miR-34a inhibits progression of neuroblastoma by targeting autophagy-related gene 5
miR-34a inhibits progression of neuroblastoma by targeting autophagy-related gene 5
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DOI：
10.1016/j.ejphar.2019.01.071
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发表时间：
2019-05-05
影响因子：
5
通讯作者：
Zhang, Qian
中科院分区：
文献类型：
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作者：
Cheng, Xinru;Xu, Qianya;Zhang, Qian
神经母细胞瘤是一种常见的儿童恶性肿瘤，儿童期死亡率高。尽管已经获得了许多关注，但仍需要新的NB诊断和预后生物标志物。microRNAs （miRNAs）在NB的进展中发挥重要作用，miR-34a是NB的肿瘤抑制因子。然而，miR-34a调控NB细胞增殖、迁移、侵袭和自噬的机制尚不清楚。本研究采用MTT法和集落法研究细胞增殖。用caspase 3活性测定法检测细胞凋亡。跨井分析检测细胞迁移和侵袭。采用GFP-LC3斑点荧光法和western blots （WB）检测细胞自噬情况。采用实时荧光定量PCR （qRT-PCR）检测miR-34a的表达。采用qRT-PCR和WB检测miR-34a对自噬相关基因5 （autophagy-related gene 5, ATG5）的调控作用。通过荧光素酶活性和RNA免疫沉淀（RIP）检测miR-34a与ATG5的相互作用。结果显示，miR-34a在低存活率NB组织和细胞中表达受到抑制。miR-34a的添加抑制了NB细胞的增殖、迁移、侵袭和自噬，但促进了NB细胞的凋亡，而miR-34a缺乏在NB细胞的进展中起相反的作用。有趣的是，ATG5被miR-34a直接靶向。此外，ATG5修复可减弱mir -34a介导的增殖、凋亡、迁移、侵袭和自噬的抑制作用。这些结果表明miR-34a通过靶向ATG5抑制NB细胞的增殖、凋亡、迁移、侵袭和自噬，为NB治疗提供了新的治疗途径。
Neuroblastoma (NB) is a common pediatric malignancy with high mortality in childhood. Although many attentions have been gained, novel biomarkers for NB diagnosis and prognosis are still needed. microRNAs (miRNAs) played important roles in NB progression and miR-34a is a tumor suppressor in NB. However, the mechanism that underlies miR-34a regulating proliferation, migration, invasion and autophagy in NB remains poorly understood. In this study, cell proliferation was investigated by MTT and colony assay. Cell apoptosis was measured by caspase 3 activity assay. Cell migration and invasion were detected by trans-well analysis. Autophagy was measured via GFP-LC3 puncta fluorescence assay and western blots (WB). The expression of miR-34a was examined by quantitative real-time PCR (qRT-PCR). The regulatory effect of miR-34a on autophagy-related gene 5 (ATG5) was detected by qRT-PCR and WB. The interaction between miR-34a and ATG5 was probed by luciferase activity and RNA immunoprecipitation (RIP) assay. Results showed that miR-34a expression was inhibited in NB tissues and cells with low survival rate. Addition of miR-34a suppressed cell proliferation, migration, invasion and autophagy but promoted apoptosis in NB cells, whereas miR-34a deficiency played opposite roles in NB progression. Intriguingly, ATG5 was directly targeted by miR-34a. Moreover, ATG5 restoration attenuated miR-34a-mediated inhibitory effect on proliferation, apoptosis, migration, invasion and autophagy. These results indicated miR-34a suppressed proliferation, apoptosis, migration, invasion and autophagy in NB cells by targeting ATG5, providing a novel therapeutic avenue for NB treatment.