磷酸核糖氨基咪唑羧化酶,磷酸核糖氨基咪唑琥珀酰胺合成酶——骨肉瘤肿瘤微环境相关代谢基因的生物信息学分析及验证

Phosphoribosyl aminoimidazole carboxylase,phosphoribosyl aminoimidazole succino carboxamide synthetase-Bioinformatics analysis and validation of metabolic genes related to the tumor microenvironment of osteosarcoma

目的研究既往骨肉瘤患者和正常样本的代谢基因,探讨骨肉瘤肿瘤微环境中代谢基因的特性。方法通过生物信息学分析参与肿瘤微环境(TME)免疫功能以及骨肉瘤患者生存和预后的代谢基因。将磷酸核糖氨基咪唑羧化酶,磷酸核糖氨基咪唑琥珀酰胺合成酶(PAICS基因)作为潜在的研究对象,探讨其在骨肉瘤细胞系中对细胞增殖、迁移和凋亡中的作用。结果通过生物信息学分析,筛选出876个与软骨肉瘤OS(overall survival)相关的代谢基因和206个免疫相关代谢基因,将其相同基因进行聚类分析,分为三类:C1、C2和C3,其中C1集群的OS低于C2和C3(P<0.01),C3集群的免疫评分高于C2和C3(P<0.01)。流式细胞实验结果示HOS和MG-63细胞系si-PAICS-1和si-PAICS-2组的凋亡率均高于对照组,HOS细胞系si-PAICS-1组、si-PAICS-2组与对照组差异有统计学意义(P<0.01);MG-63细胞系si-PAICS-1组、si-PAICS-2组与对照组差异有统计学意义(P<0.01)。Transwell细胞迁移实验结果示,HOS和MG-63细胞系si-PAICS-1和si-PAICS-2组细胞数量低于对照组,HOS细胞系si-PAICS-1组、si-PAICS-2组与对照组差异有统计学意义(P<0.01);MG-63细胞系si-PAICS-1组、si-PAICS-2组与对照组差异有统计学意义(P<0.01)。伤口愈合实验中,HOS和MG-63细胞系si-PAICS-1和si-PAICS-2组细胞迁移距离均低于对照组,HOS细胞系si-PAICS-1组、si-PAICS-2组与对照组差异有统计学意义(P<0.01);MG-63细胞系si-PAICS-1组、si-PAICS-2组与对照组差异有统计学意义(P<0.01)。对照组细胞群落形成优于HOS和MG-63细胞系si-PAICS-1和si-PAICS-2组,HOS细胞系si-PAICS-1组、si-PAICS-2组与对照组差异有统计学意义(P<0.01);MG-63细胞系si-PAICS-1组、si-PAICS-2组与对照组差异有统计学意义(P<0.01)。最后,构建骨肉瘤患者预后的预测模型,结果显示,高表达组比低表达组的OS差,ROC曲线分析验证显示验证队列中1年时的AUC为0.86;通过Lasso Cox回归分析,发现8个枢纽基因可能是潜在预后因素。结论代谢基因特别是PAICS在骨肉瘤TME免疫细胞浸润以及癌症发展和转移中起着不可或缺的作用。

Objective To study the metabolic genes of previous osteosarcoma patients and normal samples,and determine the characteristics of metabolic genes in the tumor microenvironment(TME)of osteosarcoma.Methods Computational technique was applied to identify metabolic genes involved in the immune function of TME and survival and prognosis of the osteosarcoma patients.Potential candidate gene,phosphoribosyl aminoimidazole carboxylase and phosphoribosyl aminoimidazole succino carboxamide synthetase(PAICS),was chosen for further studies in osteosarcoma cell lines for its role in cell proliferation,migration and apoptosis.Results Through bioinformatics analysis,876 metabolic genes associated with Chondrosarcoma OS(overall survival)and 206 immune-related metabolic genes were screened,and their same genes were clustered and analyzed into three categories:C1,C2 and C3,of which the OS of the C1 cluster was lower than that of C2 and C3(P<0.01),and the immune score of the C3 cluster was the highest than that of C2 and C3(P<0.01).The results of flow cytometry showed that the apoptosis rates of the SI-PAICS-1 and si-PAICS-2 groups of the HOS and MG-63 cell lines were higher than those in the control group,and the differences between the SI-PAICS-1 group and the si-PAICS-2 group and the control group were statistically significant(P<0.01),and the MG-63 cell line si-PAICS-1 group and the control group were statistically significant(P<0.01),and the MG-63 cell line si-PAICS-1 group and The difference between the si-PAICS-2 group and the control group was statistically significant(P<0.01).Transwell cell migration experiments showed that the number of cells in the si-PAICS-1 and si-PAICS-2 groups of HOS and MG-63 cell lines was lower than that in the control group,and the differences between the SI-PAICS-1 group and the si-PAICS-2 group and the control group of the HOS cell line were statistically significant(P<0.01);The difference between the si-PAICS-2 group and the control group was statistically significant(P<0.01).In wound healing experiments,the cell mobility of the SI-PAICS-1 and si-PAICS-2 groups of HOS and MG-63 cell lines was lower than that of the control group,and the differences between the SI-PAICS-1 group and the si-PAICS-2 group of the HOS cell line were statistically significant(P<0.01),and the MG-63 cell line si-PAICS-1 group and the control group were statistically significant(P<0.01),The difference between the si-PAICS-2 group and the control group was statistically significant(P<0.01).The colony formation in the control group was superior to that of the SI-PAICS-1 and si-PAICS-2 groups of the HOS cell lines,and the differences between the SI-PAICS-1 group and the si-PAICS-2 group and the control group were statistically significant(P<0.01),and the MG-63 cell line si-PAICS-1 group,The difference between the si-PAICS-2 group and the control group was statistically significant(P<0.01).Finally,by constructing a predictive model for the prognosis of osteosarcoma patients,the results show that the OS of the high-expression group is worse than that of the low-expression group.The accuracy was further verified by ROC curve analysis,and the results showed that the AUC was 0.86 at 1 year in the validation cohort,and 8 pivot genes were found to be potential prognostic factors by Lasso Cox regression analysis.Conclusion The metabolic genes specifically PAICS play an integral role in the immune cell infiltration in osteosarcoma TME,as well as cancer development and metastasis.