The Mechanism Of DRR1 On The Differentiation Of Neuroblastoma Cells

Objective Neuroblastoma(NB)is an embryonal cancer of the sympathetic nervous system,which can occur around the paraspinal or aorta of sympathetic nervous system.It is also called "the king of children’s tumors".In recent years,the prevalence of the neuroblastoma increasing,accounting for approximately 8%~10% of all childhood malignant tumor diseases.The incidence of neuroblastoma is the third ranking in childhood tumors.Neuroblastoma is becoming the most common pediatric extracranial solid tumor.Especially the high-risk patients showed high degree of malignancy,rapid disease progresses and poor prognosis.The five-year survival rate of the high-risk patients is less than 50%.Therefore,the study on the pathogenesis and novel therapeutic targets in neuroblastoma are urgently needed.Down-regulated renal cell carcinoma 1(DRR1)is widely expressed in normal tissues.DRR1 is a tumor suppressor gene,and its decreased expression has been found in various types of tumors.Recently,DRR1 has been confirmed as an actin-binding protein,and found to inhibit neuroblastoma cell proliferation by interacting with actin.However,the mechanism underlying its effect is still largely unclear.In this study,we aim to evalue the effect of DRR1 on neuroblastoma cell differentiation,and investigate the underlying mechanisms and signaling pathways during neuroblastoma development.Our results provides new theoretical basis,the novel therapeutic targets,as well as novel therapeutic strategies for neuroblastoma patients.Methods1.Neuroblastoma cells were treated with low serum or retinoic acid(RA),or transfected with DRR1 sh RNA or DRR1 expression vector.Then the morphological changes of the cells were observed,and the expression of DRR1 and the differentiation markers tyrosine hydroxylase(TH)and growth-associated protein-43(GAP43)were determined by real-time PCR.2.Neuroblastoma cells were transfected with different fragments of DRR1 vectors,the morphological changes were observed,and the expression of DRR1 and the differentiation markers were detected.The interaction of the different fragments of DRR1 and actin were analyzed with co-immunoprecipitation and Western blot.3.The downstream genes of DRR1 were screened with Ch IP-seq.Neuroblastoma cells were transfected with DRR1 sh RNA or DRR1 expression vector,and the expression of the DRR1 downstream gene CREB was examined by real-time PCR.Neuroblastoma cells were transfected with CREB sh RNA or expression vector,the morphological changes were observed,and the expression of DRR1 and differentiation markers was detected with real-time PCR.4.The correlation between the expression of CREB and the expression of the differentiation markers,and the expression of CREB and the prognosis of neuroblastoma patients were analyzed by using a clinical patient database.5.In the DRR1 sh RNA treated neuroblastoma cells with the DRR1 downstream gene overexpression,then the expression of the differentiation markers,the distribution of the cell cycle and tumor growth in vivo was evaluated,respectively.Results1.The expression of DRR1 and the differentiation markers were increased in differentiated neuroblastoma cells.Downregulation of DRR1 inhibited TH and GAP43 expression,whereas overexpression of DRR1 enhanced the expression of TH and GAP43 in neuroblastoma cells.2.Real-time PCR results showed that only the DRR1(1-120)fragment and the full length DRR1(1-144)induced the expression of differentiation markers.The results of immunoprecipitation showed that only the DRR1(1-120)fragment and the full length of DRR1 were co-precipitated with actin.3.CREB was screened as a candidate downstream gene of DRR1 by Ch IP-seq assay.The expression of DRR1 affected the expression of CREB in neuroblastoma cells.Compared to the control group,downregulation of CREB expression inhibited TH and GAP43 expression,whereas overexpression of CREB enhanced the expression of TH and GAP43 in neuroblastoma cells.4.The clinic patients’ database confirmed the positive correlation between the CREB expression and the expression of the differentiation markers TH and GAP43 in neuroblastoma cells,and the positive correlation between the low expression of CREB and the high risk in neuroblastoma patients.5.Overexpression of CREB rescuesd the effect of DRR1 shRNA on neuroblastoma cell differentiation,cell cycle distribution,and tumor growth,respectively.Conclusion1.DRR1 promotes neuroblastoma cell differentiation by interacting with F-actin.2.DRR1 promotes neuroblastoma cell differentiation by regulating CREB expression.3.DRR1-CREB axis inhibits G1/S phase transition of neuroblastoma cells and tumor growth of neuroblastoma.4.DRR1-CREB axis is associated with the prognosis of neuroblastoma patients.