DataSheet_1_Role of proteoglycan synthesis genes in osteosarcoma stem cells.docx

Osteosarcoma stem cells (OSCs) contribute to the pathogenesis of osteosarcoma (OS), which is the most common malignant primary bone tumor. The significance and underlying mechanisms of action of proteoglycans (PGs) and glycosaminoglycans (GAGs) in OSC phenotypes and OS malignancy are largely unknown. This study aimed to investigate the role of PG/GAG biosynthesis and the corresponding candidate genes in OSCs and poor clinical outcomes in OS using scRNA-seq and bulk RNA-seq datasets of clinical OS specimens, accompanied by biological validation by in vitro genetic and pharmacological analyses. The expression of β-1,3-glucuronyltransferase 3 (B3GAT3), one of the genes responsible for the biosynthesis of the common core tetrasaccharide linker region of PGs, was significantly upregulated in both OSC populations and OS tissues and was associated with poor survival in patients with OS with high stem cell properties. Moreover, the genetic inactivation of B3GAT3 by RNA interference and pharmacological inhibition of PG biosynthesis abrogated the self-renewal potential of OSCs. Collectively, these findings suggest a pivotal role for B3GAT3 and PG/GAG biosynthesis in the regulation of OSC phenotypes and OS malignancy, thereby providing a potential target for OSC-directed therapy.

骨肉瘤干细胞（OSCs）对骨肉瘤（OS）的发病机制具有重要贡献，而骨肉瘤是原发性恶性骨肿瘤中最常见的一种。蛋白聚糖（PGs）和糖胺聚糖（GAGs）在骨肉瘤表型和骨肉瘤恶性行为中的意义及其作用机制尚不明确。本研究旨在利用临床骨肉瘤标本的单细胞RNA测序（scRNA-seq）和全转录组RNA测序（bulk RNA-seq）数据集，探讨PG/GAG生物合成及其候选基因在骨肉瘤干细胞和骨肉瘤不良临床结局中的作用，并通过体外遗传学和药理学分析进行生物学验证。负责蛋白聚糖常见核心四糖连接区域生物合成的基因之一β-1,3-葡萄糖醛酸基转移酶3（B3GAT3）在骨肉瘤细胞群和骨肉瘤组织中表达显著上调，并与具有高干细胞特性的骨肉瘤患者的不良预后相关。此外，通过RNA干扰抑制B3GAT3的遗传活性和通过药理学抑制PG生物合成可消除骨肉瘤干细胞的自我更新潜能。综上所述，这些发现表明B3GAT3和PG/GAG生物合成在调节骨肉瘤表型和骨肉瘤恶性行为中起着关键作用，从而为针对骨肉瘤的治疗提供了潜在靶点。