【作者】 李博；

【导师】 周松；

【作者基本信息】 南昌大学 ， 外科学（骨科）（专业学位）， 2023， 硕士

【摘要】 背景和目的:骨肉瘤(Osteosarcoma,OS)是临床上相对常见的一种恶性骨肿瘤,发病趋势逐年上升。骨肉瘤细胞容易扩散,转移的发生是大多数患者的死亡原因。与其他恶性肿瘤相比,骨肉瘤具有其独特的特征,包括基因组背景的复杂性和不稳定性,以及多数量的突变驱动基因。在分子水平上,导致恶性肿瘤转移的机制中存在许多未知因素,目前关于驱动骨肉瘤转移的潜在生物学信息很少。因此,本研究的目的是从基因层面上阐明驱动骨肉瘤转移的相关机制研究,以开发更加具有特异性的治疗方法并降低骨肉瘤患者的死亡率。方法:1、基因的筛选:基于高通量基因表达(Gene Expression Omnibus database,GEO)数据库获取骨肉瘤转移相关的组织的基因表达情况。通过权重基因共表达网络分析(Weighted Gene Co-expression Network Analysis,WGCNA)找到具有最显著差异的模块,对差异基因(Differentialy Expressed Genes,DEGs)的功能和通路富集分析,可以确定某些基因参与的功能节点。将获得的基因序列图谱用于构建蛋白-蛋白互作网络(Protein-Protein Interaction Networks,PPI)以及寻找相应的核心基因。2、敲低核心基因LEPRE1(Leucine Proline-enriched Proteoglycan 1)探索对人骨肉瘤细胞影响:通过siRNA技术靶向下调LEPRE1基因在人骨瘤细胞中的表达,利用RT-qPCR及Western blot检测相关基因的mRNA及蛋白质表达情况。利用CCK-8实验、平板细胞克隆形成实验检测下调相关基因后的骨肉瘤细胞的增殖能力;细胞划痕实验、Transwell实验检测骨肉瘤细胞的迁移及侵袭能力。结果:1、我们对GSE21257数据集进行了WGCNA相关分析,找到了基因差异最显著的灰色模块。分析另一个数据集GSE99671中的转移和非转移样本,我们最终鉴定了5个与骨肉瘤转移相关的核心基因(LEPRE1、LTF、ELANE、LCN2、PLOD1)。2、我们通过进一步的实验验证了我们的推测,发现它们与骨肿瘤的远处转移密切相关。差异基因的富集分析结果显示,与骨肉瘤转移相关的基因主要位于细胞外区及特定颗粒管腔;其分子功能主要为氧转运蛋白活性、过氧化物酶活性、与结合珠蛋白结合等;涉及的生物学进程主要为细菌防御反应、二氧化碳输送、氧气输送及红细胞分化等。提示肿瘤发生转移与涉及的这些机制密切相关。3、干扰人骨肉瘤细胞株143B和MG63中LEPRE1的表达后,CCK-8以及平板细胞克隆形成实验结果显示骨肉瘤细胞的增殖活性以及克隆形成的能力受到了抑制;细胞划痕实验、Transwell实验进一步显示骨肉瘤细胞的迁移和侵袭能力在敲低LEPRE1基因的表达后也受到了抑制。结论:我们通过生物信息学筛选的基因与骨肉瘤转移密切相关。我们进一步的实验说明敲低LEPRE1基因,可抑制骨肉瘤细胞的增殖、迁移和侵袭能力。以上研究提示LEPRE1可能是骨肉瘤细胞的重要潜在靶标。更多还原

【Abstract】 Background:Osteosarcoma(OS)is a relatively common malignant bone tumor with an increasing incidence trend.OS cells are prone to spread,and metastasis is the cause of death in most patients.Compared with other malignant tumors,OS has its unique characteristics,including the complexity and instability of the genomic background,as well as a large number of mutations driven genes.At the molecular level,there are many unknown factors in the mechanisms leading to malignant tumor metastasis,and there is currently little potential biological information about driving OS metastasis.Therefore,the purpose of this study is to elucidate the relevant mechanisms driving the metastasis of OS at the genetic level,in order to develop more specific treatment methods and reduce the mortality rate of OS patients.method:Based on the Gene Expression Omnibus(GEO)database,we obtained the gene expression profile of OS metastasis related tissues.Weighted gene co-expression network analysis(WGCNA)was used to identify the modules with the most significant differences.Analysis of the function and pathway enrichment of differentialy expressed genes(DEGs)can identify the functional nodes in which certain genes participate.The obtained gene sequence map is used to construct protein-protein interaction networks(PPI)and search for corresponding hub genes.Knocking down the core gene Leucine Proline-enriched Proteoglycan 1(LEPRE1)to explore the impact on human Osteosarcoma cells.Targeted downregulation the expression of LEPRE1 gene in human osteosarcoma cells through siRNA technology and detect the mRNA and protein expression of related genes by RT qPCR and Western blot.The proliferation ability of Osteosarcoma cells after down-regulation of related genes was detected by CCK-8 test and plate cell cloning test.Wound healing assay and Transwell test were used to detect the migration and invasion ability of Osteosarcoma cells.Result:We conducted WGCNA correlation analysis on the GSE21257 dataset and found the grey module with the most significant genetic differences.After analyzing the metastatic and non-metastatic samples in another data set GSE99671,we finally identified five core genes related to Osteosarcoma metastasis(LEPRE1,LTF,ELANE,LCN2,PLOD1).We confirmed the hypothesis through further experiments,and we found that they were closely related to distant metastasis of bone malignant tumor.The enrichment analysis of differential genes showed that the genes related to OS metastasis were mainly located in extracellular regions and specific granule lumen.Its molecular functions mainly include oxygen transporter activity,peroxidase activity and haptoglobin binding.The biological processes involved mainly include defense response to bacterium,carbon dioxide transport,oxygen transport and erythrocyte differentiation.It is suggested that the occurrence and metastasis of tumors are closely related to these mechanisms involved.After interfering with the expression of LEPRE1 in human OS cells(143B and MG63),the results of CCK-8 and plate clone formation assay showed that the proliferative activity and cloning ability of OS cells were inhibited.Wound healing assay and Transwell assay further showed that the migration and invasion ability of OS cells was also inhibited after knocking down the expression of LEPRE1 gene.Conclusion:The genes we screened through bioinformatics are closely related to the metastasis of OS.Our further experiments demonstrate that knocking down the LEPRE1 gene can inhibit the proliferation,migration,and invasion of OS cells.The above studies suggest that LEPRE1 may be an important potential target for OS cells.更多还原