文章摘要

LINC00461靶向miR-519e-5p调控骨肉瘤细胞增殖、迁移和侵袭的分子机制

作者: 1黄桂林, 2华莎, 1周海振, 1王志酬, 1刘鹤鸣, 1杨团民
1 西安交通大学附属红会医院骨病肿瘤科，西安 710054
2 西安市第五医院风湿免疫科，西安 710082
通讯: 黄桂林 Email: guajih@163.com
DOI: 10.3978/j.issn.2095-6959.2022.11.002
基金: 陕西省重点研发计划项目(2021SF-125)。

摘要

目的：探讨长链非编码RNA(long non-coding RNA，lncRNA)LINC00461对骨肉瘤细胞增殖、迁移和侵袭的影响及分子机制。方法：选取40例骨肉瘤患者的瘤组织及瘤旁组织，培养人成骨细胞hFOB 1.19，骨肉瘤细胞系Saos2、U2OS和HOS，用real-time RT-PCR检测LINC00461和微RNA-519e-5p(microRNA-519e-5p，miR-519e-5p)表达水平。将Saos2细胞分为NC组、si-LINC00461组、si-NC组、miR-519e-5p组(miR-519e-5p类似物)、miR-NC组、si-LINC00461+anti-miR-519e-5p组、si-LINC00461+anti-miR-NC组。蛋白质印迹法检测蛋白质表达；CCK-8检测细胞活性；克隆形成实验检测细胞克隆形成数；Transwell检测细胞迁移和侵袭数；双荧光素酶报告实验和RNA pull-down实验检测LINC00461和miR-519e-5p的靶向关系。结果：与瘤旁组织比较，骨肉瘤组织中LINC00461高表达，miR-519e-5p低表达(P<0.05)。与hFOB 1.19细胞比较，骨肉瘤细胞系Saos2、U2OS和HOS中LINC00461表达水平升高，miR-519e-5p表达水平降低(P<0.05)。低表达LINC00461或过表达miR-519e-5p，Ki-67、基质金属蛋白酶(matrix metalloproteinase，MMP)2、MMP9蛋白表达水平，Saos2细胞活性，细胞克隆形成数及迁移侵袭数降低(P<0.05)。LINC00461靶向负调控miR-519e-5p的表达。低表达miR-519e-5p可以逆转LINC00461低表达对Saos2细胞增殖、迁移和侵袭的影响。结论：低表达LINC00461通过靶向上调miR-519e-5p抑制骨肉瘤细胞的增殖、迁移和侵袭。
关键词： lncRNA LINC00461；miR-519e-5p；骨肉瘤；增殖；迁移；侵袭

Molecular mechanism of LINC00461 targeting miR-519e-5p to regulate proliferation, migration, and invasion of osteosarcoma cells

Authors: 1HUANG Guilin, 2HUA Sha, 1ZHOU Haizhen, 1WANG Zhichou, 1LIU Heming, 1YANG Tuanmin
1 Department of Orthopedics and Oncology, Honghui Hospital, Xi’an Jiaotong University, Xi’an 710054, China
2 Department of Rheumatology and Immunology, Xi’an Fifth Hospital, Xi’an 710082, China

CorrespondingAuthor: HUANG Guilin Email: guajih@163.com

DOI: 10.3978/j.issn.2095-6959.2022.11.002

Foundation: This work was supported by the Shaanxi Province Key Research and Development Program, China (2021SF-125).

Abstract

Objective: To investigate the effect and molecular mechanism of long non-coding RNA (lncRNA) LINC00461 on the proliferation, migration, and invasion of osteosarcoma cells. Methods: The tumor tissues and adjacent tissues of 40 patients with osteosarcoma were selected, human osteoblast cell hFOB 1.19, osteosarcoma cell lines Saos2, U2OS, and HOS were cultured, and real-time RT-PCR was used to detect the expression levels of LINC00461 and microRNA-519e-5p (miR-519e-5p). The Saos2 cells were further divided into a NC group, a si-LINC00461 group, a si-NC group, a miR-519e-5p group (miR-519e-5p analog), a miR-NC group, a si-LINC00461 + anti-miR-519e-5p group, and a si-LINC00461 + anti-miR-NC group. Western blotting was used to detect protein expression; cell viability was detected by CCK-8; clone formation assay was performed to detect cell clone formation number; Transwell was utilized to detect cell migration and invasion number; the targeting relationship between LINC00461 and miR-519e-5p was detected by dual-luciferase reporter assay and RNA pull-down assay. Results: Compared with adjacent tissues, LINC00461 was highly expressed in osteosarcoma tissues, and miR-519e-5p was lowly expressed (P<0.05). Compared with hFOB 1.19 cell, the expression level of LINC00461 in osteosarcoma cell lines Saos2, U2OS, and HOS was increased, and the expression level of miR-519e-5p was decreased (P<0.05). After the underexpression of LINC00461 or overexpression of miR-519e-5p, the protein expression levels of Ki-67, matrix metalloproteinase (MMP)2 and MMP9, Saos2 cell activity, and the number of cell clone formation, migration, and invasion of Saos2 cell were decreased (P<0.05). LINC00461 targeted and negatively regulated the expression of miR-519e-5p. The underexpression of miR-519e-5p reversed the effect of underexpression of LINC00461 on the proliferation, migration, and invasion of Saos2 cells. Conclusion: The low expression of LINC00461 inhibits the proliferation, migration, and invasion of osteosarcoma cells by targeting up-regulation of miR-519e-5p.

Keywords： lncRNA LINC00461; miR-519e-5p; osteosarcoma; proliferation; migration; invasion

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