Study On The Effect Of Borna Disease Virus And EBLN1 Gene Of Glioblastoma Cells And Related Mechanism

BACKGROUNDIn various primary tumors of the adult central nervous system(CNS),glioblastoma(GBM)has the highest incidence and malignancy.Because of its highly invasive growth and insensitivity to radiotherapy and chemotherapy,it has a high recurrence rate and lethality.Despite considerable progres in the diagnosis and treatment of GBM in the past few decades,the prognosis of patients is still unsatisfactory,with an average survival period of less than 14 months.Compared with other malignancies,GBM is usually recurred in situ around the resection cavity and is limited to growth in the CNS.There is very little disant metastasis.Therefore,the current treatment methods are mainly directed at local tumors,and oncolytic viruses are one of them.Type 1 herpes simplex virus,adenovirus,measles virus,etc.have been shown to selectively infect and destroy tumor cells,but clinical trials have bueen less than optimistic.The reduction in tumor volume or the extension of the patient’s life cycle has not achieved the desired results.Finding an effective,safe method of treatment is a matter of no delay.We reported early on that a human-derived Borna disease virus(BDV),which infects oligodendroglioma cells,inhibits cell proliferation and promotes apoptosis.In neuroblastoma cells,BDV also exerts the same inhibitory effect.This suggests that BDV may interfere with the malignant phenotype of neuronal tumor cells.In addition,endogenised bornavirus-derived element 1(EBLN1)can effectively reduce endogenous DNA damage and repair exogenous DNA damage in colon cancer cells and cervical cancer cells.It plays an important role in stabilizing the genome,which is precisely one of the important mechanisms for inhibiting tumors.It suggests that EBLN1 gene has negative regulation in the formation of tumors.BDV is a highly neurotropic,non-cytolytic,negative-strand RNA virus,and is one of the oldest viruses integrated into the human genome in ancient times.Based on the fact that BDV can infect healthy populations and develop asymptomatic infections,as well as previous reports that BDV and EBLN1 have potential anti-tumor effects,we sought to study the dual inhibitory effects of BDV and EBLN1 on glioblastoma,in search of new treatments that are closer to human sources and have fewer side effects.It further provides new research methods for revealing the interaction between environmental factors and organisms in the process of tumor formation.OBJECTIVE1.To test the effect of BDV on the biological characteristics such as proliferation,apoptosis,migration and invasion of GBM cells;observe the effect of BDV on the growth of GBM xenografts in nude mice,so as to clarify the anti-tumor effect of BDV in GBM.2.Complete the transcriptome sequencing of GBM cells before and after infection with BDV.Through analysis of differential genes,screen relevant signal pathways and verify.Then explore the molecular mechanism of BDV inhibiting tumors.3.Analyze the expression differences of EBLN1 gene in different grades of glioma patients and verify the effect of EBLN1 overexpression on the growth of GBM xenografts in nude mice.Learn more about the function of the EBLN1 gene.METHODS1.Infect two GBM cells(U87MG and U251)and normal humant glial cells(HA)using BDV.The role of BDV in the biological functions of tumor cell proliferation,apoptosis,cycle,migration and invasion was examined by CCK-8 method,flow analysis technique,scratch test,and Transwell assay,U87MG cells were to construct subcutaneous tumor models and intracranial tumor models in nude mice,and the effects of BDV on tumorigenicity of GBM were observed.Western blotting(WB)and immunohistochemistry respectively were used to detect the expression of tumor-associated proteins in GBM cells and tissues.2.The transcriptomes of GBM cells were analyzed and eompared based oa mRNA-Seq technology to screen for differentially expressed genes caused by BDV infection.At the same time,using GO analysis,KEGG pathway analysis and other techniques to further interpret the differential genes,to find the main signal pathway of BDV in GBM,and combined with WB technology to verify.3,Collect glioma-related data from the cancer,genome atlas(TCGA)database and download the EBLN1 gene expression profile and clinical information.The correlation between EBLN1 expression and clinicopathological parameters and the impact on survival were analyzed.In the subcutaneous tumor model of GBM nude mice,the effect of EBLN1 overexpression on the tumorigenesis of GBM was observed,and the interaction between EBLN1 and BDV in the formation of GBM was also analyzed.RESULTS1.In vitro,after BDV infection of U87MG and U251 cells,compared with the control group,the proliferation ability of tumor cells was reduced in a dose-dependent manner,and at the same time,the migration and invasion activity of the cells was reduced and apoptosis was promoted.In HA cells,BDV infection did not affect the normal function of the cells.In vivo experiments,BDV can significantly inhibit the growth of transplanted tumors of GBM in nude mice:After injection of BDV,the subcutaneous tumor volume was reduced by nearly 63%,and the volume of intracranial tumors was reduced by nearly 37.5%;at the same time,no adverse effects of BDV on animals were observed.In BDV-infected GBM cells,the expression levels of Bcl-2,MMP-2/9 and VEGF proteins were down-regulated and the expression of BAX protein was up-regulated.In BDV-treated GBM tissues,Bcl-2,MMP-2,and CD34,The expression of PCNA and VEGF protein was significantly down-regulated,and the BAX protein expression was up-regulated.2.After BDV infection of U87MG cells,1827 genes changed significantly,of which 1250 were up-regulated and 577 were down-regulated.These differential genes affect the regulation of multiple biological processes in GBM cells.BDV reduced the expression of EGFR,and then inhibited the downstream PI3K/AKT/mTOR and ERK1/2 signaling pathway activities,and finally played a role in regulating the malignant phenotype of GBM cells.3.A total of 563 cases of gliomas with complete clinical parameters were included in the TCGA dataset.The expression level of EBLN1 in GBM pathological tissue samples was significantly lower than other types of gliomas,and was related to the survival status of patients.In in vivo experiments,subcutaneously implanted tumors of EBLN1-overexpressing nude mice grew slowly,which was approximately 42.7%smaller than that of the control group.If BDV was injected intratumorally,the tumor volume was further reduced by about 32.9%.CONCLUSION1,BDV can effectively inhibit the malignant phonotype of GBM cells and impede the formation of GBM tumors in nude mice,but it has no adverse effects on normal cells and animals.It shows that BDV can be a candidate for treatment of GBM.2.BDV infection caused a significant change in the transcriptome of GBM cells,which in turn affected the function of multiple biological processes of tumor cells.BDV inhibits GBM by decreasing the activity of EGFR/PI3K/AKT/mTOR and ERK1/2 signaling pathways.3,In patients with GBM,the expression level of EBLN1 gene is generally reduced and related to the survival status of patients.Overexpression of EBLN1 significantly inhibited the growth of subcutaneous GBM xenografts in mude mice and exerted synergistic antitumor effects with BDV.