【摘要】 目的 探究C-X-C趋化因子配体12/C-X-C趋化因子受体4（C-X-C chemokine ligand 12/C-X-C chemokine receptor 4,CXCL12/CXCR4）轴在儿童期神经纤维瘤Ⅰ型（neurofibromatosis typeⅠ，NF1）中的作用及机制。方法 从基因表达综合（Gene Expression Omnibus,GEO）数据库中获取GSE241224数据集，比较CXCL12表达在NF1儿童恶性周围神经鞘瘤（malignant peripheral nerve sheath tumour,MPNST）与良性神经纤维瘤之间的差异。通过加权基因共表达网络分析（weighted gene co-expression network analysis,WGCNA）对数据集中的前5 000个高方差基因进行分析，识别与CXCL12基因相关性最高的模块，并进行基因本体论（gene ontology,GO）功能富集分析。利用基因表达谱交互分析（gene expression profiling interactive analysis,GEPIA）数据库分析CXCR4基因在肿瘤与正常组织中的表达谱。利用人正常施万细胞（ipn02.3 2λ）、患者来源的丛状神经纤维瘤（plexiform neurofibroma,PNF）细胞系（WZJ）和MPNST细胞系（S462和sNF96.2），通过流式细胞术、Western blotting法检测CXCR4表达，并使用CXCR4特异性抑制剂AMD3100处理细胞，细胞划痕实验探究其对细胞迁移的影响。统计学方法采用方差分析和Dunnett’s t检验，采用R语言对生物信息学数据进行分析。结果 MPNST中CXCL12表达显著高于良性神经纤维瘤组（P=0.041 1），其中上调基因1 630个，下调基因877个。CXCL12的表达与微RNA基因（MIR3687）(r=0.57,P=3.72×10^-8)、E3泛素连接酶基因（RNF185）(r=0.58,P、含锌结合域的脱氢酶基因（ZADH2）(r=0.58,P=1.58×10^-8)、唾液酸转移酶基因（ST8SIA3）(r=0.59,P=1.43×10^-8)和锌指蛋白基因（ZNF791）(r=0.50,P=6.83×10^-9)等呈显著正相关；与微管切割酶基因（KATNAL1）、迁移和侵袭抑制蛋白基因（MIIP）(r=-0.52,P=1.16×10^-6)、去泛素化酶基因（USP42）(r=-0.52,P=9.40×10^-7)等呈显著负相关。WGCNA分析发现，blue模块与CXCL12相关性最高（r=-0.46,P<0.001），其基因富集于细胞周期调控、DNA代谢的正向调控、高尔基体至质膜的蛋白转运、细胞组件的解体以及细胞器定位等生物学功能。其中相关性最强的3个基因分别为自噬相关4C半胱氨酸肽酶基因（ATG4C）、血小板白细胞C激酶底物同源结构域M2蛋白基因（PLEKHM2）和锌指蛋白140基因（ZNF140）。NF1神经相关肿瘤中，CXCR4的表达显著高于正常组织（P<0.05）。流式细胞术检测结果发现，CXCR4在ipn02.3 2λ、WZJ、S462、sNF96.2这4种细胞膜表面的阳性率分别为2.52%、0.18%、2.03%、0.57%；细胞内阳性率分别为89.7%、79.3%、94.3%、96.4%。Western blotting法检测结果显示，CXCR4在MPNST细胞中高表达。使用CXCR4特异性抑制剂AMD3100进行干预，发现与ipn02.3 2λ细胞[（60.30±2.43）%]相比，其对MPNST细胞[（38.05±10.58）%]迁移有明显的抑制作用（P=0.026）。结论 CXCL12/CXCR4轴在儿童NF1中，特别是在MPNST中高表达，促进恶性细胞迁移，并可能通过MIR3687、RNF185、ZADH2、ST8SIA3、ZNF791、KATNAL1、MIIP、USP42、ATG4C、PLEKHM2、ZNF140等基因影响NF1细胞中基因的表达，影响MPNST的发生和发展。更多还原

【Abstract】 Objective To investigate the role and mechanism of the C-X-C chemokine ligand 12/C-X-C chemokine receptor 4（CXCL12/CXCR4） axis in neurofibromatosis type I（NF1） in children. Method The GSE241224 dataset was obtained from the Gene Expression Omnibus（GEO） database, and compared the expression of CXCL12 between malignant peripheral nerve sheath tumor（MPNST） and benign neurofibromas in children with NF1. Weighted gene co-expression network analysis（WGCNA） was conducted on the top 5 000 high-variance genes to identify the module most significantly correlated with the CXCL12 gene, followed by gene ontology（GO） functional enrichment analysis. The expression profile of the CXCR4 gene in tumor and normal tissues was analyzed using the gene expression profiling interactive analysis（GEPIA） database. Human Schwann cells（ipn02.3 2λ）, patient-derived plexiform neurofibroma（PNF） cell line（WZJ）, and MPNST cell lines（S462 and sNF96.2） were utilized, CXCR4 expression was detected by flow cytometry and Western blotting, and its effect in cell migration was investigated using the CXCR4-specific inhibitor AMD3100 through a cell scratch assay. Statistical analysis was performed by analysis of variance and Dunnett’s t-test, and the bioinformatics data were analyzed using R language. Result The expression of CXCL12 in MPNST was significantly higher than in the benign neurofibroma group（P=0.041 1）, with 1 630 genes upregulated and 877 genes downregulated. The expression levels of CXCL12 showed positive correlations with genes such as microRNA MIR3687(r=0.57, P=3.72×10^-8), E3 ubiquitin ligase gene RNF185(r=0.58, P=1.53×10^-8), Zinc-binding dehydrogenase gene ZADH2(r=-0.58, P=1.58×10^-8), sialyltransferase gene ST8 SIA3(r=0.59, P=1.43×10^-8), and Zinc finger protein gene ZNF791(r=0.50, P=6.83×10^-9). The expression levels of CXCL12 showed significant negative correlations with the microtubule-severing enzyme gene KATNAL1(r=-0.58, P=2.56×10^-6), migration and invasion inhibitory protein gene MIIP(r=-0.52, P=1.16×10^-6), and deubiquitinase gene USP42(r=-0.52, P=9.40×10^-7). WGCNA analysis revealed that the blue module had the highest correlation with CXCL12（r=-0.46, P<0.001）, with its genes enriched in biological functions such as regulation of the cell cycle, positive regulation of DNA metabolic processes, protein transport from the Golgi to the plasma membrane, cellular component disassembly, and organelle localization. The three genes exhibiting the strongest correlations were autophagy-related 4C cysteine peptidase（ATG4C）, pleckstrin homology domain containing, family M with Rundomia member M2（PLEKHM2）, and Zinc finger protein 140（ZNF140）. In NF1-related neural tumors, the expression of CXCR4 was significantly higher than in normal tissues（P<0.05）. The results of flow cytometry showed that the positivity rates of CXCR4 on the cell membrane surfaces of ipn02.3 2λ, WZJ, S462, and sNF96.2 were 2.52%, 0.18%, 2.03%, and 0.57%, respectively, while the intracellular positivity rates were 89.7%, 79.3%, 94.3%, and 96.4%, respectively. The results of Western blotting confirmed that CXCR4 was highly expressed in MPNST cells. Treatment with the CXCR4-specific inhibitor AMD3100 significantly inhibited the migration of MPNST cells [（38.05±10.58） % ]compared to ipn02.3 2λcells [（60.30±2.43）% ]（P=0.026）. Conclusion The CXCL12/CXCR4 axis is highly expressed in pediatric NF1, particularly in MPNST, which promoted the migration of malignant cells. It may also influence gene expression in NF1 cells through genes such as MIR3687, RNF185, ZADH2, ST8 SIA3, ZNF791, KATNAL1, MIIP, USP42, ATG4C, PLEKHM2 and ZNF140, thereby affects the development and progression of MPNST.更多还原