The Effect And Mechanism Of NeuroD1 Mediating Chordoma Proliferation And Invasion By Targeting And Regulating CA3

Experimental Objectives:Chordoma is one of relative rare malignant bone tumors,which originate from embryonic residual notochord and account for 1-4% among all the bone malignancies.Our research group showed that the incidence rate of chordoma accounts for up to 9.8% of all cases.Theoretically,chordoma is resistant to chemotherapy,and most cases are also insensitive to radiotherapy,while the surgical resection remains the treatment of choice.Although chordoma has a low degree of malignancy,it is almost impossible to avoid the "surgery-recurrence" cycle in the patient’s prognostic period.Due to the complicated anatomical structure and adjacent important blood vessels and nerve tissue after the primary surgery,revision surgery becomes increasingly difficult with unexpected complications,which may pose negative trend to the long-term prognosis,and even bring the death eventually.Therefore,it is vital to study the molecular mechanism in the malignant biological behavior of chordoma to hopefully explore new therapeutic strategies and targeted drugs for chordoma.Through transcriptome sequencing in previous work,we found that CA3 and NeuroD1 were overexpressed in chordoma compared with normal notochord tissue.CA3,a member of the carbonic anhydrase gene family,is mainly involved in key physiological processes regarding the oxidative metabolism and ion exchange in vivo.NeuroD1 is a transcription factor that is highly expressed during embryonic development.Both molecules have not been comprehensively reported in published chordoma researches.This study aims to clarify whether NeuroD1 regulates CA3 transcriptionally and their effects on the malignant biology of chordoma.Experimental Methods:1.Verified the expression of CA3 and NeuroD1 in chordoma cells and tissues by western-blot experiment,pathological specimens processing and high throughput immunohistochemical staining of tissue chips.2.Then use molecular biology techniques such as bioinformatics analysis,fragment PCR synthesis,gel electrophoresis and recovery,plasmid cloning and recombination to construct the overexpression vector of the target gene,and dual luciferase reporter genes and other methods to detect the potential regulatory effect of NeuroD1 on CA3.3.Perform the knockout of NeuroD1 and CA3,and over-expression of CA3 in chordoma cell line,and investigate the change of proliferative activity of chordoma cells following different CA3 function by CCK8 experiment,and study the change of the invasion activity of chordoma cells following different CA3 function by Transwell experiment.Transcriptome sequencing and analysis were performed on CA3 knockout chordoma cells to excavate associated tumor-related genes and signal pathways.Experimental Results:1.Tissue specimens of some chordoma patients were obtained.The expression of CA3 and NeuroD1 in chordoma tissues was significantly higher by Western blot experiments.Tissue chip immunohistochemical staining confirmed that CA3 had associations with NeuroD1 and both proteins had abnormally increased expression in chordoma cells.It’s preliminary proved that there is a certain amount of expressions of CA3 and NeuroD1 in the occurrence and development of chordoma.2.Predicted the binding site of NeuroD1 in the CA3 promoter region by bioinformatic analysis,designed the target fragment for covalent binding,constructed the overexpression luciferase reporter gene vector of both,and confirmed that NeuroD1 and CA3 genes have a targeted regulatory relationship by dual luciferin Gene validation experiments.3.Corresponding changes in the proliferation and invasion biology of chordoma cells that have overexpressed and knocked down CA3 function were confirmed by CCK8 proliferation experiments and Transwell experiments.Knockdown of NeuroD1 function can reduce the expression of CA3,further confirming that NeuroD1 has a regulatory effect on CA3.Transcriptome sequencing analysis shows that 171 differentially expressed genes and 6 signaling pathways with significant enrichment were found.Experimental Conclusions:1.We found that the expression of NeuroD1 and CA3 in chordoma tissues was higher than in normal tissues.2.The overexpression vectors of CA3 and NeuroD1 were constructed,and the molecular and protein functional level proved for the first time that NeuroD1 has a targeted pairing binding regulatory relationship to the CA3 gene.3.It was confirmed for the first time that the CA3 gene has a certain degree of regulatory effect on the tumor biological behaviors of chordoma cell.With the inhibited function of CA3 gene,171 genes showed significant expression changes and were enriched in 6 signaling pathways,among which the regulatory mechanisms still need to be further researched.