Wei Zicheng, Xia Kezhou, Liu Wenda, Huang Xinghan, Wei Zhun, Guo Weichun
Department of Orthopedics, Renmin Hospital of Wuhan University, 238 Jiefang Road, Wuhan, Hubei Province, 430060, China.
Mol Med. 2024 Dec 20;30(1):260. doi: 10.1186/s10020-024-01038-9.
Osteosarcoma, the most prevalent primary bone malignancy in children and adolescents, exhibits high heterogeneity. The CGREF1 gene encodes a novel 301 amino acid classical secreted protein that contains the presumed N-terminal signaling peptide and EF hand motif. However, its role in osteosarcoma remains unclear.
Tumor Immune Estimation Resource (TIMER), The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases were utilized for bioinformatics analysis. Western blot and immunohistochemistry (IHC) techniques were employed to detect the expression of relevant proteins. siRNA, lentivirus, and plasmid technologies were applied to modulate gene expression. The downstream pathway of CGREF1 was identified through RNA sequencing analysis. Cell counting kit-8 (CCK-8) assay, colony formation assay, flow cytometry, wound healing assay, and Transwell assay were conducted for in vitro functional experiments. In vivo experiments involved subcutaneous tumor formation in nude mice.
Our analysis of public databases and clinical samples revealed that CGREF1 is highly expressed in osteosarcoma and is associated with poor prognosis. Knockdown of CGREF1 impeded cell cycle progression and suppressed the proliferation of osteosarcoma cells. Conversely, upregulation of CGREF1 exhibited an opposing pattern. The RNA-seq data from 143B cells was subjected to analysis, revealing that the differentially expressed genes were predominantly enriched in the Wnt signaling pathway. Further experimental results demonstrated that CGREF1 affects activation of the Wnt pathway by regulating GSK3/β-catenin signaling, thereby affecting proliferation ability of osteosarcoma cells. Finally, experiments using subcutaneous transplanted tumor models in nude mice showed that CGREF1 knockdown inhibited tumor growth in vivo by inhibiting the Wnt/β-catenin signaling pathway.
The expression of CGREF1 was significantly upregulated in osteosarcoma and correlated with unfavorable prognosis. CGREF1 exerted a regulatory effect on the proliferation of osteosarcoma cells both in vitro and in vivo through modulation of the wnt/β-catenin signaling pathway. In the future, targeting CGREF1 could potentially offer a novel therapeutic strategy for treating osteosarcoma.
骨肉瘤是儿童和青少年中最常见的原发性骨恶性肿瘤，具有高度异质性。CGREF1基因编码一种新的由301个氨基酸组成的经典分泌蛋白，该蛋白包含推测的N端信号肽和EF手基序。然而，其在骨肉瘤中的作用仍不清楚。
利用肿瘤免疫评估资源（TIMER）、癌症基因组图谱（TCGA）和基因表达综合数据库（GEO）进行生物信息学分析。采用蛋白质免疫印迹法和免疫组织化学（IHC）技术检测相关蛋白的表达。应用小干扰RNA（siRNA）、慢病毒和质粒技术调节基因表达。通过RNA测序分析确定CGREF1的下游通路。进行细胞计数试剂盒-8（CCK-8）检测、集落形成检测、流式细胞术、伤口愈合检测和Transwell检测以进行体外功能实验。体内实验包括在裸鼠中形成皮下肿瘤。
我们对公共数据库和临床样本的分析表明，CGREF1在骨肉瘤中高表达，且与预后不良相关。敲低CGREF1可阻碍细胞周期进程并抑制骨肉瘤细胞的增殖。相反，CGREF1的上调则表现出相反的模式。对143B细胞的RNA测序数据进行分析，发现差异表达基因主要富集于Wnt信号通路。进一步的实验结果表明，CGREF1通过调节糖原合成酶激酶3/β-连环蛋白（GSK3/β-catenin）信号影响Wnt通路的激活，从而影响骨肉瘤细胞的增殖能力。最后，使用裸鼠皮下移植瘤模型的实验表明，敲低CGREF1可通过抑制Wnt/β-连环蛋白信号通路在体内抑制肿瘤生长。
CGREF1在骨肉瘤中的表达显著上调，且与不良预后相关。CGREF1通过调节Wnt/β-连环蛋白信号通路在体外和体内对骨肉瘤细胞的增殖发挥调节作用。未来，靶向CGREF1可能为骨肉瘤的治疗提供一种新的治疗策略。
