LINC01082的m5C修饰通过调节miR-543-TNRC6A轴抑制骨肉瘤进展。

m5C modification of LINC01082 inhibits osteosarcoma progression by modulating the miR-543-TNRC6A axis.

作者信息

Hu Yawei, Wu Jiawen, Zeng Huaping, Zhou Jianhua, Gong Ming, Guo Zengfeng, Zhang Wang, Zhang Ningfeng, Zhang Hao

机构信息

Department of Spine Surgery, People's Hospital of Longhua, Shenzhen, Guangdong, China.

Department of Ultrasound, The First Affiliated Hospital of Shenzhen University, Shenzhen Second People's Hospital, Shenzhen, Guangdong, China.

出版信息

Transl Oncol. 2025 Aug 15;61:102502. doi: 10.1016/j.tranon.2025.102502.

DOI:10.1016/j.tranon.2025.102502

PMID:40818239

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC12392324/

Abstract

BACKGROUND

Osteosarcoma (OS) is a highly malignant bone tumor primarily affecting children and adolescents, with a significant portion of patients developing metastasis, leading to poor prognosis. Recent studies have identified long noncoding RNAs (lncRNAs) as critical regulators in cancer progression. Among these, LINC01082 has shown tumor-suppressive roles in various cancers, but its function and regulatory mechanisms in OS remain unclear.

METHODS

We investigated the expression patterns and biological functions of LINC01082 in OS tissues and cell lines using RT-qPCR, western blotting, and cell viability assays. The regulatory impact of 5-methylcytosine (m5C) RNA modification on LINC01082 stability was assessed through MeRIP-qPCR, RNA immunoprecipitation (RIP), and CRISPR/dCas13b-NSUN2-mediated m5C targeting. We further explored the interaction between LINC01082, miR-543, and TNRC6A within RNA-induced silencing complexes (RISCs) using luciferase reporter assays, RNA pull-down, and functional assays.

RESULTS

LINC01082 was significantly downregulated in OS tissues and cell lines, with lower expression levels correlating with poorer patient survival. M5C modification, mediated by NSUN2, stabilized LINC01082 through its interaction with the m5C reader protein YBX1. CRISPR/dCas13b-NSUN2-mediated m5C targeting increased LINC01082 expression, resulting in reduced OS cell proliferation and migration, and increased apoptosis. Further, LINC01082 was found to positively regulate TNRC6A expression, with miR-543 modulating this interaction within RISCs. Inhibition of TNRC6A reversed the tumor-suppressive effects of LINC01082 methylation, highlighting the functional significance of the LINC01082-TNRC6A axis in OS.

CONCLUSION

Our study highlights a novel m5C-dependent regulatory mechanism of LINC01082, and demonstrates the potential of CRISPR/dCas13b-NSUN2-mediated m5C editing to functionally modulate this lncRNA and suppress osteosarcoma progression.

摘要

背景

骨肉瘤（OS）是一种主要影响儿童和青少年的高度恶性骨肿瘤，相当一部分患者会发生转移，导致预后不良。最近的研究已确定长链非编码RNA（lncRNA）是癌症进展中的关键调节因子。其中，LINC01082在多种癌症中发挥肿瘤抑制作用，但其在骨肉瘤中的功能和调控机制仍不清楚。

方法

我们使用逆转录定量聚合酶链反应（RT-qPCR）、蛋白质免疫印迹法和细胞活力测定法，研究了LINC01082在骨肉瘤组织和细胞系中的表达模式及生物学功能。通过甲基化RNA免疫沉淀定量PCR（MeRIP-qPCR）、RNA免疫沉淀（RIP）和CRISPR/dCas13b-NSUN2介导的m5C靶向作用，评估了5-甲基胞嘧啶（m5C）RNA修饰对LINC01082稳定性的调控影响。我们还使用荧光素酶报告基因检测、RNA下拉和功能检测，进一步探究了LINC01082、miR-543和TNRC6A在RNA诱导沉默复合体（RISC）中的相互作用。

结果

LINC01082在骨肉瘤组织和细胞系中显著下调，表达水平较低与患者较差的生存率相关。由NSUN2介导的m5C修饰通过与m5C阅读蛋白YBX1相互作用来稳定LINC01082。CRISPR/dCas13b-NSUN2介导的m5C靶向作用增加了LINC01082的表达，导致骨肉瘤细胞增殖和迁移减少，凋亡增加。此外，发现LINC01082正向调节TNRC6A的表达，miR-543在RISC中调节这种相互作用。抑制TNRC6A可逆转LINC01082甲基化的肿瘤抑制作用，突出了LINC01082-TNRC6A轴在骨肉瘤中的功能意义。