrosarcoma cell model by treating chondrosarcoma cells with a high concentration of JQ1.Second,CCK-8 cell proliferation experiment,flow cytometry,Western blot,cell counting assay,and colony formation experiment were used to confirm the resistance of JQ1 and cisplatin in the obtained stable drug-resistant chondrosarcoma cell line,indicating that the resistant cell has the potential ability of multiple drug resistance.The analysis of i TRAQ-based proteomics showed there are 379 differentially expressed proteins,of which,211 proteins were up-regulated differentially expressed proteins.We found JQ1-resistant chondrosarcoma cells may have enhanced migration ability and glycolysis through bioinformatics analysis such as GO functional annotation and KEGG pathway annotation.The scratch wound healing assay,Transwell assay,and Western blot were used to confirm the migration ability of drug-resistant chondrosarcoma cells is enhanced.And then,the Seahorse XF96 glycolysis capacity test,glucose deprivation test with cell count assay,and the cloning formation assay,and q PCR were used to confirm JQ1-resistant chondrosarcoma cells have enhanced glycolysis ability.We found TXNIP is under-expressed in JQ1-resistant chondrosarcoma cells and overexpression TXNIP can reduce the activity of glycolysis in JQ1-resistant chondrosarcoma cells using Seahorse XF96 glycolysis ability test,glucose deprivation assay,Western blot and q PCR.More interestingly,Glut was found highly expressed in JQ1-resistant chondrosarcoma cells,accompanied by decreased expression of TXNIP.Taken together,we established a JQ1 resistant chondrosarcoma cell line and confirmed that JQ1 resistant chondrosarcoma cells have the ability of JQ1 and cisplatin resistance.We found the migration ability and glycolysis ability of JQ1-resistant chondrosarcoma cells are enhanced.Further,we found the TXNIP/GLUT1 signaling pathway mediates the JQ1 resistance of chondrosarcoma cells by promoting glycolysis.This study may lead to the identification of molecular targets for the development of novel anti-cancer agents.更多还原