Abstract：
Long noncoding RNAs (lncRNAs) have been demonstrated to participate in neuroblastoma cisplatin resistance and tumorigenesis. LncRNA LINC00460 was previously reported to play a critical regulatory role in many cancer development. Nevertheless, its role in modulating neuroblastoma cisplatin resistance has not been explored till now. Cisplatin-resistant neuroblastoma cell lines were established by exposing neuroblastoma cell lines to progressively increasing concentrations of cisplatin for 6 months. LINC00460, microRNA (miR)-149-5p, and delta-like ligand 1 (DLL1) mRNA expression was measured through RT-qPCR. The protein levels of DLL1, epithelial-to-mesenchymal transition (EMT) markers, and the Notch signaling-related molecules were measured via western blotting. The IC50 value for cisplatin, cell growth, metastasis and apoptosis were analyzed in cisplatin-resistant neuroblastoma cells. The binding between LINC00460 (or DLL1) and miR-149-5p was validated through dual-luciferase reporter assay. The murine xenograft model was established to perform in vivo assays. LINC00460 and DLL1 levels were elevated, while miR-149-5p level was reduced in cisplatin-resistant neuroblastoma cells. LINC00460 depletion attenuated IC50 values for cisplatin, weakened cell growth, metastasis, and EMT, and enhanced apoptosis in cisplatin-resistant neuroblastoma cells. Mechanically, LINC00460 sponged miR-338-3p to increase DLL1 level, thereby activating Notch signaling pathway. DLL1 overexpression antagonized LINC00460 silencing-induced suppression on neuroblastoma cell cisplatin resistance and malignant behaviors, while such effects were further reversed by treatment with DAPT, the inhibitor of Notch pathway. Additionally, LINC00460 knockdown further augmented cisplatin-induced impairment on tumor growth in vivo. LINC00460 contributes to neuroblastoma cisplatin resistance and tumorigenesis through miR-149-5p/DLL1/Notch pathway, providing new directions to improve the therapeutic efficacy of chemotherapy drugs applied in patients with neuroblastoma.
摘要：
长非编码RNA（lncRNA）已被证明参与神经母细胞瘤顺铂耐药和肿瘤发生。LncRNALINC00460先前被报道在许多癌症发展中起着关键的调节作用。然而，其在调节神经母细胞瘤顺铂耐药中的作用至今尚未被研究。通过将神经母细胞瘤细胞系暴露于逐渐增加浓度的顺铂6个月来建立顺铂抗性神经母细胞瘤细胞系。LINC00460,microRNA(miR)-149-5p,通过RT-qPCR检测δ样配体1(DLL1)mRNA的表达。DLL1的蛋白质水平，上皮-间质转化（EMT）标志物，和Notch信号相关分子通过蛋白质印迹法测量。顺铂的IC50值，细胞生长,分析顺铂耐药神经母细胞瘤细胞的转移和凋亡。LINC00460(或DLL1)与miR-149-5p之间的结合通过双荧光素酶报告基因测定来验证。建立鼠异种移植模型以进行体内测定。LINC00460和DLL1水平升高，而miR-149-5p水平在顺铂耐药神经母细胞瘤细胞中降低。LINC00460耗竭减弱顺铂的IC50值，细胞生长减弱,转移,和EMT,增强顺铂耐药神经母细胞瘤细胞的凋亡。机械上,LINC00460海绵化miR-338-3p以增加DLL1水平，从而激活Notch信号通路。DLL1过表达拮抗LINC00460沉默抑制神经母细胞瘤细胞顺铂耐药和恶性行为,而DAPT治疗进一步逆转了这种效应，Notch通路的抑制剂。此外,LINC00460敲除进一步增强顺铂诱导的体内肿瘤生长损伤。LINC00460通过miR-149-5p/DLL1/Notch通路促进神经母细胞瘤顺铂耐药和肿瘤发生,为提高化疗药物应用于神经母细胞瘤患者的治疗效果提供了新的方向。
