| BackgroundGlioma is the most common central nervous system tumor that originates from brain glia,accounting for about 32%of primary intracranial tumors and 81%of central nervous system malignancies.The incidence of glioma is 6.21/100,000 and the mortality rate is 3.15/100,000.With the advancement of molecular biology,the 2016 edition of the WHO(World Health Organization)central nervous system tumor classification uses IDH(Isocitratedehydrogenase)mutations and 1p/19q co-deletion and other molecular typing the differences in prognosis of gliomas.However,because primary glioblastomas which accounts for more than half of primary brain malignancies rarely show the above two beneficial molecular changes,primary glioblastomas has a poor prognosis.Even with the most aggressive treatment methods,including expanded resection,radiation therapy,and chemotherapy,the median survival of the patient is still less than 15 months.Since almost all glioblastoma patients will relapse,it is urgent to find more effective prognostic factors and therapeutic targets to prevent and treat this malignant tumor.In the past two decades,with the advancement of research on new immune checkpoints,many different immune checkpoint families have been discovered.Among them,the B7 family is one of the important immune checkpoint families.The B7 family consists of structurally related cell surface proteins that regulate the immune response by ligands transmitting co-stimulatory or co-suppressive signals.The B7 family consists of eight family members,including B7-H4,B7-H6 and CD276(B7-H3)etc.Some members of the B7 family,such as CD274,were approved for immunotherapy of non-small cell lung cancer,urothelial cancer,and triple-negative breast cancer in 2016,2017,and 2019,respectively.Some neuro-oncologists have tried to use the CD274 gene blocking strategy to treat recurrence of glioblastoma,but the effect is not ideal.So other members of the B 7 family came into our sight.CD276(B7-H3)is one of the most conservative B7 family members in evolution.CD276,as a costimulatory/cosuppressive molecule,was first reported by Chapoval et al in 2001.CD276 is located on human chromosome 15 and has a co-stimulatory effect on CD4+and CD8+cells,inducing cellular immunity.CD276 encodes a type I transmembrane glycoprotein composed of 316 amino acids with a molecular weight of 45-66 kDa.The protein includes an intracellular structure,a transmembrane region and an extracellular structure,and its extracellular domain includes two immunoglobulin constants(IgC)and a variable domain(IgV).CD276 mRNA is widely expressed in many important organs.Although its mRNA is widely expressed,its post-transcriptional expression is strictly regulated.The protein expression is low in a variety of normal tissues,but it is widely overexpressed in many human tumors.The overexpression of CD276 in tumors suggests an important role in tumor progression.A large amount of evidence shows that CD276 is involved in biological processes such as tumor proliferation,migration,and invasion.Many researchers have confirmed that CD276 expression is closely related with tumor grade and negatively correlated with tumor prognosis.However,some reaserches showed that CD276 is the good prognosis of some tumors.However,the role of CD276 in glioma-related biological properties is not fully understood.With the advancement of molecular biology and bioinformatics,genome sequencing and bioinformatics technology has provided new ideas for cancer genome research.In particular,the application of data in the TCGA(The Cancer Genome Atlas)and CGGA(the Chinese Glioma Genome Atlas)databases provides new insights into the prognosis and treatment of gliomas.By analyzing the clinical information related to CD276 in the TCGA database and the CGGA database and enriching its related proteins,it is possible to speculate its possible molecular biological function.The purpose of this study is to explore the biological role of CD276 in glioma invasion and clinical prognosis and its possible molecular mechanism using information data science and related experiments in vivo and in vitro.Chapter One:TCGA and CGGA test CD276 can be used as independent prognostic molecules of gliomaObjective:Use TCGA and CGGA databases to verify whether CD276 can be used as an independent prognostic factor in gliomas,and use enrichment analysis to infer the possible related factors of CD276.Method:1.Download all mRNAs and clinical data of gliomas and normal brain tissue from the TCGA public database,screen out and verify differentially expressed gene CD276,and draw survival curve.2.Download all mRNA and clinical data of glioma from the CGGA public database,screen out and verify differentially expressed gene CD276,and draw survival curve.3.CD276 gene was analyzed by univariate and multivariate prognostic analysis in CGGA database,and draw ROC curve.4.Clinical correlation analysis of CD276 gene-related clinical information in CGGA database.5.Enrichment analysis of related genes of CD276 gene in TCGA and CGGA database.Results:1.In TCGA database CD276 differentially expressed in brain tissue and glioma tissue,logFC>2,FDR value<0.052.In CGGA database the prognosis of glioma with high expression of CD276 is poor(P<0.05).3.In CGGA database univariate analysis of CD276 gene P<0.001,HR>1.The results show that CD276 gene is related to survival and is a high risk factor for survival.4.In CGGA database multivariate analysis of CD276 gene P<0.001,HR value>1.The results show that CD276 gene is related to survival and is a high risk factor for survival.5.Area under the ROC curve of the CD276 gene in the CGGA database,the one-year AUC of the CD276 gene is 0.721,the three-year AUC is 0.773,and the five-year AUC is 0.77,it is suggested that CD276 has high reliability as a prognostic factor for gliomas.6.In CGGA database CD276 is positively correlated with glioma grade,age,Chemo-status,PRS-type(P<0.001),and negatively correlated with IDH mutation and 1p/19q co-deletion(P<0.001).7.The enrichment results of CD276 in the CGGA database show:CD276 is positively correlated with Collagen cataboli process,collagen containing extracellular matrix,P53-mediated DNA damage response,extracellular matrix(extracellular matrix),extracellular matrix disassembly,G1 DNA damage checkpoint(G1 DNA damage checkpoint)related proteins;CD276 is negatively correlated Glutamate receptor activity(Glutamate receptor signaling),Glutamate receptor signaling Pathway(glutamate receptor signaling pathway)related proteins.8.TCGA database enrichment results show that CD276-related proteins are in cell adhesion molecule binding,actin binding,cadherin binding,cadherin binding,carbohydrate binding,cytokine receptor binding(cytokine receptor binding)and other functional enrichment.Conclusions:1.CGGA and TCGA databases have verified that CD276 can be used as an independent prognostic molecule for gliomas:CD276 expression is negatively correlated with glioma survival rate;CD276 is positively correlated with glioma grade;CD276 is negatively associated with IDH mutations and 1p/19q co-deletion Related.2.CD27 6 enrichment results showed that it was positively related to protein molecules such as extracellular matrix,collagen breakdown,and cell adhesion molecules,suggesting that it was closely related to tumor aggressiveness.Chapter Two:The expression of CD276 in glioma tissue and its clinical relationship with glioma biological characteristicsObjective:To further evaluate the clinical significance of CD276 expression in gliomas and its relationship with clinical features such as living situation,the benign prognostic factor IDH 1 mutation and 1p/19q co-deletion,the P53 mutation,the Ki-67 proliferation.And study the correlation between CD276 and MMP9,to find the CD276 influence the invasion of glioma by MMP9 whether or not.Methods:1.147 cases of glioma wax blocks from the Fifth Affiliated Hospital and Second Affiliated Hospital of Zhengzhou University were collected,and the expression of CD276,IDH1,MMP9,P53,Ki-67 in wax blocks was detected by immunohistochemistry.2.1p/19q fluorescence in situ hybridization detection of specimens3.SPSS21 software was used for statistical method.The correlation between CD276 expression and glioma classification,IDH1 mutation,1p/19q co-deletion,MMP9 expression,P53 mutation and Ki-67 proliferation index were analyzed by Spearman correlation analysis.P<0.05 is statistically significant.Results:1.The higher the glioma grade,the higher the CD276 expression rate,and the expression of CD276 in glioma was positively correlated with the glioma grade(P<0.001).2.The expression of CD276 in glioma was negatively correlated with IDH 1 mutation(P<0.001).3.The expression of CD276 in glioma was negatively correlated with 1p/19qco-deletion(P<0.01).4.The expression of CD276 in gliomas was positively correlated with P53 mutations(P<0.001).5.CD276 expression in gliomas was positively correlated with Ki-67 proliferation index(P<0.001).6.The expression of CD276 in gliomas was positively correlated with the expression of MMP9(P<0.001).7.CD276 expression correlates with poor prognosis of glioma(P<0.001).Conclusions:1.The expression of CD276 in gliomas was positively correlated with poor prognostic factors such as tumor grade,P53 mutation,and Ki-67 proliferation index;it is negatively correlated with good prognostic factors such as IDH1 mutation and 1p/19q co-deletion.It is suggested that CD276 is a poor prognostic factor for gliomas.2.The expression of CD276 in gliomas was positively correlated with MMP9,suggesting that its poor prognosis is related to tumor aggressiveness.Chapter Three:Biological effects of CD276RNAi on U87 glioma cellsObjective:Detect CD276mRNA in an in vitro cell tumor cell line,and pick the right cell line,interfere its CD276mRNA.Observe the effects of glioblastoma cell line after CD276RNAi on cell invasion,cell cycle,etc.,and the effect on the expression ofβ-catenin,TNFR1,MMP9,explore the possible mechanism of CD276 on the aggressiveness of glioma.Method:1.CD276 gene was explored in U87,U251,U118 cell lines,and appropriate cell lines were selected for subsequent experiments.2.The best MOI value of lentivirus infected U87 cells was explored and the lethal curve of antibiotic BSD was explored.3.Construction of Lentiviral Expression Vector Interfered by CD276 Gene.4.CD276 gene interfered with lentiviral packaging,U87-CD276-KD and U87-NC tumor cells were cultured in stable transformants.5.CCK8 detect the cell proliferation 1～5 days curves of U87-CD276-KD tumor cells and U87-NC tumor cells.6.Flow cytometric detect the changes in cell cycle levels of U87-CD276-KD tumor cells and U87-NC tumor cell.7.Transwell migration and invasion detection of U87-CD276-KD tumor cells and U87-NC tumor cells.8.RT-PCR was used to detect changes in mRNA levels of CD276,β-catenin,TNFR1,and MMP9 in U87-CD276-KD tumor cells and U87-NC tumor cells.9.Western-blotting was used to detect changes in the protein expression levels of CD276,β-catenin,TNFR1,and MMP9 in U87-CD276-KD tumor cells and U87-NC.10.The protein interacting with CD276 in U87 cells was enriched by immunoprecipitation,and Western-blotting was used to detect whether β-catenin,TNFR1,MMP9 protein combined with CD276 protein.11.SPSS21 software was used for statistical methods.The t-test was used for gene expression intensity,survival time,and tumor size.The statistical results were statistically significant at P<0.05.Results:1.U87 cell line CD276 mRNA △ CT value is between 2-13,which can be used for subsequent interference experiments.2.U87-CD276-KD tumor cells and U87-NC tumor stable transfectants were successfully constructed.3.Compared with U87-NC tumor cells,U87-CD276-KD tumor cells detected by CCK8 showed weaker cell proliferation ability(P<0.05),4.Compared with U87-NC tumor cells,U87-CD276-KD tumor cells showed no obvious arrest of cell cycle detected by flow cytometry(P>0.05).5.Transwell test results showed that U87-CD276-KD tumor cells had less migration than U87-NC tumor cells(P<0.05).6.Transwell test results showed that U87-CD276-KD tumor cells were less aggressive than U87-NC tumor cells(P<0.05).7.RT-PCR showed that compared with U87-NC tumor cells,U87-CD276-KD tumor cells had reduced mRNA of CD276,β-catenin,TNFR1 and MMP9(P<0.01).8.Western-blotting test showed that compared with U87-NC tumor cells,U87-CD276-KD tumor cells had reduced expression of CD276,β-catenin,TNFR1 and MMP9 proteins(P<0.01).9.After CD276 protein immunoprecipitation enrichment,western-bloting detection showed that β-catenin and MMP9 protein bands were positive,and TNFR1 protein bands were negative.Conclusions:1.Down-regulating the expression of CD276 in U87 cells can inhibit cell proliferation.2.Down-regulated the expression of CD276 in U87 cells,which basically did not affect the cell cycle,and there was no cell cycle arrest.3.Down-regulating the expression of CD276 in U87 cells can inhibit cell migration.4.Down-regulating the expression of CD276 in U87 cells can inhibit cell invasion.5.Interfering with the expression of CD276 can inhibit the expression ofβ-catenin,TNFR1 and MMP9 in U87 cells at the mRNA and protein levels.It is suggested that CD276 may affect MMP9 through β-catenin and TNFR1 signaling pathways to affect the biological functions of glioma invasion.6.The immunoprecipitation enrichment showed that CD276 in U87 cells combined with β-catenin and MMP9.Chapter Four:Effects of CD276RNAi on intracranial orthotopic implant tumors in nude miceObjective:To study the effect of U87 cell line CD276RNAi on tumor formation in situ in nude mice,compare the survival time of nude mice and the change of tumor maximum diameter after CD276 silencing,and compare the effects of β-catenin and MMP9 expression to further verify the effect of CD276 on the tumor in vivo The biological function of the tumor.Method:1.U87-CD276-KD stably transfected tumor cells and empty virus control U87-NC tumor cells were implanted in the intracranial of nude mice,and observe the effects of CD276RNAi on the biological behavior of gliomas in vivo.2.The performance of U87-CD276-KD group and U87-NC group was observed and the survival time was recorded.The size of the tumor was observed with the naked eye,and the brain tissue was fixed and embedded with HE staining.The maximum diameter of the tumor was measured gross and by the microscope.3.Detect the expression of CD276 in U87-CD276-KD group and U87-NC group by immunohistochemistry.4.RT-PCR was used to detect the expression of CD276,β-catenin,TNFR1,MMP9 mRNA in tumors of nude mice in U87-CD276-KD group and U87-NC group.5.Western-blotting test was used to detect the expression of CD276,β-catenin,TNFR1 and MMP9 protein in tumors of mice in U87-CD276-KD group and U87-NC group.6.SPSS21 software was used for statistical methods.The t-test was used for gene expression intensity,survival time,and tumor size.The statistical results were statistically significant at P<0.05.Results:1.Compared with the U87-NC group with the empty virus control group,the survival time of nude mice in the U87-CD276-KD stable transfer group was significantly increased(P<0.05).2.Compared with the U87-NC group with the empty virus control group,the expression of CD276 in the U87-CD276-KD stable-transition group was reduced(P<0.05),and the tumor diameter was significantly smaller than the control group(P<0.05).3.The results of immunohistochemistry showed that the expression of CD276 in the U87-CD276-KD stable transfer group was less than the U87-NC group.(P<0.05).4.RT-PCR results showed that compared with U87-NC group,CD276 had a lower mRNA level in U87-CD276-KD group,and the mRNA levels of β-catenin,TNFR1 and MMP9 also decreased.The difference was statistically significant(P<0.01).5.Western-blotting results showed that compared with U87-NC group,CD276 protein level was reduced in U87-CD276-KD group,and the protein levels ofβ-catenin,TNFR1 and MMP9 were also decreased.The difference was statistically significant(P<0.05).Conclusions:1.In vitro experiments confirmed that CD276 RNAi glioma cell line reduced tumor formation in nude mice brains,weakened tumor invasiveness,and prolonged survival time.2.In vivo experiments,the expression of β-catenin and MMP9 decreased after CD276 siRNA,suggesting that CD276 affects the invasion of microglioma and other biological functions through β-catenin/MMP9.Conclusions1.Bioinformatics confirms that CD276 is an independent prognostic factor of glioma,and its expression is positively correlated with the grade of glioma,and its high expression is closely related to the poor prognosis of patients,suggesting that CD276 plays a role in promoting the occurrence and development of glioma;Enrichment analysis shows that CD276 is closely related to the biological behavior of gliomas such as tumor aggressiveness.2.Histological verification showed that CD276 expression was positively correlated with histological grade,negatively correlated with IDH1 mutation and 1p/19q co-deletion,positively correlated with P53 mutation,Ki67 proliferation index,and MMP9 expression,and negatively correlated with survival.3.After silencing the glioma U87 cell CD276,the proliferation,migration and invasiveness of the tumor were weakened,and the tumorigenesis in nude mice was weakened,the tumor diameter was reduced,and the survival time of experimental animals was prolonged.4.In vivo and in vitro experiments confirmed that silencing CD276 can decrease the expression of β-catenin,TNFR1,and MMP9.5.IP enrichment shows that CD276 binds to β-catenin and MMP9 in U87 cells,suggesting that CD276 may form protein complexes with β-catenin and MMP9 or affect the invasion of microgliomas and other biological functions through β-catenin signaling pathway.6.Based on bioinformatics,histology,in vitro experiments and in vivo experiments,it has been confirmed that the high expression of CD276 can be used as a clinical indicator of poor prognosis of glioma.CD276RNAi can reduce the proliferation,migration and invasion of glioma cells,suggesting that CD276 has the potential to become a therapeutic target for glioma. |
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