Long noncoding RNA KCNQ1OT1 promotes apoptosis in neuroblastoma cells by regulating miR-296-5p/Bax axis
Long noncoding RNA KCNQ1OT1 promotes apoptosis in neuroblastoma cells by regulating miR-296-5p/Bax axis
复制标题
长非编码RNA KCNQ1OT1通过调节miR-296-5p/Bax轴促进神经母细胞瘤细胞凋亡
DOI：
10.1111/febs.15047
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发表时间：
2019-09-06
期刊：
影响因子：
5.4
通讯作者：
Liu, Xue-Yuan
中科院分区：
文献类型：
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作者：
Li, Meng-Meng;Liu, Xiao-Hui;Liu, Xue-Yuan
长非编码 RNA (lncRNA) 正在成为细胞侵袭、生长、凋亡和分化等多种细胞过程的重要调节因子。 LncRNA 可以作为竞争性内源 RNA (ceRNA) 发挥作用，吸收和隔离 microRNA (miRNA) 以调节特定靶点。此前，我们发现FADD、Fas、Casp和Bax等多个miRNA的靶基因与神经细胞凋亡相关，并形成调控网络。在多个因素中，发现 microRNA-296-5p 表达与 caspase 活性和细胞凋亡呈负相关。在这里，我们的目的是研究 miR-296-5p 在神经母细胞瘤 (NB) 细胞中的作用。通过进行定量实时 PCR (qRT-PCR)、蛋白质印迹和流式细胞术分析，我们分析了转染 miR-296-5p 模拟物或抑制剂的 NB 细胞中凋亡标志物的表达。通路特异性 PCR 阵列使我们能够鉴定 miR-296-5p 的靶基因。使用 LncBase 在线工具，我们预测 lncRNA KCNQ1 相反链/反义转录本 1 (KCNQ1OT1) 作为 miR-296-5p 的上游调节因子。 KCNQ1OT1 和 miR-296-5p 的结合通过 RNA 免疫沉淀和生物素 Pull-down 测定进行验证。我们还证明 miR-296-5p 在体外和体内抑制 NB 细胞的凋亡。从机制上讲，miR-296-5p直接结合Bax mRNA的3'UTR，从而在mRNA和蛋白质水平上抑制Bax。此外，通过生物信息分析和分子实验，我们发现KCNQ1OT1吸收miR-296-5p并削弱其对NB细胞凋亡的影响。总之，KCNQ1OT1 是一种有效的细胞凋亡促进因子，通过海绵 miR-296-5p 和上调 Bax 发挥作用。我们的研究结果确定了 NB 细胞中细胞命运的调节轴。
Long noncoding RNAs (lncRNAs) are emerging as important regulators of multiple cellular processes such as cell invasion, growth, apoptosis and differentiation. LncRNAs can function as competing endogenous RNAs (ceRNAs) which sponge and sequester microRNA (miRNA) to regulate specific targets. Previously, we found that the target genes of several miRNAs, including FADD, Fas, Casp and Bax, are related to neuronal apoptosis and form a regulatory network. Among several factors, microRNA-296-5p expression was found to be negatively correlated with caspase activity and apoptosis. Here, we aimed to investigate the role of miR-296-5p in neuroblastoma (NB) cells. By performing quantitative real-time PCR (qRT-PCR), western blot and flow cytometry assays we analysed the expression of apoptotic markers in NB cells transfected with miR-296-5p mimics or inhibitor. Pathway-specific PCR array allowed us to identify the target genes of miR-296-5p. Using LncBase online tool, we predicted lncRNA KCNQ1 opposite strand/antisense transcript 1 (KCNQ1OT1) as an upstream regulator of miR-296-5p. The binding of KCNQ1OT1 and miR-296-5p was validated via RNA immunoprecipitation and Biotin pull-down assays. We also demonstrate that miR-296-5p suppresses apoptosis of NB cells in vitro and in vivo. Mechanistically, miR-296-5p directly bound the 3 ' UTR of Bax mRNA, thus repressing Bax at the mRNA and protein level. Moreover, through bioinformatic analysis and molecular experiments, we showed that KCNQ1OT1 sponged miR-296-5p and impaired its effect on NB cell apoptosis. In summary, KCNQ1OT1 is a potent promoting factor of cell apoptosis, which acts by sponging miR-296-5p and upregulating Bax. Our findings identify a regulatory axis of cell fate in NB cells.