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高通量测序分析骨肉瘤患者血清外泌体中的 microRNA 谱。

MicroRNA profiling of serum exosomes in patients with osteosarcoma by high-throughput sequencing.

机构信息

Department of Orthopedics, Ningbo City Sixth Hospital, Ningbo, China.

Department of Hematology, The Third Affiliated Hospital of Soochow University, Changzhou, China.

出版信息

J Investig Med. 2020 Apr;68(4):893-901. doi: 10.1136/jim-2019-001196. Epub 2020 Feb 13.

Abstract

The microRNA expression profile of plasma exosomes in osteosarcoma needs to be further explored. The present study intends to investigate the practicality of plasma exosomal miRNAs as novel biomarkers of osteosarcoma. In the study, exosome-like vesicles were purified from the plasma of patients with osteosarcoma and healthy control. Differential centrifugation was used. The purified vesicles which ranged from 50 to 100 nm in size were identified as exosomes by transmission electron microscopy and western blot. Validating assays in vitro and in vivo were performed via CCK8, reverse transcription-quantitative PCR, flow cytometry, transwell and wound healing assays and xenograft model. High-throughput sequencing identified that 57 miRNAs, 20 of which were upregulated and 37 downregulated, were differentially expressed in patients with osteosarcoma and healthy control (p<0.01; fold change ≥3). In comparison to the controls, the expression levels of miR-92a-3p, miR-130a-3p, miR-195-3 p, miR-335-5 p, let-7i-3p were upregulated in the exosomes from patients with osteosarcoma with statistical significance. Studies in vitro and in vivo have proved that osteosarcoma-secreted exosomes from miR-195-3 p upregulated 143B osteosarcoma cells promote cell proliferation and invasion. Overall, the present study identified exosomal miRNAs with dysregulated expression in patients with osteosarcoma, and they may have potential as targets for the treatment of patients with osteosarcoma.

摘要

血浆外泌体中的微小 RNA 表达谱在骨肉瘤中有待进一步探索。本研究旨在探讨血浆外泌体 miRNA 作为骨肉瘤新型生物标志物的实用性。在研究中,从骨肉瘤患者和健康对照者的血浆中纯化出类外泌体小泡。采用差速离心法。通过透射电子显微镜和 Western blot 鉴定,大小在 50 至 100nm 之间的纯化小泡为外泌体。通过 CCK8、逆转录定量 PCR、流式细胞术、Transwell 和划痕愈合试验及异种移植模型进行体外和体内验证试验。高通量测序鉴定出 57 个 miRNA,其中 20 个上调,37 个下调,在骨肉瘤患者和健康对照者中表达差异显著(p<0.01;倍数变化≥3)。与对照组相比,骨肉瘤患者的外泌体中 miR-92a-3p、miR-130a-3p、miR-195-3p、miR-335-5p、let-7i-3p 的表达水平显著上调。体外和体内研究均证实,骨肉瘤分泌的 miR-195-3p 上调了 143B 骨肉瘤细胞的增殖和侵袭能力。总的来说,本研究鉴定出骨肉瘤患者中存在表达失调的外泌体 miRNA,它们可能成为治疗骨肉瘤患者的潜在靶点。

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