Department of Neurology, Shanghai Tenth People's Hospital, Tongji University School of Medicine, Shanghai, China.
FEBS J. 2020 Feb;287(3):561-577. doi: 10.1111/febs.15047. Epub 2019 Sep 6.
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.
长链非编码 RNA（lncRNA）作为细胞侵袭、生长、凋亡和分化等多种细胞过程的重要调节因子而出现。lncRNA 可以作为竞争性内源性 RNA（ceRNA）发挥作用，其可以吸收和隔离 microRNA（miRNA）来调节特定的靶标。以前，我们发现包括 FADD、Fas、Casp 和 Bax 在内的几个 miRNA 的靶基因与神经元凋亡有关，并形成一个调控网络。在几个因素中，miRNA-296-5p 的表达与 Caspase 活性和凋亡呈负相关。在这里，我们旨在研究 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）、western blot 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。通过 LncBase 在线工具，我们预测了 lncRNA KCNQ1 反义链/反义转录本 1（KCNQ1OT1）作为 miR-296-5p 的上游调节因子。通过 RNA 免疫沉淀和生物素下拉测定验证了 KCNQ1OT1 和 miR-296-5p 的结合。我们还证明 miR-296-5p 抑制了 NB 细胞在体外和体内的凋亡。在机制上，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，通过生物信息学分析和分子实验，我们表明 KCNQ1OT1 吸收 miR-296-5p，从而损害其对 NB 细胞凋亡的影响。总之，KCNQ1OT1 是一种促进细胞凋亡的有力因素，它通过吸收 miR-296-5p 并上调 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。
