背景：脑胶质瘤是中枢神经系统中最常见的肿瘤疾病，目前针对其一线治疗手段包括手术切除及术后放化疗，但总体疗效并不理想，因此亟需出现更多、更精准的诊疗靶点进一步推动胶质瘤的个体化靶向治疗。线粒体是细胞内氧化磷酸化及合成ATP的场所，在癌细胞代谢过程中发挥着重要生物学功能。巨噬细胞极化是指在特定微环境刺激下，巨噬细胞的功能表型发生转变的过程，在不同抗癌途径中扮演着重要角色。而线粒体是调节巨噬细胞极化的主要细胞器。因此，结合转录组数据筛选关键目的基因，对其进行生物信息学分析获取相关生物学特征，并对目的基因调控胶质瘤的机制进行深入探究，有助于为胶质瘤靶向治疗提供精准靶点及理论支撑。

研究内容和方法：首先，从胶质瘤相关转录组数据集中获取差异表达基因，将其与WGCNA识别出的巨噬细胞极化评分相关基因及线粒体相关基因取交集，获得48个重叠基因；进一步通过Cytoscape插件中的MCODE聚类分析得到5个目的基因，构建胶质瘤预后模型；并进行独立预后分析、肿瘤微环境分析、列线图构建、免疫检查点分析、功能富集、亚细胞定位、临床病理特征分析及基因调控网络构建；并通过验证集数据、患者组织样本及免疫组化数据库对预后基因进行验证。之后，我们纳入单细胞数据集，通过细胞类型鉴定和细胞分群注释的方法，结合预后基因获取关键细胞；并对关键细胞进行了拟时序分析和细胞通讯分析。接着，我们选取目的基因SUCLG2进行深入的机制探究。首先，在胶质瘤细胞系及动物模型中，敲除SUCLG2观察其对胶质瘤发生发展的影响；接着对线粒体功能相关标记物进行了检测，以此判断SUCLG2对线粒体功能的作用；之后，对线粒体功能相关蛋白的表达、琥珀酰化水平及酶的活性进行了检测，探究了SUCLG2对线粒体相关代谢酶的作用机制。最后，我们对泛素化的水平、途径及去琥珀酰化酶的调控作用进行了探究，详细阐述了目的基因SUCLG2通过线粒体功能对胶质瘤的调控机制。

研究结果：

第一部分：我们通过对转录组数据集的处理，筛选出了表达具有差异、线粒体相关及巨噬细胞极化相关的交集预后基因，分别为OXCT1、ECI2、MCCC2、CPT2及SUCLG2，并以此为基础构建了胶质瘤预后模型。接着通过生物信息学的方法，对5个预后基因的生物学特征进行了全方位的综合分析。最后结合验证集、HPA数据库及组织样本进行了验证。

第二部分：我们对单细胞数据集GSE138794进行了数据质量统计及预处理，获得了高变异基因中排名前十的标记基因，接着对其进行了细胞分群注释及类型鉴定，结合5个预后基因获取了关键细胞，分别为骨髓源性巨噬细胞、少突胶质细胞和神经祖细胞。最后对三种细胞进行了拟时序分析和细胞通讯分析，揭示了其在胶质瘤发展过程中的功能状态改变及细胞间的相互作用关系。

第三部分：选取目的基因SUCLG2探究其在胶质瘤中的调控机制。我们构建了SUCLG2敲除的胶质瘤细胞系和动物模型，发现敲除后可显著抑制胶质瘤细胞增殖。通过对线粒体功能标记物进行了检测，证明了敲除SUCLG2导致线粒体功能受损。最后通过对线粒体功能代谢酶的活性及琥珀酰化水平的检测，验证了SUCLG2提高代谢酶的活性进而抑制线粒体功能蛋白的琥珀酰化。

第四部分：我们对目的基因SUCLG2调控机制中的泛素化进一步深入探索。通过在胶质瘤细胞系中敲除和过表达实验，论证了去琥珀酰化酶SIRT5可以促进SUCLG2泛素化，然后通过溶酶体途径进行降解。此外，SUCLG2也介导了SIRT5对胶质瘤细胞增殖的调控作用。

结论：我们基于线粒体和巨噬细胞极化筛选出了胶质瘤预后基因，构建了预后模型，并分析了其相关生物学特征。结合单细胞数据集获取了关键细胞，并对其功能状态改变过程及细胞间相互作用关系进行了阐述。针对胶质瘤诊疗新靶点SUCLG2进行了深入探究，论证了其介导线粒体功能相关蛋白的琥珀酰化及通过溶酶体途径泛素化的降解模式在胶质瘤中的调控机制，为胶质瘤个体化治疗提供了有效靶点及充分的理论支撑。

BACKGROUND: Glioma is the most common tumor in the central nervous system. Currently, the first-line treatments for glioma include surgical resection and postoperative radiotherapy, but the overall therapeutic efficacy is unsatisfactory. Therefore, there is an urgent need for more and more precise diagnostic and therapeutic targets to further promote the individualized targeted therapy of glioma. Mitochondria are the site of intracellular oxidative phosphorylation and ATP synthesis, and perform important biological functions in the metabolic process of cancer cells. Macrophage polarization refers to the process of macrophage functional phenotypic shift under specific microenvironmental stimuli, and takes a significant role in different anticancer pathways. And mitochondria are the main organelles that regulate macrophage polarization. Therefore, combining transcriptomic data to screen key target genes, bioinformatics analysis to obtain relevant biological features, and in-depth investigation of the mechanism of target genes regulating gliomas can help to provide precise targets and theoretical support for the targeted therapy of gliomas.

RESEARCH CONTENT AND METHODS: Firstly, differentially expressed genes were obtained from the glioma-related transcriptome dataset, and 48 overlapping genes were retrieved by taking the intersection with macrophage polarization score-related genes and mitochondria-related genes identified by WGCNA; further, 5 target genes were obtained by MCODE clustering analysis in Cytoscape plug-in to construct the prognosis model of gliomas; and independent prognostic analyses, tumor micro environment analysis, nomogram construction, immune checkpoint analysis, functional enrichment, subcellular localization, clinicopathological characterization and gene regulatory network construction; and the prognostic genes were validated by validation set data, patient tissue samples and immunohistochemistry database. After that, we incorporated single-cell datasets and obtained key cells in combination with prognostic genes by means of cell type identification and cell subgroup annotation; and performed pseudotemporal analysis and cell communication analysis on key cells. Then, we selected the target gene SUCLG2 for in-depth mechanism exploration. First, we knocked down SUCLG2 in glioma cell lines and animal models to observe its effect on glioma development; then, we observed the morphology of intracellular mitochondria by transmission electron microscopy and detected mitochondrial function-related markers to determine the role of SUCLG2 on mitochondrial function; and then, the expression of mitochondrial function-related proteins, the level of succinylation, and enzyme activities were examined to explore the role of SUCLG2 in mitochondrial function. After that, the expression of mitochondrial function-related proteins, succinylation levels and enzyme activities were checked, and the mechanism of SUCLG2's action on mitochondrial-related metabolic enzymes was explored. Finally, we investigated the level and pathway of ubiquitination and the regulatory role of desuccinylase, and elaborated the regulatory mechanism of the target gene SUCLG2 on glioma through mitochondrial function.

RESULTS:

Section One: We screened the intersecting prognostic genes with differential expression, mitochondria-related and macrophage polarization-related, namely OXCT1, ECI2, MCCC2, CPT2 and SUCLG2, through the processing of transcriptomic datasets, and constructed a glioma prognostic model based on them. Then the biological characteristics of the five prognostic genes were comprehensively analyzed by bioinformatics. Finally, the validation was carried out by combining the validation set, HPA database and tissue samples.

Section Two: We performed data quality statistics and preprocessing on the single-cell dataset GSE138794 to obtain the top ten marker genes among the highly variable genes, followed by cellular subset annotation and type identification, and combined with five prognostic genes to acquire the key cells, which were bone marrow-derived macrophages, oligodendrocytes and neural progenitor cells. Finally, the three types of cells were analyzed by pseudotemporal analysis and cellular communication analysis, which revealed their altered functional status and inter-cellular interactions during the development of gliomas.

Section Three: The target gene SUCLG2 was selected to explore its regulatory mechanism in glioma. We constructed SUCLG2 knockdown glioma cell lines and animal models and found that the knockdown significantly inhibited glioma cell proliferation. Mitochondrial morphology was observed by transmission electron microscopy and mitochondrial function markers were examined, demonstrating that knockdown of SUCLG2 resulted in impaired mitochondrial function. Finally, by detecting the activity of mitochondrial functional metabolic enzymes and the level of succinylation, it was verified that SUCLG2 increased the activity of metabolic enzymes and thus inhibited the succinylation of mitochondrial functional proteins.

Section Four: We further explored in depth the ubiquitination in the regulatory mechanism of the target gene SUCLG2. Through knockdown and overexpression experiments in glioma cell lines, it was shown that the desuccinylase SIRT5 promotes SUCLG2 ubiquitination followed by degradation through the lysosomal pathway. In addition, SUCLG2 mediated the regulatory effect of SIRT5 on glioma cell proliferation.

CONCLUSION: We screened glioma prognostic genes based on mitochondrial and macrophage polarization, constructed a prognostic model, and analyzed its related biological features. Combined with single-cell dataset, we acquired key cells, and elaborated on the process of their functional state change and intercellular interaction relationship. The new target of SUCLG2 for glioma diagnosis and treatment was explored in depth, and the mechanism of SUCLG2 mediating the succinylation of mitochondrial function-related proteins and the degradation mode of ubiquitination through the lysosomal pathway in glioma was demonstrated, which provides an effective target and sufficient theoretical support for the individualized treatment of glioma.