Study On The Mechanism Of Human Herpesvirus Type 6 U94 Gene Inhibiting Glioma

Human herpesvirus 6A and human herpesvirus 6B(HHV-6A and HHV-6B)are two members of the Betaherpesvirinae subfamily of herpesviruses.It is reported that HHV-6 is related with Roseola infantum,acute graft versus host disease,viral myocarditis,multiple sclerosis,post hematopoietic cell transplantation(HCT)infection(encephalitis,pneumonitis,and hepatitis),etc.Glioma is a highly malignant brain tumor.We reported previously that the DNA and protein of human herpesvirus 6 could be detected in glioma tumor tissues.However,the effects of HHV-6 U94,which is abundantly expressed during the virus' latency period,on glioma progression remain unknown.In this study,human glioma cell line U87 was used as the research obj ect to investigate the role and molecular mechanism of HHV-6 U94 in the progression of glioma.Firstly,U87 cells was transfected with HHV-6 U94 gene by lentivirus,and the expression of U94 was confirmed using immunofluorescence assay.Secondly,we observed the effect of HHV-6 U94 gene on the proliferation of U87 cells by CCK-8 assay,plate colony formation assay and soft agar colony formation assay.The results showed that U94 gene can significantly inhibit the proliferation of U87 cells.In addition,U87 cell migration and invasion abilities were evaluated by wound healing assay,Transwell migration assay and invasion assay,respectively.The results revealed that HHV-6 U94 gene also significantly inhibited the migration and invasion of U87 cells compared with that of the control cells.Thirdly,we explored the effect of HHV-6 U94 gene on gene expression in U87 cells.The mRNA of U87 cells transfected with U94 gene(u87-u94)was extracted and assessed using a microarray.Data analysis using unsupervised hierarchical clustering showed that the expression levels of RAP1A,SLC7A11,FOXP1,and TCF4 were downregulated in U87-U94 cells compared 262 with those in the control cells.The expression levels of these genes were further verified by qRT-PCR and Western blot.The results showed that the expression levels of mRNA in RAP1 A,SLC7A11,FOXP1,and TCF4 of U87-U94 cells were decreased compared with those in the control U87 cells,and the SLC7A11 and TCF4 protein levels were also reduced in U94-expressing U87 cells.In addition,the levels of phosphorylated AKT and GSK3? were also reduced in the U87-U94 cells.The effects of HHV-6 U94 gene on glioma tumor growth in vivo were evaluated through a xenograft nude mouse model.The average tumor weights of the U87-U94 cells inj ection group were much lower than those of the control group.The results suggest that the growth of the tumors formed from the U87-U94 cells was dramatically inhibited.Taken together,these data indicated that HHV-6 U94 gene could suppress U87 tumor cell proliferation,migration and invasion,and tumorigenesis in vivo by inhibiting AKT/GSK3? signaling pathway in glioma.This study provides a theoretical basis and new ideas for the search for new therapeutic targets for glioma.