Abnormal Expression Of GSG2 In Human Glioma:A Function And Preliminary Mechanism Study

ObjectiveThis study aimed to investigate the functional manifestations and potential mechanisms of GSG2 gene expression in human gliomas.MethodThis study is divided into two parts.The first part explores the expression and function of GSG2 in human glioma cells in vitro and in vivo.The second part analyzes the potential mechanism of GSG2 affecting glioma cell function by gene chip technology.In the first part,gene expression was screened in 179 human gliomas and normal brain tissue samples by tissue microarray technique,and its correlation with clinical data was further discussed.Furthermore,the GSG2 gene was used as a template to design the RNA interference target sequence,and the target gene RNA interference lentiviral vector was constructed and transfected into U251 and U87 cell lines.The transfection efficiency was detected by Western blot.On this basis,we divided the cells into the knockdown group(KD)and the negative control group(NC),and verified the function by MTT,apoptosis detection and colony formation experiments.In the second part,U251 cells were divided into KD group and NC group to extract mRNA,and the expression difference was analyzed by gene chip.The Ingenuity Pathway Analysis(IPA)tool was used to find potential targets downstream of the GSG2 gene,and the expression of differential genes was verified by qPCR and Western Blot techniques.ResultGene expression screening was performed on 179 human gliomas and normal brain tissue samples using tissue microarray technology.It was found that GSG2 protein was expressed in both glioma and normal tissues,and the expression was significantly different(P<0.0001).At the same time,the expression of GSG2 was also associated with pathological grade and recurrence(P = 0.0054,P = 0.034).Subsequently,we transfected the U251 and U87 cell lines with lentivirus and constructed a GSG2 knockdown model.Western blot analysis confirmed that the expression of GSG2 in U251 and U87 cell lines was significantly inhibited.MTT,apoptosis detection and colony formation experiments were performed in the KD group and the NC group,respectively.The results showed that the proliferation of KD group was slower than that of NC group(P<0.05),the number of apoptosis was increased(P<0.05),and the number of cell clones was decreased(P<0.05).The U251 cell line was divided into KD group and NC group to make a gene chip to analyze the expression of downstream pathway.In the KD group,the interferon pathway was significantly activated(Z-score =3.153).The molecules such as FITM1,OAS1,IFITM1,and MX1 were significantly up-regulated,and BAX was significantly down-regulated.Upstream regulatory factor analysis showed that IFNG was predicted to be strongly activated,and there were 157 co-activated genes in the gene;MAPK1 was predicted to be strongly inhibited,and there were 78 genes with consistent inhibition.In this project,the first regulatory network such as ACKR2 has an activation effect on antiviral response by ADAR and other genes,Consistency Score = 83.711;replication of mouse herpesvirus-4,herpetic stomatitis virus and viral replicon has an inhibitory effect.Quantitative analysis of downstream differentially expressed genes in U251 cells revealed that the genes up-regulated in the KD group were STAT2,IRF1,PSMB8,IFITM2,STAT1,IFITM3,IFI35,TAP1,ISG15,IFIT1,IFIT3,OAS1,MX1(P <0.05).The down-regulated genes were CCNE1,E2F1,CDK4,CCNA2,CCND1,CDK2,CCNA1(P<0.05).The expression of Cyclin E1,NF-?B and E2F1 in KD group was down-regulated by Western Blot,and the expression of Cyclin D1 protein was not changed.ConclusionThis study found that GSG2 gene is differentially expressed in glioma cells and normal brain tissue,and the expression level increases with the pathological grade and recurrence of glioma.Interference with the expression of the target gene GSG2 in the human glioma cell lines U87 and U251 has the effect of inhibiting proliferation and promoting apoptosis.After gene chip analysis,qPCR and Western Blot,the expression of Cyclin E1,NF-?B and E2F1 was down-regulated after Lentivirus infection,which laid a foundation for exploring its molecular mechanism and signal transduction pathway.