Study On Mechanism Of GBP5 Feedback Regulation Of Ifns Mediating Tumor Resistance To HSV-1

Research background and purpose:Malignant tumors are common diseases that affect human health.In recent years,the incidence of malignant tumors is on the rise and is one of the major causes of human death.Although traditional treatment options for malignant tumors such as surgery,radiotherapy and chemotherapy can alleviate the malignant development of some tumors,they still suffer from low curative power,poor prognosis and toxic side effects.Oncolytic virus(OV)can directly and specifically infect and lyse tumor cells,release tumor antigens,activate the body’s anti-tumor immune response,and produce offspring viruses to continuously infect surrounding tumor cells.With the advantages of high efficiency,safety and low side effects,it has become a hot research topic in malignant tumor treatment.Oncolytic herpes simplex virus 1(oHSV-1)is a class of OV in which HSV-1 has been modified with enhanced tumor-targeting ability,lytic activity and anti-tumor immunity.Classical oHSV-1 modification strategies have mostly focused on knocking down the ICP34.5 gene of HSV-1 and have achieved good therapeutic results in tumor cells with deficient or incomplete interferon response.However,for normal or tumor cells,HSV-1 is a foreign invasive pathogen,the body responds toHSV-1 invasion by generating a series of intrinsic immune responses to recognize viral particles and infected cells and to clear them.Interferons(IFNs)are important components of the body’s intrinsic immune system and are important limiting factors released by host cells in response to infection by viruses or other pathogens.Effective replication of viruses in tumor cells is a key factor in OV therapy and a major reason for tumor cell tolerance to OV,and IFNs and Interferon-Stimulated genes(ISGs)have the function of inhibiting viral replication,so the activation or absence of IFNs and their signaling pathways are directly related to the tumor-killing effect of OV.IFNs are known to inhibit viral infection and replication by inducing the expression of ISGs,and the guanylate-binding protein5(GBP5)gene is an important component of ISGs that mediates antiviral immune responses.Studies have reported that GBP5 can inhibit the proliferation and replication of many viruses,such as HIV,IAV,and RSV,etc.GBP5 can act at various stages of the viral replication cycle to directly inhibit viral replication,or indirectly exert antiviral biological effects by stimulating the activation of signaling pathways related to antiviral immunity.At the same time,GBP5 plays an important role in the activation of inflammatory vesicles and the regulation of the tumor immune microenvironment.In addition,several studies have shown that GBP5 expression is upregulated in a variety of malignant tumor cells compared to normal tissues,which can be used as a marker to reflect malignant progression and poor prognosis of tumors.However,the molecular mechanisms underlying the activation of intracellular signaling pathways by GBP5 to mediate tumor cell tolerance toHSV-1 have not been reported in detail.Based on the above information,this study analyzed the relationship between the expression level of GBP5and tumor cell tolerance toHSV-1 by screening tumor cells sensitive toHSV-1 and the constitutive expression differences of GBP5 between different tumor cells,constructing a eukaryotic expression vector plasmid of GBP5 gene to explore the molecular mechanism of GBP5 inhibition of HSV-1 killing tumor cells,in elucidating the molecular mechanism of GBP5 Based on the elucidation of the molecular mechanism of the tumorolytic effect of GBP5 inhibition on HSV-1,then explored the tumorolytic potentiation effect of HSV-1 on tumor cells after silencing GBP5 and the mechanism,with a view to providing new R&D ideas for the modification of novel tumorolytic oHSV-1 and new targets for anti-tumor therapy.Materials and methods:1.The viral titer of HSV-1 was determined using the half tissue culture infectious dose method.Cell proliferation after HSV-1 action on nasopharyngeal carcinoma cell line(6-10B),neuroglia cell line(HMC-3),glioblastoma cell line(LN229),hepatocellular carcinoma cell line(Hep G2,Huh7,Hep3B),and oral squamous carcinoma cell line(HSC-3,HSC-4)was detected using CCK-8 proliferation inhibition assay.Using viral vacancy formation assay,microscopic imaging and flow cytometry(FCM)to detect changes in cytopathy,cell cycle ratio,apoptosis ratio,Mitochondrial Membrane Potential(MMP)and intracellular Reactive Oxygen Species(ROS)levels after HSV-1 action on sensitive tumor cells.Differential expression of GBP5constitutively between different tumor cells and differential expression of ISGs in virus-sensitive cell lines after HSV-1 action detected by RT-q PCR.The expression of antiviral proteins in HSV-1-sensitive cell lines was examined by Western blot.2.Using molecular cloning techniques,GBP5 eukaryotic expression plasmids were constructed and identified by product sequencing,EGFP fluorescence tracing,RT-q PCR and Western blot.After transfection of tumor cells with GBP5 expression plasmid and infection with HSV-1,cell proliferation inhibition,apoptosis rate,expression levels of ISGs,ISGs-related antiviral proteins,and activation of c GAS-STING signaling pathway were examined by microscopic imaging,viral null meiosis assay,FCM,RT-q PCR and Western blot.3.Validation of the knockdown efficiency of si GBP5 by RT-q PCR.Morphological changes of HSV-1 cells and expression of intracellular HSV-1 replication-critical genes after HSV-1 action on knockdown GBP5 tumor cells using microscopic imaging,viral null meiotic assay and RT-q PCR.The changes of cell cycle ratio,apoptosis occurrence ratio,MMP and cellular ROS level were examined by FCM.The expression levels of intracellular ISGs-related antiviral proteins and the activation of c GAS-STING signaling pathway after si GBP5 transfection of tumor cells and re-infection with HSV-1were detected by Western blot.Result:1.The viral titer of the test HSV-1 was 1.31×10~7TCID50/m L.The results of CCK-8 proliferation inhibition assay showed that HSV-1 could effectively inhibit a variety of tumor cell lines,and significant inhibition of tumor cell proliferation was observed after 24 h of action,among which the most significant inhibition effect was observed in oral squamous carcinoma HSC-3 cells.Cell imaging showed that HSV-1action led to lesion,lysis and death of HSC-3 cell morphology.Viral phagocytic spot formation assay showed that the killing effect of HSV-1 on HSC-3 cells was MOI dose-dependent.The results of FCM showed that HSV-1 caused cell cycle arrest,apoptosis,and altered MMP and intracellular ROS levels in HSC-3 cells.The results of RT-q PCR showed that GBP5 was constitutively least expressed in HSC-3 cells and HSV-1 stimulated the increase of GBP5 expression in HSC-3 cells.The results of Western blot showed that HSV-1 significantly downregulated the expression level of ISGs-related antiviral proteins.2.The results of double digestion identification,product sequencing,EGFP fluorescence tracing,RT-q PCR and Western blot showed that the overexpression plasmid of GBP5 was successfully constructed.The results of microscopic imaging and Viral phagocytic spot formation assay showed that GBP5 overexpression could reduce the proliferation inhibition of tumor cells by HSV-1.RT-q PCR,FCM and Western blot results showed that overexpression of GBP5 reduced the proportion of HSV-1-induced apoptosis,cell cycle arrest phenomenon and the expression levels of viral own-related genes,upregulated the expression levels of IFNs and ISGs,and activated the c GAS-STING signaling pathway.3.Cell imaging results showed that HSV-1 and si GBP5 combined to promote the morphological changes of HSV-1 on tumor cells.Viral vacancy formation assay and RT-q PCR results showed that low expression of GBP5 promoted HSV-1 in tumor cells.The results of FCM showed that low expression of GBP5 promoted HSV-1-induced cell cycle arrest,apoptosis,and altered MMP,while increasing intracellular ROS levels.Western blot showed that si GBP5 inhibited the activation of c GAS-STING signaling pathway and down-regulated the expression of cellular ISGs-related antiviral proteins.Conclusion:In this study,we found that HSC-3,a cell line with constitutively low expression of GBP5,was more sensitive to the effects of HSV-1.HSV-1 exerted its tumor cell-killing effects by inducing cycle arrest and apoptosis in tumor cells,decreasing cellular MMP and increasing intracellular ROS levels.HSV-1 infection stimulated increased expression of GBP5 and inhibited the expression of some ISGs-related antiviral proteins.Over-expression of GBP5 reduces the expression of HSV-1 key genes in tumor cells,activates the c GAS-STING signaling pathway and upregulates the expression of IFNs and ISGs,which in turn mediates tumor cells’tolerance toHSV-1.The combination of HSV-1 and si GBP5 could enhance the killing effect of HSV-1 on tumor cells.Taken together,the above findings suggest that GBP5 can be used as a potential target for combined strategy or modification of novel lysing virus oHSV-1 for the treatment of malignant tumors.