The Role And Mechanism Of Transcription Factor TCF4 In Neuroblastoma

Objective:Neuroblastoma(NB)is a common malignant solid tumor of the sympathetic nervous system in children.Its clinical manifestations are heterogeneous.In some infants,such as 18 months old,it can spontaneously subside.However,with the age of the patient,it may show rapid progress or even death.With the development of molecular biological technologies such as genome and epigenome,the molecular characteristics of neuroblastoma have been studied more and more deeply.Studies have found that some specific transcription factors are expressed in neuroblastoma.It is well known that some specific transcription factors in malignant tumor cells can maintain their own tumor characteristics by regulating the expression of a variety of pathogenic genes.Studying the mechanism of specific transcription factors in neuroblastoma will help to provide new ideas and directions for the diagnosis and treatment of neuroblastoma.Therefore,this study mainly screened the neuroblastoma-specific transcription factor gene,and studied the function and mechanism of this transcription factor in neuroblastoma.Methods:1.Part 1:The expression of TCF4 in neuroblastoma and its clinical significance:Through the comprehensive analysis of the ChiIP-Seq results of histone H3K27ac of neuroblastoma cell line,the important genes regulated by super enhancer in neuroblastoma were screened;Use Cancer Cell Line Encyclopedia(CCLE)bioinformatics database to query the expression of transcription factor TCF4 screened from various tumors;The clinical database R2:Genomics Analysis and Visualization Platform was used to query the expression of TCF4 in children with neuroblastoma and the information of clinical prognosis.2.Part 2:Effects of TCF4 on the function of neuroblastoma cells in vivo and in vitro:First,the sh-TCF4 gene interference plasmid with stable knockdown of TCF4 gene was designed and synthesized,and the SK-N-BE(2)and IMR-32 cell lines with stable knockdown of TCF4 gene were obtained by lentivirus packaging technology,and then the knockdown effect of TCF4 gene was detected by RT-PCRh and western-blot technology at the gene and protein levels respectively;The effects of knockdown of TCF4 gene on the proliferation of neuroblastoma cells SK-N-BE(2)and IMR-32 were detected by CCK-8 cell proliferation test,colony formation test and subcutaneous tumorigenesis test in nude mice;The effect of knockdown of TCF4 gene on the apoptosis of neuroblastoma cell SK-N-BE(2)and IMR-32 was detected by flow cytometry;The effect of knockdown of TCF4 gene on the metastasis and colonization of neuroblastoma cells IMR-32 and SK-N-BE(2)was detected by tumor metastasis test in nude mice.3.Part 3:Study on the mechanism of TCF4 in neuroblastoma:collect the RNA of the control group and sh-TCF4 group cells transfected with sh-NC,carry out RNA-Seq detection,calculate and screen the significant change genes and the enriched signal pathway,and carry out experimental verification;Combined with CHIP-Seq experiment,we searched for the downstream gene of TCF4 transcription.Results:1.Part 1:In neuroblastoma,we screened TCF4 as a transcription factor regulated by super enhancer,and it has high transcription activity in neuroblastoma;CCLE data showed that the expression of TCF4 in neuroblastoma ranked first among various tumor cells;R2datebase analysis of clinical database showed that patients with neuroblastoma with high expression of TCF4 had poor prognosis.2.Part 2:After knocking down the expression of TCF4 gene in neuroblastoma cells,the results of CCK-8 experiment and clonogenesis experiment showed that the proliferation ability of neuroblastoma cells decreased compared with the control group;The results of flow cytometry showed that compared with the control group,the apoptosis rate of knockdown group was significantly increased;The results of subcutaneous tumorigenesis experiment in nude mice showed that compared with the nude mice in the control group,the tumor volume in the knockdown group was significantly decreased;The results of tumor metastasis model in nude mice showed that compared with the control group,the metastasis degree of liver,kidney,spine and other organs in the knockdown group was significantly reduced;Immunohistochemical results showed that compared with the control group,the tumor tissue in the knockdown group had lower positive staining of the proliferation index Ki-67 and higher positive staining of the apoptosis index Bax.3.Part 3:The KEGG analysis of RNA-seq results showed that the PI3K-AKT signal pathway decreased significantly after the knockdown of TCF4 gene expression,and the PCR results confirmed the decline of the important related genes of the pathway;Through the analysis of the results of RNA-seq and CHIP-seq,it is found that the key gene of lipid metabolism SCD receives the transcriptional regulation of TCF4.Conclusions:1.TCF4 is a transcription factor with high specific expression in neuroblastoma,and its expression is related to the poor clinical prognosis of patients with neuroblastoma.2.Inhibiting the expression of TCF4 can inhibit the growth and proliferation of neuroblasts and promote their apoptosis in vivo and in vitro.3.TCF4 participates in the regulation of PI3K-AKT signal pathway in neuroblastoma and regulates the transcription of SCD,a key gene of lipid metabolism.