| Neuroblastoma(NB)is the most common extracranial solid malignant tumor in children under 5 years old.Its characteristics includes complex biological behavior,great individual differences,diversity of diagnostic assessment systems.The evaluation systems and targeted treatment models for NB will open up a new chapter,with the progression of molecular biology research in NB diagnosis and treatment,as well as the development of multidisciplinary integrated treatment.In recent years,long non-coding RNA(lnc RNA)has gradually become one of the most popular molecules in biomedical researches.lnc RNA had been proved to be a key factor in the regulation of gene expression such as transcription,epigenetics,and its dysfunction is closely related to many human diseases.Large number of studies have confirmed that lnc RNA dysregulation plays an irreplaceable role in the occurrence and evolution of cancer.Further study of the lnc RNAs can provide functional annotation for tumor-related transcripts,so that these molecules can provide ideal targets for tumor diagnosis and treatment.In this study,we performed high throughput total transcriptome sequencing on clinical specimens of ganglioneuroma,ganglioneuroblastoma and high risk neuroblastoma respectively.The results showed that lnc RNA ADAMTS9-AS2 was highly expressed in ganglioneuroma and ganglioneuroblastoma compared with NB.In SK-N-AS and SK-N-SH cells,the results showed that overexpression of ADAMTS9-AS2 could significantly inhibit the proliferation and metastasis of NB cells,while down-regulated expression of ADAMTS9-AS2 had the opposite effect.In SK-N-SH and SH-SY5 Y cells treated with retinoic acid,further differentiation and axon were observed in the cytoplasm obviously.The expression of ADAMTS9-AS2 in both groups of NB cells was examined by RT-q PCR and the expression increased significantly with further differentiation.In summary,ADAMTS9-AS2 expression correlated with the proliferation,differentiation and metastasis ability of NB cells.In order to better understand the molecular mechanisms of ADAMTS9-AS2 in NB,we investigated the downstream factors,pathways and related proteins regulated by ADAMTS9-AS2 through various molecular biology experimental techniques.Firstly,the main signaling pathways affected were found to be the cell adhesion pathway,cell morphology changes,ERK pathway,cell migration and chemokine regulatory pathways by RNA-seq in the control and ADAMTS9-AS2 overexpression groups.Subsequently,we applied RT-q PCR and western blot assays to confirm that overexpression of ADAMTS9-AS2 significantly inhibited the activation of MEK and ERK and suppressed the expression of MMP-9.While down-regulated expression of ADAMTS9-AS2 promoted the phosphorylation of MEK and ERK,as well as the expression of MMP-9.Furthermore,in NB cells,with overexpression of ADAMTS9-AS2,the expression of differentiation indicators,including NSE,Syn and Tau,increased significantly.Nestin,a stemness indicator,decreased distinctly.Conversely,the opposite result will be obtained.Thus,ADAMTS9-AS2 can suppressed proliferation and metastasis of neuroblastoma cells by regulating the MEK/ERK and metastasis-related genes.And ADAMTS9-AS2 expression correlates with the degree of differentiation and cell morphology of NB cells.In addition,many studies have found that lnc RNA can perform specific biological functions in vivo mainly by interacting with proteins,RNA or DNA.Lnc RNA-protein interactions have been a hot topic in recent years.We confirmed that the interaction of ADAMTS9-AS2 with LIN28 B mainly by RNA pull-down assay and RNA RIP,which can effect the expression of MYCN.In conclusion,the regulatory mechanism and potential clinical value of ADAMTS9-AS2 in neuroblastoma may provide new perspectives for deeply studies on its pathogenesis as well as diagnosis and treatment. |
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