TTF1 Suppresses Neuroblastoma Malignant Biological Behavior And Induce Differentiation By Targeting TrkA And MiR-204/TrkB Axis

BackgroundNeuroblastoma(NB)is one of the most common pediatric extracranial solid malignancies.NB exhibits extraordinary clinical heterogeneity.Very low-risk group NB does not need any treatment,and the tumor can regress spontaneously.High-risk group NB requires intensive treatment,despite the use of multi-disciplinary treatment modalities,including preoperative chemotherapy,surgery,postoperative chemotherapy,radiotherapy,peripheral blood stem cell transplant,and immunotherapy,the outcome of high-risk NB is still disappointed,and the mortality rate is as high as 40%-50%.In-depth study of the mechanism of NB occurrence and differentiation is of great significance for the treatment of NB.TTF1(Thyroid Transcription Factor 1)is an important transcription factor regulating neural differentiation.Studies have shown that TTF1 can promote the proliferation and differentiation of neural stem cells,and regulate the specialization and distribution of neurons.At the same time,TTF1 can also interact with other transcription factors to regulate the expression of neural differentiation-related genes.In addition,TTF1 plays an important regulatory role in the occurrence and differentiation of various tumors.So far,the role of TTF1 in NB development and differentiation has not been study.PurposesThis study aims to investigate the role and mechanism of TTF1 in regulating NB growth and differentiation,in order to provide potential targets and theoretical basis for NB treatment.Methods1.Collect 9 cases of NB tissues with different degrees of differentiation(gangliocytoma(GN),differentiated NB and undifferentiated NB),and use high through-put RNA sequencing technique to screen differentially expressed genes(DEGs)and miRNA,and use GO(Gene Ontology)and KEGG(Kyoto Encyclopedia of Genes and Genomes)to analyze genes;TTF1 and its downstream expression of TrkA,miR-204 and TrKB were analyzed and verified by Real time PCR.2.Predict and verify the mutual regulatory relationship among TTF1,TrkA,miR-204 and TrkB through bioinformatics analysis,luciferase reporter assay and RNA immunoprecipitation(RIP)experiments.Collect 60 cases of NB tissues with different degrees of differentiation.Immunohistochemical(IHC)staining,FISH in situ hybridization,PCR and Western blot(WB)were used to verify the expression and correlations of TTF1,TrkA,miR-204 and TrkB among different type of NB tissue.3.Construct TTF1 overexpression and low expression NB cells respectively,and use PCR and WB to detect the expression of TTF1,TrkA,miR-204 and TrkB in TTF1 NB cells.Use CCK8 assay,EdU assay,clone formation assay,flow cytometry,wound healing assay and Transwell experiment to investigate the impact of TTF1 on NB cell proliferation,migration,invasion and anti-apoptosis and other malignant biological behavior.4.Inhibit miR-204,silence TrkA or overexpress TrkB in TTF1 overexpressed NB cells,respectively.Use colony formation assay,EdU assay,wound healing assay,Transwell experiments,and flow cytometry to investigate whether inhibiting miR-204,silencing TrkA or overexpressing TrkB can reverse the effect of TTF1.5.Use NGF(Nerve Growth Factor)to pretreated TTF1 overexpressed NB cells,EdU assay was performed to detect NB cell proliferation,PCR,WB and immunofluorescence were used to detect expression of neurogenic differentiation-related molecules,such as MAP2,NSE and TAU.6.Produce subcutaneous xenograft NB model in nude mice,and observe whether TTF1 overexpression prohibit the NB growth in nude mice;RT-PCR,WB and IHC staining were used to detect the expression of TTF1 and its downstream genes and P53.NGF is used to treat NB tumor in the nude mice xenograft model,and observe whether the overexpression of TTF1 enhances the impact of NGF on tumor growth inhibition and tumor differentiation.Results1.GO and KEGG analysis indicated that DEGs in NB tissues were related to cell metabolism,cell cycle regulation,and tumor signaling pathways,such as P53 and PI3K.TTF1,TrkA and miR-204 were differentially expressed genes and miRNA in NB tissues.2.TrkA and miR-204 are transcription downstream genes of TTF1,while TrkB is the direct target gene of miR-204.The expression of TTF1,TrkA and miR-204 were significantly down-regulated in undifferentiated NB tissues,and the TrKB protein but not mRNA was highly expressed in undifferentiated NB tissues.3.TTF1 overexpression up-regulated TrKA mRNA expression in NB cells,but had no significant effect on TrkB mRNA expression.Knockdown of TTF1 down-regulates TrkA protein expression and up-regulates TrkB protein expression.Overexpression of TTF1 inhibits NB cell proliferation,NB cell migration and NB cell invasion,induces cell apoptosis and cell cycle arrest;while knockdown of TTF1 produces the opposite biological effect.TTF1 inhibits malignant biological behaviors of NB cells.4.Inhibition of miR-204 in TTF1 overexpressed NB cells up-regulate TrkB protein expression,induce migration,invasion,and proliferation of NB cells,and increase NB cell cycle.In addition,silencing TrkA or overexpressing TrkB can also promote cell proliferation,reduce apoptosis and down-regulate the expression of pro-apoptosis proteins.Inhibiting miR-204,silencing TrkA and overexpressing TrkB reversed the inhibitory effect of TTF1 on NB cells.5.TTF1 overexpression can promote the differentiation of NGF pretreated NB cells,inhibit cell proliferation and increase the expression of neurogenic differentiation-related markers.6.Subcutaneous xenograft NB model in nude mouse confirmed that overexpression of TTF1 inhibited tumor growth,up-regulated the expression of miR-204,TTF1,TrkA and p53,and decreased the expression of TrkB.TTF1 overexpression combined with NGF treatment can significantly inhibit tumor growth and promote the expression of MAP2,NSE and TAU.In vivo experiments confirmed that overexpression of TTF1 inhibits NB growth and promote NB differentiation.ConclusionsTTF1 inhibits NB cell proliferation,migration,invasion and other malignant biological behaviors,and induces neurogenic differentiation in vitro and vivo,through regulating TrkA and miR-204/TrkB axis.TTF1 could be a promising target for NB treatment.