The Function And Mechanism Of Toll Like Receptor 4 And B7-H6 In The Tumorgenesis,Progression And Immune Evasion Of Glioma

Glioma occurs in neuroderm and is the most common primary tumor of the central nervous system,accounting for about 80%of malignant brain tumors[1,2].According to the classification of WHO,glioma is divided into grade Ⅰ-Ⅳ.Grade Ⅰand grade Ⅱ gliomas are low-risk tumors with relatively good prognosis.Ⅲ and Ⅳgrade gliomas are high-risk malignant tumors with poor prognosis[3,4].Malignant glioma is highly invasive and highly malignant,with high morbidity and mortality in all age groups[4].The symptoms of glioma patients are not obvious at early stage.As the tumor grows,the symptoms of intracranial hypertension and compression of brain tissue gradually appear,which are easily ignored and delay the best treatment time.So far,there is no effective clinical treatment for malignant glioma.Surgical resection is the main treatment strategy at present.However,there is no definite boundary between malignant glioma and normal brain tissue,and it is difficult to remove the tumor tissues completely.Moreover,even if the tumor is completely removed,the recurrence easily occurs and the mortality is high.In addition,the presence and function of the blood-brain barrier blocks the entry of chemotherapeutic agents into the tumor tissue,causing glioma to be insensitive to chemotherapeutic agents,and the efficacy of various preventive and therapeutic drugs is not satisfactory or toxic and side effects are great.Malignant glioma is also less sensitive to radiotherapy.Therefore,the prognosis of glioma patients is poor and the recurrence rate is high[5].The mean survival time of glioma patients is 1.5 years.The 5 years survival rate for glioblastoma is less than 3%[3,5].According to the 2014 National Cancer Center,glioma is the ninth most lethal tumor in China.Therefore,the treatment of malignant glioma remains challenging.It is urgent to explore new target and therapy strategy to improve the sensitivity to therapies,kill tumors effectively,and finally improve the life quality and survival time of glioma patients.Part 1 The function and mechanism of LPS activated toll like receptor 4 signal pathway in the tumorgenesis,progression and immune evasion of glioma CD133+ cancer stem cellsObjective:Cancer stem cells(CSCs)is found in the malignant tumor tissue of glioma.Glioma CSCs exhibit high tumorigenic activity and has many characteristics of stem cells,such as multilineage differentiation,unlimited proliferation and self-renewal.Most studies now use CD133 as a common marker for glioma CSCs.It is reported that glioma CSCs plays an important role in glioma growth,invasion,angiogenesis,resistance to chemotherapy and radiotherapy,and immune escape.Toll-like receptor 4(Toll-like receptor 4,TLR4)is the most studied Toll like receptors(Toll-like receptors,TLRs)at present.Recent studies have found that many tumor tissues abnormally express high levels of TLR4,and TLR4 plays an important role in the development and progression of tumors.However,whether glioma CD133+ CSCs expresses TLR4?And what is the specific functions of TLR4?It’s not clear yet.Therefore,this part uses human glioma cell lines and primary cells isolated from patients’ tumor pathology tissue to induce glioma CSCs,and obtains glioma CD 133+ CSCs and uses them as the research object.Lipopolysaccharide(lipopolysaccharide,LPS)was used to activate TLR4 signaling pathway and the CD 133+ glioma CSCs reactive CTL was established in order to study the roles and possible mechanisms of TLR4 signal transduction pathway in development and immune escape of glioma CD133+ CSCs.Methods:Human glioma cell lines SF295,U251 and four primary tumor cells,isolated from human glioma samples(pTl-4),were used to induce and generate glioma CSCs.The expression levels of molecular markers of glioma CSCs were identified via flow cytometry.Six kind of glioma CD 133+ CSCs were separated and purified by CD 133 magnetic-activated cell separation purification system.The expression of TLR4 in glioma CD133+ CSCs and patient tissues were detected via flow cytometry,Western Blot and immunohistochemical staining(IHC).After LPS stimulation,the proliferation,expression of molecular markers,cytokine secretion and expression of related molecules in signal pathways are investigated via Cell Counting Kit-8(CCK-8),real time quantity polymerase chain reaction(qRT-PCR),flow cytometry,enzyme linked immunosorbent assay(ELISA)and Western Blot.Mononuclear cells were isolated from peripheral blood of healthy donors,and CD3+ T cells were obtained by magnetic bead sorting.Glioma CD 133+ CSCs was irradiated and cultured with CD3+T cells to establish glioma CD133+ CSCs reactive cytotoxic T lymphocyte(CTL).lactate dehydrogenase(LDH)cytotoxicity test was used to detect the cytotoxic lysis of glioma CD133+ CSCs reactive CTL to different target cells,and its phenotype was identified by flow cytometry.Results:Glioma CSCs expressed CD133,Nanog,SSEA-1,Msil and Nestin.TLR4 protein was expressed in gliomas CD133+ CSCs and patient tissues.LPS promoted the proliferation of glioma CD133+ CSCs and protects them against doxorubicin induced apoptosis.LPS up-regulated the expression of CD133,Nanog and Nestin at the gene and protein levels in glioma CD133+CSCs.LPS promoted the secretion of cytokines including monocyte chemotactic protein-1(MCP-1),macrophage inflammatory protein-1α(MIP-1α),tumor necrosis factor-α(TNF-α),interleukin(IL)-1β,IL-6 and IL-10.LPS activated TLR4 signal pathway and induced the expression of cyclin-dependent kinase(CDK)4,CDK6,Cyclin E,B-cell lymphoma-2(bcl-2),nuclear factor κB(NF-κB),p-p38,p-Jun amino-terminal kinase(JNK),p-extracellular signal related kinases(ERK)and p-protein kinase B(Akt)in glioma CD133+CSCs.The capacity of glioma CD 133+ CSC-reactive cytotoxic T lymphocyte to selectively kill glioma CD 133+ CSCs was reduced by LPS,and this effect was not apparent after TLR4 knockdown in glioma CD133+ CSCs.Memory effect T cells play roles in the immune evasion of glioma CD133+CSCs mediated by TLR4 signaling pathway.Conclusion:TLR4 is expressed in glioma CD133+ CSCs expression TLR4.LPS acitivated TLR4 signaling pathway,promoted the proliferation,drug resistance,molecular marker expression and cytokine secretion of glioma CD 133+ CSCs,and protected glioma CD133+ CSCs from glioma CD133+ CSCs reactive CTL induced cytolysis.Therefore,TLR4 signaling pathway is an important factor in the development and immune escape of glioma CD 133+ CSCs,and blocking TLR4 signaling pathway may provide a new therapeutic strategy for glioma patients.Part 2 The role and mechanism of B7-H6 in tumorigenesis and progression ofgliomaObjective:Although great progress has been made in treatment regimens,gliomas are still incurable and the 5-year survival remains poor.Studies focusing on molecules that regulate tumorigenesis or immunity may provide potential therapeutic strategies for patients with glioma.It is reported that,different from the normal organization,B7-H6 is selectively expressed in tumor cells and plays vital roles in tumorigenesis and tumor immunity.However,B7-H6 has been less studied in gliomas.Therefore,we investigate glioma cell lines and primary cells obtained from glioma patient tissues via molecular biological and immunological methods.We explored the expression level of B7-H6 in gliomas;and the expression of B7-H6 in glioma cells after stimulated with cytokines and inflammatory mediators,trying to elucidate the specific regulatory mechanisms of B7-H6 expression in gliomas.We analyzed the role and possible mechanism of B7-H6 in tumorigenesis and progression of glioma.We aimed to provide a new target for the effective treatment of glioma and a new approach for the design of new biological therapy strategies.Methods:The expression levels of B7-H6 in glioma cell lines,primary cells and tissue samples were detected by PCR and IHC.The expression levels of B7-H6 in glioma cells were detected by qRT-PCR and Western Blot after LPS stimulation at different time points.The expression level of B7-H6 in glioma cells was knocked down by small interfering RNA(siRNA).The proliferation of glioma cells was detected via CCK-8,clone formation assay and cell counting.The migration and invasion capacity of glioma cells were detected via transwell migration and invasion assay.The expression levels of related proteins in human glioma tumor cells were detected by Western Blot,and the signal transduction pathway and related protein molecules involved in the development and progression of glioma caused by B7-H6 were analyzed.Results:The PCR results demonstrated that B7-H6 was expressed in glioma cell lines and primary cells isolated from human glioma tissues.IHC detection showed that B7-H6 was expressed in glioma patient tissues.After stimulated with LPS at different time pointss,the expression of B7-H6 increased at mRNA and protein levels.siRNA was used to knockdown B7-H6 expression levels in glioma cells.After B7-H6 knockdown,the proliferation,migration and invasion capacity of glioma cells were inhibited,possibly via decreasing the expression of vimentin,N-cadherin,matrix metalloproteinase(MMP)2,MMP9 and survivin,and increasing the expression of E-cadherin and bcl-2 associated x protein(Bax).Conclusion:B7-H6 was expressed in glioma cell lines,primary cells and tissues of patients.LPS upregulated the expression levels of B7-H6 in glioma cells.B7-H6 played important roles in the tumorigenesis and progression of glioma.B7-H6 promote tumor proliferation,migration and invasion via regulating the expression of related proteins.Therefore,targeting B7-H6 may provide a novel therapeutic strategy for glioma patients.