酪蛋白激酶Fam20C小分子抑制剂的发现及其抗肿瘤初步机制研究

发布时间：2018-06-05 06:19

本文选题：Fam20C + Fam20C抑制剂(FL-1607)　；参考：《暨南大学》2017年硕士论文

【摘要】：三阴性乳腺癌(triple negative breast cancer,TNBC)是一种侵袭转移能力更强,预后更差的特殊类型乳腺癌。Fam20C是三阴性乳腺癌细胞迁移的关键因子。本文旨在考察Fam20C作为TNBC治疗的潜在靶点的可行性和寻找Fam20C抑制剂作为抗TNBC的新途径。通过从PrePPI蛋白相互作用数据库收集蛋白相互作用数据,构建Fam20C蛋白质相互作用网络,结合GO功能注释,从系统生物学角度研究Fam20C在TNBC中的作用机制。通过对TCGA和METABRIC两个癌症数据库的信息挖掘,考察不同亚型乳腺癌样本Fam20C的mRNA表达量是否存在显著差异。通过同源建模和蛋白质晶体叠合确定选择性抑制剂的设计结合位点,运用分子对接方法、MTT细胞毒活性实验和分子动力学模拟方法筛选Fam20C抑制剂。利用荧光显微镜观察、蛋白免疫印迹、流式细胞计数、划痕实验研究抑制剂抗肿瘤初步机制。网络分析表明,Fam20C蛋白相互作用总网络包含672节点和974相互作用,结合GO功能注释初步阐明了Fam20C在癌症中的作用机制。转录分析表明,在TCGA数据库,Fam20C的mRNA表达量在TNBC样本组比Non-TNBC样本组更高(Wilcoxon秩和检验P=6.79E-16),与METABRIC数据库结果一致(Wilcoxon秩和检验P=2.77E-19)。MTT细胞毒活性试验得出FL-1607对MDA-MB-468细胞具有一定的抗肿瘤活性,并诱导细胞产生凋亡细胞形态学变化(染色质浓缩),流式细胞仪结果显示,细胞凋亡率随FL-1607给药剂量增加而增加,呈剂量依赖性。蛋白免疫印迹发现,FL-1607促进Bax、Cyto.C、Caspase3蛋白表达上调,并导致Bcl-2蛋白表达下调,表明药物可诱导细胞发生线粒体途径的细胞凋亡。划痕实验表明,与空白组相比,FL-1607处理组MDA-MB-468细胞划痕愈合程度更低。综上,Fam20C是治疗三阴性乳腺癌的有效靶点,FL-1607可以诱导TNBC细胞系(MDA-MB-468)细胞凋亡和潜在抑制细胞迁移。
[Abstract]:Tri-negative breast cancer TNBCis is a special type of breast cancer, which has stronger ability of invasion and metastasis and worse prognosis. Fam20C is the key factor in the migration of tri-negative breast cancer cells. The purpose of this study was to investigate the feasibility of Fam20C as a potential target for TNBC therapy and to explore a new approach to Fam20C inhibitor as anti TNBC. Through collecting protein interaction data from PrePPI protein interaction database, constructing Fam20C protein interaction network and combining with go functional annotation, the mechanism of Fam20C in TNBC was studied from the point of view of system biology. Based on the information mining of TCGA and METABRIC cancer databases, we investigated whether there were significant differences in mRNA expression of Fam20C in different subtypes of breast cancer samples. The design binding sites of selective inhibitors were determined by homology modeling and protein crystal superposition. Fam20C inhibitors were screened by molecular docking assay and molecular dynamics simulation. Fluorescence microscopy, Western blotting, flow cytometry and scratch test were used to study the mechanism of inhibitor antitumor. Network analysis showed that the total network of Fam20C protein interaction included 672 nodes and 974 interactions. Combined with go functional annotation, the mechanism of Fam20C in cancer was preliminarily clarified. Transcriptional analysis showed that the mRNA expression of Fam20C in the TCGA database was higher in the TNBC sample group than in the Non-TNBC sample group. The results of the Wilcoxon rank sum test and METABRIC database showed that FL-1607 had a certain anti-tumor activity on MDA-MB-468 cells. Morphological changes of apoptotic cells were induced. The results of chromatin concentration and flow cytometry showed that the apoptosis rate increased in a dose-dependent manner with the increase of the dosage of FL-1607. Western blot showed that FL-1607 promoted the up-regulation of BaxanCyto.Caspase3 protein expression and down-regulated the expression of Bcl-2 protein, suggesting that drugs could induce apoptosis in mitochondria pathway. Scratch test showed that the degree of scratch healing of MDA-MB-468 cells in FL-1607 group was lower than that in control group. FL-1607, an effective target for the treatment of triple negative breast cancer, can induce apoptosis and potentially inhibit cell migration in TNBC cell line MDA-MB-468.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R91;R96

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