The Function And Molecular Mechanism Of TRIP4 In Glioblastoma

Objective: Glioblastma is the most common primary tumor of the central nervous system.It has high malignancy,strong invasiveness and poor prognosis.Traditional treatment methods have little effect,so it is urgent to find new and effective molecular therapeutic targets.TRIP4 was identified as a hallmarker of tumorigenesis,but its precise function in glioblastoma progression is completely unknown.Here,we explored the carcinogenic role of TRIP4 in glioblastoma.The high expression of TRIP4 was observed in human glioblastoma cells and tissues.Its knocked suppressed glioblastoma progression in vitro,including glioblastoma cell proliferation,migration and invasion inhibition and apoptosis induction.Identification of TRIP4 downstream targeting regulatory gene DDIT4 by RNAseq analysis.In this study,we will further study the regulation of TRIP4 on DDIT4 expression,luciferase activity,cell growth and invasion,tumor formation,and the related signaling pathway in glioblastoma cell lines and animal models,thereby identifying the biological functions and molecular mechanisms of DDIT4.Moreover,we will also analyze the expression of TRIP4 and DDIT4 in tumor tissues and evaluate their correlation with glioblastoma development,clinical stage,metastasis and prognosis,identifying the clinical significance of TRIP4 and DDIT4.This program will provide new understanding for revealing the novel mechanisms involved in TRIP4 expression regulation.Methods:1.Analyze the expression of TRIP4 in GBM and its relationship with pathological staging by TCGA and GEO databases.2.Western blot and RT-PCR were used to detect the expression of TRIP4,and the location of TRIP4 in subcellular cells was detected by immunofluorescence staining in glioblastoma cells.3.TRIP4 was knocked down by siRNA,and the effects of TRIP4 on the biological functions of proliferation,migration,invasion and apoptosis of glioblastoma cells were detected by MTT,clone formation,scratch,Transwell and flow cytometry,respectively.4.Through the method of lentiviral packaging,establish a stably transfected cell line of glioblastoma TRIP4 stably knockdown,and screen the effective TRIP4 downstream targeting factor after RNAseq,.the DDIT4 was identified.Firstly,the expression of downstream target genes at the protein and mRNA levels after TRIP4 knockdown or overexpression was detected by Western blot and RT-PCR.5.Rescue experiment: After knocking down TRIP4 in glioma cells,DDIT4 was overexpressed,and MTT and Transwell were performed to verify Reconfirmation of the correlation between TRIP4 and the downstream target gene DDIT4 Results:1.TCGA and GEO database analysis showed that TRIP4 is highly expressed in glioblastoma tissue and positively correlated with pathological stage.2.In glioblastoma cell lines,TRIP4 is expressed at higher levels of protein and mRNA.3.Knockdown TRIP4 can inhibit the proliferation,migration and invasion of glioblastoma cells and promote the apoptosis.4.The RNAseq results were screened for the downstream target gene TRIT4 of TRIP4.The correlation between them was firstly identified by Western blot and RT-PCR.The results showed that the expression of DDIT4 at the protein and mRNA levels after knockdown(or overexpression)of TRIP4 There is also a corresponding decrease(or increase),which indicates a positive correlation between the expression levels of TRIP4 and DDIT4.5.Rescue results show that the inhibitory effect of knockdown(or overexpression)TRIP4 on proliferation and invasion of glioblastoma cells can be restored by overexpression(or knockdown)of DDIT4,confirming the positive correlation between TRIP4 and DDIT4.relationship.Conclusion:The expression level of TRIP4 was higher in glioblastoma,and TRIP4 was positively correlated with DDIT4 expression.In vitro cell-level experiments showed that TRIP4 can inhibit the development of glioblastoma,and this function is mediated by the downstream target gene DDIT4.It is suggested that the TRIP4/DDIT4 pathway may be a new target for the treatment of glioblastoma.