Exploration Of Prognostic Evaluation Markers Of Neuroblastoma And Related Mechanisms

Neuroblastoma is the most common pediatric extracranial solid malignancy.It is mostly in stage III/IV upon diagnosis,with a high degree of malignancy,rapid development and early metastasis.Despite the combination of surgery,radiotherapy and chemotherapy,the survival rate of patients in advanced stage is still very low.Therefore,early diagnosis,targeting therapeutics and accurate prognosis prediction of NB to improve its survival rate are clinical problems waiting to be solved.The occurrence and development of NB is considered to be the result of multi-genes involvement and microenvironment.Several genetic alterations have been found in NB to be associated with tumor progression and prognosis,such as MYCN gene amplification,as well as mutations in reverse transcriptase TERT gene and ALK gene.The next generation sequencing found that alterations of ARID1 gene are associated with poor prognosis,suggesting that the ARID1 gene may play a key role in the progression of NB,with potential as a prognostic marker,but the specific mechanism is unknown.Circulating blood exosomes,as an important pathogenic agent in the tumor microenvironment,also play an important role in the progression of NB.Exosomal miRNAs have been used in the diagnosis and prognosis assessment of a variety of tumors.By detecting and analyzing differentially expressed miRNAs in circulating plasm of NB patients,we clarified the role of exosome miNRA in the diagnosis and prognosis of NB.We further explored the intrinsic mechanism of the exosomal miRNAs in NB.Objectives(1)To detect the expression of arid1 proteins in clinical neuroblastoma tissues and analyze their correlations with the pathological type,clinical grade and COG risk prognosis;(2)To explore the effects of ARID1 gene on cell proliferation,metastasis,invasion and other aspects,and to investigate the underlying mechanisms by RNAseq.(3)To explore the significance of exosome miRNA diagnosis and prognosis evaluation in NB and the internal mechanism of action.Methods 154 cases of untreated NB pathological specimen and clinical data from Shanghai Children's Medical Center were enrolled in this study.The expression levels of arid1 proteins were detected by immunohistochemistry,and their clinical correlations were analyzed.ARID1-knockdown NB cell lines were set up,and functional studies were performed.Differentially expressed genes and pathways were analyzed after high-throughput sequencing and then verified in vitro.Exosomal miRNAs were isolated from NB patients and healthy controls,and differentially expressed miRNAs were analyed and verified.Its mechanism of action in NB was further studied.Results(1)In 154 cases of NB,75.3% were loss of arid1 a protein expression,while 16.2% were loss of arid1 b,which was related with poor prognosis.(2)Knockdown of ARID1 gene in vitro can increase the invasion of NB tumor cells.and knockdown of ARID1 B gene can active the oxidative phosphorylation of tumor cells.(3)miR199a-3p was highly expressed in the plasma exosomes of NB patients,which can promote the proliferation and migration of NB cells by inhibiting the function of NEDD4 gene.Conclusions The loss of arid1 b protein is associated with poor histological classification,high clinical stage,and high risk COG of NB and can be used as an independent risk factor for NB prognosis evaluation.Knockdown of ARID1 B gene in vitro can promote the invasion of tumor cells,most likely by the activation of oxidative phosphorylation.The loss rate of arid1 a protein is higher,and it is more likely to be associated with the benign and malignancy of tumors.A series of tumor-related pathways and genes are up-regulated by knocking down the ARID1 A gene,and the invasion of tumor cells is also enhanced.The circulating plasma exosomal miR199a-3p is highly expressed in NB patients,and it promotes proliferation and metastasis of NB cells by inhibiting the NEDD4 gene,which has a guiding role in the diagnosis and prognosis evaluation of NB.