Jiang Hongyuan, Zhao Xuliang, Zang Jinhui, Wang Ruijiao, Gao Jiake, Chen Jinli, Yu Tengbo
Department of Sports Medicine, Affiliated Hospital of Qingdao University, Qingdao, Shandong, China.
Qingdao Medical School, Qingdao University, Qingdao, Shandong, China.
Front Pharmacol. 2024 Apr 24;15:1399625. doi: 10.3389/fphar.2024.1399625. eCollection 2024.
To investigate the immune mechanism of osteosarcoma (OS)-specific markers to mitigate bone destruction in the aggressive OS, prone to recurrence and metastasis. Gene expression patterns from the Gene Expression Omnibus (GEO) database (GSE126209) were analyzed using weighted gene co-expression network analysis (WGCNA), protein-protein interaction (PPI) analysis, least absolute shrinkage and selection operator (LASSO) modeling, and survival analysis to identify charged multivesicular body protein 4C (CHMP4C). Subsequently, its role in regulating the immune system and immune cell infiltration was explored. CHMP4C expression and signaling molecules in OS were assessed in osteosarcoma cell lines (MG63, U2OS, HOS) and hFOB1.19 cells using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and immunofluorescence staining. The impact of CHMP4C upregulation and interference on OS-related signaling molecules in MG63 cells was studied. Functional validation of CHMP4C in MG63 OS cells was confirmed through cell counting Kit-8 (CCK-8), transwell, and colony formation assays. experiments were conducted using Specific Pathogen Free (SPF)-grade male BALB/C nude mice for OS xenograft studies. Based on the gene expression profiles analysis of six osteosarcoma samples and six normal tissue samples, we identified 1,511 upregulated DEGs and 5,678 downregulated DEGs in normal tissue samples. A significant positive correlation between the "yellow-green" module and OS was found through WGCNA analysis. Expression levels of CHMP4C, phosphorylated Glycogen Synthase Kinase 3β (p-GSK3β), and β-catenin were notably higher in U2OS, HOS, and MG63 OS cells than in hFOB1.19 human osteoblasts. Overexpressing CHMP4C in MG63 OS cells upregulated CHMP4C, p-GSK3β, and β-catenin while downregulating GSK3β, leading to increased proliferation and migration of MG63 cells. Conversely, interrupting CHMP4C had the opposite effect. High expression of CHMP4C significantly accelerated the growth of OS in nude mice, resulting in substantial upregulation of CHMP4C, p-GSK3β, and β-catenin expression and suppression of Glycogen Synthase Kinase 3β (GSK3β) expression in OS tissues. CHMP4C may serve as a specific immunomodulatory gene for OS. Its activation of the Wnt/β-catenin signaling pathway, mainly by increasing the phosphorylation echelon of GSK3β, promotes the invasion and spread of OS.
为研究骨肉瘤（OS）特异性标志物减轻侵袭性骨肉瘤骨破坏的免疫机制，该骨肉瘤易于复发和转移。使用加权基因共表达网络分析（WGCNA）、蛋白质-蛋白质相互作用（PPI）分析、最小绝对收缩和选择算子（LASSO）建模以及生存分析对来自基因表达综合数据库（GEO）（GSE126209）的基因表达模式进行分析，以鉴定带电多囊泡体蛋白4C（CHMP4C）。随后，探讨其在调节免疫系统和免疫细胞浸润中的作用。使用逆转录定量聚合酶链反应（RT-qPCR）和免疫荧光染色在骨肉瘤细胞系（MG63、U2OS、HOS）和hFOB1.19细胞中评估OS中CHMP4C的表达和信号分子。研究了CHMP4C上调和干扰对MG63细胞中OS相关信号分子的影响。通过细胞计数试剂盒-8（CCK-8）、Transwell和集落形成试验证实了CHMP4C在MG63骨肉瘤细胞中的功能验证。使用无特定病原体（SPF）级雄性BALB/C裸鼠进行OS异种移植研究。基于对六个骨肉瘤样本和六个正常组织样本的基因表达谱分析，我们在正常组织样本中鉴定出1511个上调的差异表达基因（DEG）和5678个下调的DEG。通过WGCNA分析发现“黄绿”模块与OS之间存在显著正相关。CHMP4C、磷酸化糖原合酶激酶3β（p-GSK3β）和β-连环蛋白的表达水平在U2OS、HOS和MG63骨肉瘤细胞中明显高于hFOB1.19人成骨细胞。在MG63骨肉瘤细胞中过表达CHMP4C可上调CHMP4C、p-GSK3β和β-连环蛋白，同时下调GSK3β，导致MG63细胞增殖和迁移增加。相反，干扰CHMP4C则产生相反的效果。CHMP4C的高表达显著加速了裸鼠中骨肉瘤的生长，导致骨肉瘤组织中CHMP4C、p-GSK3β和β-连环蛋白表达大幅上调，糖原合酶激酶3β（GSK3β）表达受到抑制。CHMP4C可能作为骨肉瘤的一种特异性免疫调节基因。其对Wnt/β-连环蛋白信号通路的激活，主要通过增加GSK3β的磷酸化水平，促进了骨肉瘤的侵袭和扩散。
