MYCN Amplified Neuroblastoma Cells-derived Exosomal Oncogenic MiRNAs Promote Proliferation And Migration Of Non-MYCN Amplified Cells

Objective:Neuroblastoma(NB)is the most common extracranial solid tumor in children.MYCN amplification is found in 20-30% of children with NB,which has been shown to be closely related to the malignant progression of NB and poor prognosis of children.The regulatory role of miRNAs carried by exosomes in the process of disease progression has attracted more and more research attention.Therefore,our study was performed to investigate whether MYCN amplified neuroblastoma cells-derived exosomes containing miRNAs could exert effects on proliferation and migration of non-MYCN amplified cells in neuroblastoma.Methods:Exosomes were isolated from MYCN amplified SK-N-BE(2)cells and characterized via Transmission electron microscopy(TEM),western blot(WB)and nanoparticle tracking analysis(NTA).After isolation,exosomes were co-cultured with non-MYCN amplified SHSY5 Y cells.Mi R-17-5p expression in SK-N-BE(2)cells,SH-SY5 Y cells,and exosomes was quantified by quantitative real-time PCR(RT-q PCR).The expression changes of target genes after over-expression and down-regulation of miR-17-5p expression were detected by RT-q PCR and WB.Luciferase assay was performed to determine whether miR‐17-5p target PTEN‐3′‐UTR.The effects of miR-17-5p on SH-SY5 Y cells viability and migration were evaluated by CCK8,colony formation assay and Transwell.Results:The expression of miR-17-92 cluster in MYCN amplified SK-N-BE(2)cells was significantly higher,while PTEN was significantly lower than that in non-MYCN amplified SH-SY5 Y cells.Transfection of miR-17-5p mimics or inhibitors can significantly increase or down-regulate the expression of miR-17-5p in SH-SY5 Y.Functional experiments show that the overexpression of miR-17-5p can promote the proliferation and migration of SHSY5 Y.In addition,the results of dual luciferase reporter gene detection confirmed that miR-17-5p can specifically bind to and inhibit the expression of PTEN.The results of WB further confirmed that the PTEN protein level of SH-SY5 Y cells in the miR-17-5p mimic group was significantly reduced,and the p-Akt/Akt ratio was significantly increased.TEM results showed that the extracted exosomes had a typical cup-shaped structure and were covered by a phospholipid membrane with a diameter of 50-150 nm.The results of NTA showed that the particle size of exosomes reached a peak at 50-150 nm.In addition,Western-blot results found that the markers of exosomes(the protein TSG101 located inside the exosomes and the four-transmembrane protein molecule CD63)are rich in exosomes.The overexpression of miR-17-5p were observed in SH-SY5 Y co-cultured with SK-NBE(2)-exosomes,and the expression of PTEN was significantly decreased while the pAkt/Akt ratio was significantly increased.Moreover,SH-SY5 Y co-cultured with exosomes with overexpressed miR-17-5p presented with enhanced SH-SY5 Y proliferation and migration ability.Conclusions:Our study demonstrates that miR-17-5p was up-regulated in MYCN amplified SK-NBE(2)cells and tissues.Mi R-17-5p activated the PI3K/Akt signaling pathway by targeting PTEN.Overexpression of miR-17-5p led to the promotion of proliferation and migration in vitro.In addition,SK-N-BE(2)cells-derived exosomes overexpressing miR-17-5p contributed to enhanced proliferation and migration of SH-SY5 Y cells in vitro through inhibition of PTEN.