Circ_0051079沉默通过TRIM66/Wnt/β-连环蛋白途径以miR-625-5p依赖性方式抑制骨肉瘤细胞的恶性表型。

Circ_0051079 silencing inhibits the malignant phenotypes of osteosarcoma cells by the TRIM66/Wnt/β-catenin pathway in a miR-625-5p-dependent manner.

作者信息

Wang Weilin, Wang Jianhua, Li Yingyi, Zhao Yongxu

机构信息

Department 2 of Orthopedics, The Fifth people's Hospital Of Dalian, NO. 890 Huanghe Street, Shahekou District, Dalian, Liaoning, China.

出版信息

J Bone Oncol. 2022 Jun 2;35:100436. doi: 10.1016/j.jbo.2022.100436. eCollection 2022 Aug.

DOI:10.1016/j.jbo.2022.100436

PMID:35733786

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC9207668/

Abstract

BACKGROUND

Circular RNA (circRNA) is a newly-discovered endogenous transcript that has been reported to participate in osteosarcoma (OS) progression. However, the underlying mechanism of circ_0051079 modulating OS development remains unclear.

METHODS

RNA expressions of circ_0051079, miR-625-5p and tripartite motif containing 66 (TRIM66) were detected by quantitative real-time polymerase chain reaction. Protein expression was checked by Western blot analysis. The functional effects of circ_0051079 on OS cell malignancy were investigated by cell counting kit-8, clonogenicity, transwell, tube formation and flow cytometry assays. The interactions among circ_0051079, miR-625-5p and TRIM66 were identified by dual-luciferase reporter and RNA immunoprecipitation assays. Mouse xenograft model assay was performed to elucidate the effects of circ_0051079 knockdown on tumor formation .

RESULTS

Circ_0051079 and TRIM66 expressions were significantly upregulated, but miR-625-5p was downregulated in OS tissues and cells compared with control groups. Circ_0051079 expression was significantly associated with tumor-node-metastasis stage and tumor size of OS patients. Circ_0051079 knockdown inhibited OS cell proliferation, migration and invasion, repressed angiogenesis but induced cell apoptosis, accompanied by the decreases of PCNA and Bcl-2 production and an increase of Bax production. MiR-625-5p, a target miRNA of circ_0051079, participated in regulating circ_0051079-induced effects. Also, TRIM66 was identified as a target mRNA of miR-625-5p, and partially attenuated the inhibitory effects of miR-625-5p in OS cells. Circ_0051079 modulated the Wnt/β-catenin pathway through TRIM66 . Importantly, circ_0051079 silencing reduced TRIM66 expression by interacting with miR-625-5p. Further, circ_0051079 depletion inhibited tumor formation .

CONCLUSION

Circ_0051079 regulated OS development by the miR-625-5p/TRIM66/Wnt/β-catenin pathway, providing a novel therapeutic target for OS.

摘要

背景

环状RNA（circRNA）是一种新发现的内源性转录本，据报道其参与骨肉瘤（OS）的进展。然而，circ_0051079调控OS发展的潜在机制仍不清楚。

方法

采用定量实时聚合酶链反应检测circ_0051079、miR-625-5p和含三联基序蛋白66（TRIM66）的RNA表达。通过蛋白质印迹分析检测蛋白质表达。采用细胞计数试剂盒-8、克隆形成、Transwell、管腔形成和流式细胞术检测circ_0051079对OS细胞恶性程度的功能影响。通过双荧光素酶报告基因和RNA免疫沉淀试验确定circ_0051079、miR-625-5p和TRIM66之间的相互作用。进行小鼠异种移植模型试验以阐明circ_0051079敲低对肿瘤形成的影响。

结果

与对照组相比，circ_0051079和TRIM66在OS组织和细胞中的表达显著上调，但miR-625-5p下调。circ_0051079表达与OS患者的肿瘤-淋巴结-转移分期和肿瘤大小显著相关。circ_0051079敲低抑制OS细胞增殖、迁移和侵袭，抑制血管生成但诱导细胞凋亡，同时PCNA和Bcl-2产生减少，Bax产生增加。miR-625-5p是circ_0051079的靶标miRNA，参与调节circ_0051079诱导的效应。此外，TRIM66被鉴定为miR-625-5p的靶标mRNA，并部分减弱了miR-625-5p对OS细胞的抑制作用。circ_0051079通过TRIM66调节Wnt/β-连环蛋白通路。重要的是，circ_0051079沉默通过与miR-625-5p相互作用降低TRIM66表达。此外，circ_0051079缺失抑制肿瘤形成。