Abstract
BackgroundNeuroblastoma (NB) is the most common malignant tumor originating from the extracranial sympathetic nervous system in children. The molecular mechanisms underlying this disease are complex, and not completely understood.MethodsQuantitative real-time PCR (qRT-PCR) was applied to quantify the expression of miR-20a-5p and its target gene ATG7 in clinical NB tissues. The biological function of miR-20a-5p and ATG7 in SH-SY5Y cells was investigated through in vitro studies (Real-Time cell kinetic analyzer, colony formation assay, caspase-Glo 3/7 assay and western blotting). The luciferase reporter assay was conducted to verify the biological relationship between miR-20a-5p and ATG7.ResultsHere we found that miR-20a-5p expression was significantly downregulated whereas its target autophagy-related gene 7 (ATG7) was increased along with clinical staging of NB progression. Correlation analysis showed that miR-20a-5p had a negative correlation trend with ATG7. In SH-SY5Y cells, forced expression of miR-20a-5p suppressed ATG7 expression, autophagy initiation and cellular proliferation while promoted apoptosis, suggesting a potential association between miR-20a-5p and ATG7. Further bioinformatic target prediction combined with protein expression and luciferase reporter assay verified that miR-20a-5p inhibited ATG7 by directly binding to its 3′-UTR, confirming the involvement of miR-20a-5p in the regulation of ATG7 in NB.ConclusionsThese results clarified that miR-20a-5p inhibited cell proliferation and promoted apoptosis through negative regulation of ATG7 and thus autophagy suppression in SH-SY5Y cells. Therefore, defining the context-specific roles of autophagy in NB and regulatory mechanisms involved will be critical for developing autophagy-targeted therapeutics against NB. Both miR-20a-5p and ATG7 would be potential therapeutic targets for future NB treatment.
Highlights
Neuroblastoma (NB) is the most common malignant tumor originating from the extracranial sympathetic nervous system in children
Both miR‐20a‐5p and autophagyrelated gene 7 (ATG7) was dysregulated in NB samples and correlated with tumor stage To investigate the role of miR-20a-5p and ATG7 in NB progression, quantified expression was detected in 35 NB specimens by RT-PCR
Since ATG7 is crucial in autophagy activation and autophagosome formation [30], these results suggest that miR-20a-5p may inhibit cell proliferation through autophagy via negative regulation of ATG7 in SH-SY5Y cells
Summary
Neuroblastoma (NB) is the most common malignant tumor originating from the extracranial sympathetic nervous system in children. The molecular mechanisms underlying this disease are complex, and not completely understood. Neuroblastoma (NB) is the most common extracranial solid tumour occurring in children [1], which accounts for more than 15% of all pediatric oncology deaths. Half of neuroblastoma cases are classified as high-risk for disease relapse, with long-term survival less than 40% [2]. The unclear pathogenesis of neuroblastoma impedes the development of therapeutic drugs discovery and effective tumor therapy. Only Unituxin (dinutuximab) was approved by the US Food and Drug Administration as a novel targeted drug in 2015, which can prolong the survival of patients with high-risk NB [6]. Clear clarification of mechanisms underlying NB progression is urgently needed
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