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KIF14 3'-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。
Abstract：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 KIF14 3'-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+miR-NC 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 colony formation ability (180.0±11.36 vs 136.0±14.53, P<0.05). In rescue experiments, compared with the shOIP5-AS1+miR-186-5p inhibitor+siNC group, the shOIP5-AS1+miR-186-5p inhibitor+siKIF14 group showed significantly decreased cell viability and colony formation ability (139.33±8.96 vs183.03±18.0, P<0.05).Conclusion: The expression level of OIP5-AS1 is significantly upregulated in SK-N-SH cells and acts as a competing endogenous RNA (ceRNA) to competitively inhibit miR-186-5p, thereby upregulating KIF14 expression and promoting NB progression.
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2024, Vol. 30 
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