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m6A-dependent upregulation of TRAF6 by METTL3 is associated with metastatic osteosarcoma
Journal of Bone Oncology ( IF 3.4 ) Pub Date : 2022-01-19 , DOI: 10.1016/j.jbo.2022.100411
Jing Wang 1 , Wentao Wang 2 , Xing Huang 1 , Jiashi Cao 1 , Shuming Hou 1 , Xiangzhi Ni 1 , Cheng Peng 1 , Tielong Liu 1
Affiliation  

Objectives

RNA N6-methyladenosine (m6A) is associated with tumorigenesis. The importance of methyltransferase-like 3 (METTL3) has been reported in cancer progression and metastasis. However, its role and molecular mechanism in osteosarcoma (OS), the most common primary bone tumor, is poorly studied. In this study, we aimed to investigate the functional role and underlying mechanism of METTL3 in the metastasis of OS.

Methods

The expression differences of METTL3 between metastatic and non-metastatic OS tissues and patients with different Enneking stages were detected using RT-qPCR. METTL3 was artificially downregulated in the cells, followed by wound healing assay, Matrigel assay, immunofluorescence, in vivo tumorigenic assay, HE staining, and western blot. Transcriptome sequencing and m6A-seq was conducted to identify the downstream genes of METTL3, and RIP and dual-luciferase assays were performed for validation. The expression of TRAF6 in OS tissues was detected using RT-qPCR. Finally, the rescue experiments were conducted.

Results

METTL3 was overexpressed in metastatic OS tissues, and downregulation of METTL3 decreased cell migration, invasion, epithelial-mesenchymal transition, and tumorigenic and metastatic activities. The m6A site was highly enriched in cells poorly expressing METTL3, and the m6A peak was mainly enriched in the exon region. METTL3 was positively correlated with TRAF6 in metastatic OS, and depletion of METTL3 resulted in the loss of TRAF6 expression in OS cells. Upregulation of TRAF6 contributed to metastases in vitro and in vivo.

Conclusion

METTL3 is highly expressed in OS and enhances TRAF6 expression through m6A modification, thereby promoting the metastases of OS cells.



中文翻译:

METTL3对TRAF6的m6A依赖性上调与转移性骨肉瘤有关

目标

RNA N6-甲基腺苷 (m6A) 与肿瘤发生有关。据报道,甲基转移酶样 3 (METTL3) 在癌症进展和转移中的重要性。然而,其在最常见的原发性骨肿瘤骨肉瘤(OS)中的作用和分子机制研究很少。在本研究中,我们旨在研究 METTL3 在 OS 转移中的功能作用和潜在机制。

方法

使用 RT-qPCR 检测转移性和非转移性 OS 组织以及不同 Enneking 阶段患者之间 METTL3 的表达差异。METTL3 在细胞中被人为下调,然后是伤口愈合试验、基质胶试验、免疫荧光、体内致瘤试验、HE 染色和蛋白质印迹。进行转录组测序和 m6A-seq 以鉴定 METTL3 的下游基因,并进行 RIP 和双荧光素酶测定以进行验证。使用 RT-qPCR 检测 OS 组织中 TRAF6 的表达。最后进行了救援实验。

结果

METTL3 在转移性 OS 组织中过表达,METTL3 的下调降低了细胞迁移、侵袭、上皮-间质转化以及致瘤和转移活性。m6A位点在METTL3表达不佳的细胞中高度富集,m6A峰主要富集在外显子区域。METTL3 与转移性 OS 中的 TRAF6 呈正相关,METTL3 的消耗导致 OS 细胞中 TRAF6 表达的丧失。TRAF6 的上调导致体外体内转移。

结论

METTL3在OS中高表达,通过m6A修饰增强TRAF6表达,从而促进OS细胞的转移。

更新日期:2022-01-26
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