Research On The Expression And Function Of LncRNA XIST In The Development And Progression Of Neuroblastoma

BackgroundNeuroblastoma(Neuroblastoma,NB)is the most common extracranial solid tumor in childhood,originating from embryonic neural crest cells.NB accounts for nearly 10%of all childhood cancers,but it accounts for up to 15%of all childhood cancer deaths,and is the main cause of death from cancer in children aged 1 to 5.Due to the concealment of the primary site,difficulty in early diagnosis,high malignancy,rapid progression,and early metastasis of NB,the prognosis of children is poor.According to tumor histology,clinical stage,tumor cell ploidy,MYCN oncogene amplification and other parameters,the risk stratification of NB patients was divided into low,medium,and high-risk groups.At present,the most advanced and intensive treatment methods are surgery,myeloablative chemotherapy,and radiotherapy combined with immunotherapy.Despite continuous updates and improvements in diagnosis and treatment,the five-year survival rate of high-risk and relapsed NB patients is still less than 40%.The various clinical manifestations and course of NB depend on tumor biology.Due to the interaction between different epigenetic and genetic factors,the detailed pathogenesis has not yet been clarified.Long non-coding RNA(LncRNA)refers to non-protein coding transcripts with a length of more than 200 nt.In recent years,the regulatory role in cell biology has gradually attracted attention.LncRNA can bind to DNA,RNA and protein to regulate the expression of related genes in terms of epigenetics,transcriptional regulation and post-regulation.LncRNA,as a key node molecule in the process of regulating mRNA(message RNA,mRNA)expression,participates in the occurrence and development of many tumors.LncRNA has been confirmed to participate in the occurrence and development of NB through abnormal expression.Since LncRNA is easier to detect in tissues and serum,it is considered to be an emerging marker for tumor diagnosis and prognosis.LncRNA X-inactive specific transcript(XIST)is the main regulator of X chromosome inactivation in mammals.The biological role and underlying mechanism of XIST in neuroblastoma are still unclear.This research group collected clinical specimens from NB patients,and used qPCR to detect the relative levels of XIST in NB tissues and NB cell lines to assess the correlation with clinical pathology.The effects of XIST on cell growth,invasion and migration,and the occurrence and development of NB in vitro were studied.ChIP-qPCR was used to analyze the level of histone H3 trimethyllysine 27(H3K27me3)in the DKK1 promoter.The interaction between XIST and Zeste homolog 2(Enhancer of zeste homolog 2,EZH2)was verified by RNA immunoprecipitation(RIP)and RNA pull-down experiments.In this project,we aim to clarify the expression intensity and pervasiveness of XIST in NB tumor tissues,and conduct a preliminary study on XIST’s involvement in the proliferation,apoptosis,invasion and metastasis of NB cells in vivo(in vitro),and clarify the corresponding functions.Molecular biological mechanism to determine whether it will become a potential biomarker of NB,and provide new ideas for the development of NB therapeutic targets.Method1.Collect 30 samples of NB tissues and paired normal adrenal gland tissues adjacent to cancer,extract tissue RNA and detect the expression of the target in NB and normal tissues adjacent to cancer and NB cell lines by real-time fluorescent quantitative PCR technology(qRT-PCR)Level,analyze the correlation between the expression of XIST and the pathological characteristics of clinical patients.2.Design siRNA targeting XIST and transfect human neuroblast SK-N-BE(2)cells and SH-SY5Y cells,extract RNA and test knockdown efficiency,soft agar clone formation experiment,CCK-8(Cell count kit)experiment to detect cell proliferation ability,Transwell experiment to detect its migration and invasion ability,and to study the influence of XIST on the biological function of NB cells.3.ChIP-qPCR was used to analyze the level of histone H3 trimethyllysine 27(H3K27me3)in the DKK1 promoter.4.The interaction between XIST,EZH2 and DKKI was verified by RIP analysis,RNA pulldown experiment,qPCR and Western bolt experiment.Result1.XIST expression was increased in neuroblastoma tissues and cell lines.2.Downregulation of XIST inhibited the growth,migration and invasion of neuroblastoma cells.3.EZH2 inhibits DKK1 expression by inducing H3K27me3 methylation in the DKK1 promoter.4.XIST interacts with EZH2 to increase the level of H3K27Me3 in the DKK1 promoter.5.Downregulation of XIST can inhibit the growth,invasion and migration of neuroblastoma cells by increasing the secretion of DKK1.Conclution1.LncRNA XIST induces H3 histone methylation through EZH2 to down-regulate DKK1,which promotes the growth,migration and invasion of neuroblastoma cells.2.As an oncogene,LncRNA XIST can be used as a molecular marker in the early diagnosis,treatment and prognosis evaluation of neuroblastoma.