衰老基因在骨肉瘤治疗、预后及肿瘤微环境中的作用研究

Role of senescent genes in the treatment,prognosis and tumor microenvironment for osteosarcoma

目的利用生物信息学技术分析并验证衰老基因在成骨型骨肉瘤患者治疗、预后及肿瘤微环境(TME)中的作用。方法从中国国家基因组科学数据库(https://ngdc.cncb.ac.cn/aging/index)中获取衰老相关基因,从TARGET数据库(https://ocg.cancer.gov/programs/target)中获取骨肉瘤患者的基因表达谱和临床信息,并从基因表达数据库(GEO)中获取骨肉瘤的单细胞数据集GSE162454用于下游分析。通过单细胞数据集对骨肉瘤细胞进行分型,寻找成骨细胞/成软骨细胞与其他细胞亚型的差异化表达基因(DEGs),并与衰老相关基因进行比对。采用单因素、多因素Cox回归分析确定预后相关衰老DEGs基因,构建骨肉瘤衰老相关模型(OSRM)并计算风险评分,分析OSRM在骨肉瘤治疗、预后及TME中的特征。结果GSE162454共包含6个骨肉瘤样本,经过滤质控后共有19933个细胞。经统一流形逼近与投影(UMAP)聚类后确定17个细胞亚群。成骨细胞/成软骨细胞与其他亚群间共有4821个DEGs,与衰老基因集比对后共获取132个衰老相关DEGs。在TARGET数据库中,单因素、多因素Cox分析获取了4个预后基因:ADH5(醇脱氢酶5)、ARHGAP1(Rho GTPase激活蛋白1)、APOE(载脂蛋白E)、ATF4(激活转录因子4)用于构建OSRM。依据风险评分将84例成骨型骨肉瘤患者分为高风险组(n=42)与低风险组(n=42),低风险组预后较好(HR=0.13,95%CI 0.06~0.28,P<0.001),且对免疫检查点抑制剂有较强的应答敏感性。qRT-PCR和Western blotting证实PD-1过表达的K7M2细胞中ADH5(P<0.01)、APOE(P<0.001)、ATF4(P<0.05)呈持续高表达,可预测抗PD-1对骨肉瘤的免疫治疗效果。结论ADH5、ARHGAP1、APOE、ATF4是成骨型骨肉瘤中潜在的衰老相关促癌或抑癌基因,基于这些基因构建的OSRM可判断骨肉瘤的治疗应答、预后和TME特征。OSRM关键基因促进了骨肉瘤的分子病理诊断,并可作为预测抗PD-1免疫治疗应答的潜在生物标志物。

Objective To analyze and verify the role of senescent genes in the treatment,prognosis,and tumor microenvironment(TME)characteristics of osteoblastic osteosarcoma,bioinformatic methods were employed.Methods Senescent genes were obtained from the China National Genome Science database(https://ngdc.cncb.ac.cn/aging/index).The gene expression profile and clinical information of osteosarcoma patients were sourced from the TARGET database(https://ocg.cancer.gov/programs/target),while single-cell RNA-sequencing(scRNA-seq)data was collected from GSE162454 on the Gene Expression Omnibus(GEO)for downstream analysis.Osteosarcoma cells were classified based on scRNA-seq,and differential expression analysis between osteoblasts/chondroblasts and other cell types was conducted to identify differently expressed genes(DEGs).After matching with the senescent genes,prognostic senescent DEGs were identified through univariable and multivariable Cox regression analysis.Subsequently,the osteosarcoma senescent-related model(OSRM)was constructed,and the risk score was calculated.The role of OSRM in treatment,prognosis,and TME of osteosarcoma was further investigated.Results The analysis revealed that GSE162454 contained 6 osteosarcoma samples,with 19933 cells identified after filtering,quality control,and normalization.Seventeen cellular subtypes were identified using uniform manifold approximation and projection(UMAP)methods.A total of 4821 DEGs were found between osteoblasts/chondroblasts and other subtypes,with 132 senescent DEGs obtained after matching with the senescent gene set.In the TARGET database,4 prognostic senescent DEGs[ADH5(alcohol dehydrogenase 5),ARHGAP1(Rho GTPase activating protein 1),APOE(apolipoprotein E),and ATF4(activating transcription factor 4)]were identified through univariable and multivariable Cox analyses to construct OSRM.Based on risk score,patients were stratified into high-and low-risk groups,with the latter showing better prognosis(HR=0.13,95%CI 0.06-0.28,P<0.001)and higher sensitivity to immune checkpoint inhibitors.qRT-PCR and Western blotting confirmed the high expression of senescent genes ADH5(P<0.01),APOE(P<0.01),and ATF4(P<0.05)in the K7M2 osteosarcoma cell line,suggesting the potential for predicting the response to anti-PD-1 immunotherapy for osteosarcoma.Conclusions scRNA-seq facilitated the division of osteosarcoma into 17 cell subtypes.ADH5,ARHGAP1,APOE,and ATF4 emerged as potential cancer-promoting or suppressing senescent genes in osteosarcoma.OSRM was found to be associated with treatment response,prognosis,and TME characteristics,thereby promoting the molecular pathological diagnosis of osteoblastic osteosarcoma and prediction for anti-PD-1 immunotherapy.