LncRNA OIP5-AS1通过调节miR-186-5p/KIF14信号轴来促进神经母细胞瘤的增殖  

Effect of LncRNA OIP5-AS1 on Proliferation of Neuroblastoma by Regulating the miR-186-5p/KIF14 Signaling Axis

作　　者：安艳晓 焦晗亮 祁艳卫[1] 仲智勇 AN Yanxiao;JIAO Hanliang;QI Yanwei(Hebei Children's Hospital,Hebei Shijiazhuang 050000,China)

机构地区：[1]河北省儿童医院普外二科,河北石家庄050000

出　　处：《河北医学》2024年第8期1290-1296,共7页Hebei Medicine

基　　金：河北卫生健康委员会科研计划项目,(编号:20240636)。

摘　　要：目的:神经母细胞瘤(NB)是儿童最常见的实体瘤。本研究旨在确定长链非编码RNA(LncRNA)Opa相互作用蛋白5反义RNA 1(OIP5-AS1)在NB中的作用及其可能的分子机制。方法:体外培养神经母细胞瘤细胞系SK-N-SH和人神经母细胞BE2C。通过定量实时聚合酶链式反应测定OIP5-AS1、miR-186-5p和驱动蛋白分子14(KIF14)的表达。使用CCK-8测定法、平板克隆形成法和EdU掺入法评估细胞增殖。蛋白质印迹法检测KIF14蛋白水平。通过在线数据库Starbase3.0预测靶基因,并通过双荧光素酶报告基因测定进行验证。结果:OIP5-AS1在SK-N-SH细胞中的表达水平较BE2C细胞显著上调(1.88±0.14 vs 1.00±0.07),P<0.05。shOIP5-AS1组下调OIP5-AS1表达后,SK-N-SH细胞活力、克隆形成能力(111.33±15.57个vs 154.67±20.74个vs 149.33±17.01个,P<0.05)和DNA合成能(EdU阳性细胞比例:35.09±8.02%vs 67.87±7.21%vs 63.33±7.17%,P<0.05)弱于NC组和shCtrl组。经生物信息学预测和双荧光素酶报告基因验证,OIP5-AS1与miR-186-5p以及miR-186-5p与KIF143'-UTR具有靶向调控作用。与NC组和shCtrl组相比,shOIP5-AS1组KIF14 mRNA相对表达量(0.41±0.09,P<0.05)和KIF14蛋白表达(0.20±0.02,P<0.05)均下调。相反,shOIP5-AS1+miR-186-5p inhibitor组KIF14 mRNA相对表达量和KIF14蛋白表达量分别为0.83±0.12和0.62±0.04,较shOIP5-AS1+miR-NC组升高。RNA结合蛋白免疫共沉淀结果也显示,与对照(IgG)相比,Ago2抗体可以富集OIP5-AS1和miR-186-5p。与shOIP5-AS1+miR-NC组相比,shOIP5-AS1+miR-186-5p inhibitor组细胞活力和克隆形成能力(180.0±11.36 vs 136.0±14.53)显著增加(P<0.05)。在挽救性实验中,与shOIP5-AS1+miR-186-5p inhibitor+siNC组相比,shOIP5-AS1+miR-186-5p inhibitor+siKIF14组细胞活力和克隆形成能力(139.33±8.96 vs 183.03±18.0,P<0.05)明显降低(P<0.05)。结论:SK-N-SH细胞中OIP5-AS1表达水平显著上调,并且作为ceRNA竞争性地抑制miR186-5p,上调KIF14表达,从而促进NB进展。Objective:To determine the role of long non-coding RNA(LncRNA)Opa-interacting protein 5 antisense RNA 1(OIP5-AS1)in NB and its potential molecular mechanisms.Methods:Neuroblastoma cell lines SK-N-SH and human neuroblastoma BE2C were cultured in vitro.The expression of OIP5-AS1,miR-186-5p,and kinesin family member 14(KIF14)was measured by quantitative real-time PCR.Cell proliferation was assessed using the CCK-8 assay,colony formation assay,and EdU incorporation assay.KIF14 protein levels were detected by Western blot.Target genes were predicted using the online database Starbase3.0 and verified by dual-luciferase reporter assay.Results:The expression level of OIP5-AS1 in SK-N-SH cells was significantly higher than that in BE2C cells(1.88±0.14 vs 1.00±0.07,P<0.05).After downregulating OIP5-AS1 expression in the shOIP5-AS1 group,SK-N-SH cell viability,colony formation ability(111.33±15.57 vs 154.67±20.74 vs 149.33±17.01,P<0.05),and DNA synthesis ability(EdU positive cell ratio:35.09±8.02%vs 67.87±7.21%vs 63.33±7.17%,P<0.05)were weaker than those in the NC group and shCtrl group.Bioinformatics prediction and dual-luciferase reporter assay confirmed the targeting regulation between OIP5-AS1 and miR-186-5p as well as miR-186-5p and KIF143'-UTR.Compared with the NC group and shCtrl group,the shOIP5-AS1 group showed downregulated KIF14 mRNA relative expression(0.41±0.09,P<0.05)and KIF14 protein expression(0.20±0.02,P<0.05).Conversely,the shOIP5-AS1+miR-186-5p inhibitor group exhibited increased KIF14 mRNA relative expression and KIF14 protein expression(0.83±0.12 and 0.62±0.04,respectively)compared to the shOIP5-AS1+miRNC group.RNA-binding protein immunoprecipitation results also showed that,compared with the control(IgG),the Ago2 antibody could enrich OIP5-AS1 and miR-186-5p.Compared with the shOIP5-AS1+miR-NC group,the shOIP5-AS1+miR-186-5p inhibitor group had significantly increased cell viability and colo-ny formation ability(180.0±11.36 vs 136.0±14.53,P<0.05).In rescue experiments,compared with the sh

关 键 词：神经母细胞瘤 长链非编码RNA Opa相互作用蛋白5反义RNA 1 驱动蛋白分子14 

分 类 号：R739.4[医药卫生—肿瘤]