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Circ_0081001下调miR-494-3p以增强BACH1表达并促进骨肉瘤进展。

Circ_0081001 down-regulates miR-494-3p to enhance BACH1 expression and promotes osteosarcoma progression.

作者信息

Liu Shizhang, Duan Keke, Zhang Xiaoxia, Cao Xiane, Wang Xixia, Meng Fanbin, Liu Huitong, Xu Bingqiang, Wang Xi

机构信息

Department of Orthopedics, Shaanxi Provincial People's Hospital, Xian 710068, Shanxi, China.

Department of Orthopedics, Linyi People's Hospital, Linyi 276100, Shandong, China.

出版信息

Aging (Albany NY). 2021 Jun 30;13(13):17274-17284. doi: 10.18632/aging.203207.

Abstract

The study was aimed at deciphering the function and mechanism of circ_0081001 in osteosarcoma (OS). In this study, quantitative real-time polymerase chain reaction (qRT-PCR) was utilized for quantifying circ_0081001, miR-494-3p, and BTB domain and CNC homolog 1 (BACH1) mRNA expressions in OS tissues and cells. Cell counting kit-8 (CCK-8) assay, together with 5-Ethynyl-2'-deoxyuridine (EdU) assay, was performed for evaluating cell proliferation; the alterations in apoptosis were analyzed utilizing flow cytometry; Transwell assay was conducted for examining cell migration and invasion; moreover, Western blot was utilized for the quantification of BACH1 protein expression; bioinformatics, dual-luciferase reporter gene, and RNA-binding protein immunoprecipitation assays were executed for validating the binding relationships between circ_0081001 and miR-494-3p, and between miR-494-3p and BACH1. As shown, circ_0081001, whose expression was elevated in OS tissues, had a negative association with miR-494-3p expression and a positive correlation with BACH1 expression. After circ_0081001 was overexpressed, the proliferation, migration, and invasion of OS cells were boosted but the apoptosis was reduced, whereas miR-494-3p exhibited opposite effects. The binding sites between circ_0081001 and miR-494-3p, and between miR-494-3p and the 3'UTR of BACH1 were experimentally verified. In conclusion, circ_0081001/miR-494-3p/BACH1 axis promoted the malignant biological behaviors of OS cells.

摘要

该研究旨在阐明环状RNA_0081001(circ_0081001)在骨肉瘤(OS)中的功能及机制。本研究采用定量实时聚合酶链反应(qRT-PCR)检测circ_0081001、微小RNA-494-3p(miR-494-3p)及BTB结构域和CNC同源蛋白1(BACH1)mRNA在OS组织和细胞中的表达。采用细胞计数试剂盒-8(CCK-8)法和5-乙炔基-2'-脱氧尿苷(EdU)法评估细胞增殖;运用流式细胞术分析细胞凋亡的变化;采用Transwell法检测细胞迁移和侵袭能力;此外,利用蛋白质免疫印迹法检测BACH1蛋白表达水平;通过生物信息学、双荧光素酶报告基因及RNA结合蛋白免疫沉淀实验验证circ_0081001与miR-494-3p以及miR-494-3p与BACH1之间的结合关系。结果显示,circ_0081001在OS组织中表达上调,其与miR-494-3p表达呈负相关,与BACH1表达呈正相关。过表达circ_0081001后,OS细胞的增殖、迁移和侵袭能力增强,但凋亡减少,而miR-494-3p则表现出相反的作用。实验验证了circ_0081001与miR-494-3p以及miR-494-3p与BACH1的3'非翻译区(3'UTR)之间的结合位点。综上所述,circ_0081001/miR-494-3p/BACH1轴促进了OS细胞的恶性生物学行为。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/84e0/8312427/72d0a36ae9d1/aging-13-203207-g001.jpg

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