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长链非编码 RNA NORAD 通过靶向 miR-410-3p 调节骨肉瘤的耐药敏感性。

LncRNA NORAD targets miR-410-3p to regulate drug resistance sensitivity of osteosarcoma.

机构信息

Department of Teaching and Research Center, Liaoning University of Traditional Chinese Medicine, Shenyang, 110000, China.

Foot and Ankle Surgery, Central Hospital Affiliated To ShenYang Medical College, Shenyang, 110000, China.

出版信息

Cell Mol Biol (Noisy-le-grand). 2020 Jun 5;66(3):143-148.

Abstract

The current experiment was performed to investigate the effect of LncRNA NORAD on the sensitivity of miR-410-3p to drug resistance of osteosarcoma cells. The cisplatin-resistant cell line HOS/DDP was induced; si-NC, si-NORAD, miR-NC, miR-410-3p, pcDNA-NC, and pcDNA-NORAD were transfected into HOS/DDP cells, respectively; record as a si-NC group, si-NORAD group, miR-NC group, miR-410-3p group, pcDNA-NC group, pcDNA-NORAD group; si-NORAD was co-transfected into HOS/DDP cells with anti-miR-NC, anti-miR-410-3p, recorded as an anti-miR-NC+si-NORAD group and anti-miR-410-3p+si-NORAD group. Real-time quantitative PCR (RT-qPCR) was used to detect LncRNA NORAD, miR-410-3p and multidrug resistance protein 1 (MRP1) mRNA expression levels; Western blot was used to detect cyclin D1 (CyclinD1), MRP1, phosphorylated (p-p65), phosphorylated IкBα (p-IкBα) protein expression; cell counting kit 8 (CCK-8) was used to detect cell viability; dual luciferase report assay to detect targeting relationship between LncRNA NORAD and miR-410-3p. Compared with osteosarcoma cells HOS, the expression levels of LncRNA NORAD, MRP1 mRNA and protein in osteosarcoma resistant cells HOS/DDP were significantly increased, and miR-410-3p expression levels were significantly reduced (P<0.05). Low expression of NORAD or high expression of miR-410-3p, CyclinD1, MRP1 expression levels were significantly reduced, the cell survival rate was significantly reduced, and the half inhibitory concentration of cisplatin was significantly reduced (P<0.05). LncRNA NORAD targets and regulates miR-410-3p, and low expression of miR-410-3p can reverse the effects of low NORAD expression on HOS/DDP cell proliferation and cisplatin resistance. Low expressions of NORAD, p-p65, p-IкBα protein expression levels were significantly reduced; low expression of miR-410-3p could reverse the inhibitory effect of low NORAD expression on p-p65, p-IкBα protein expression. Inhibition of LncRNA NORAD expression can inhibit the proliferation of osteosarcoma HOS/DDP cells through targeted regulation of miR-410-3p, increasing its sensitivity to cisplatin, and it may be related to the NF-κB signaling pathway.

摘要

当前的实验旨在研究长链非编码 RNA(LncRNA)NORAD 对骨肉瘤细胞对 miR-410-3p 耐药性的敏感性的影响。诱导顺铂耐药细胞系 HOS/DDP;转染 si-NC、si-NORAD、miR-NC、miR-410-3p、pcDNA-NC 和 pcDNA-NORAD 分别进入 HOS/DDP 细胞,分别记录为 si-NC 组、si-NORAD 组、miR-NC 组、miR-410-3p 组、pcDNA-NC 组、pcDNA-NORAD 组;将 si-NORAD 共转染入 HOS/DDP 细胞中,同时转染 anti-miR-NC、anti-miR-410-3p,记录为 anti-miR-NC+si-NORAD 组和 anti-miR-410-3p+si-NORAD 组。实时定量聚合酶链反应(RT-qPCR)检测 LncRNA NORAD、miR-410-3p 和多药耐药蛋白 1(MRP1)mRNA 表达水平;Western blot 检测细胞周期蛋白 D1(CyclinD1)、MRP1、磷酸化(p-p65)、磷酸化 IκBα(p-IκBα)蛋白表达水平;细胞计数试剂盒 8(CCK-8)检测细胞活力;双荧光素酶报告实验检测 LncRNA NORAD 与 miR-410-3p 的靶向关系。与骨肉瘤细胞 HOS 相比,骨肉瘤耐药细胞 HOS/DDP 中 LncRNA NORAD、MRP1mRNA 和蛋白的表达水平明显增加,miR-410-3p 的表达水平明显降低(P<0.05)。NORAD 低表达或 miR-410-3p 高表达,CyclinD1、MRP1 表达水平明显降低,细胞存活率明显降低,顺铂的半数抑制浓度明显降低(P<0.05)。LncRNA NORAD 靶向并调节 miR-410-3p,低表达 miR-410-3p 可逆转 NORAD 低表达对 HOS/DDP 细胞增殖和顺铂耐药的影响。NORAD 低表达,p-p65、p-IκBα 蛋白表达水平明显降低;低表达 miR-410-3p 可逆转 NORAD 低表达对 p-p65、p-IκBα 蛋白表达的抑制作用。抑制 LncRNA NORAD 表达可通过靶向调节 miR-410-3p 抑制骨肉瘤 HOS/DDP 细胞的增殖,增加其对顺铂的敏感性,可能与 NF-κB 信号通路有关。

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