The Mechanism Of Pyroptosis-related Gene CASP6 In Glioma Progression Based On Bioinformatics

Glioma is the most common tumor in the Central Nervous System(CNS),with high mortality and poor treatment outcomes.The World Health Organization(WHO)classifies it into four histopathological grades(WHO grades I-IV)according to the degree of progression.Grades I and II are defined as low grade gliomas(LGG),while grades III and IV were defined as high grade gliomas.In general,surgical resection followed by radiotherapy and chemotherapy is the main treatment strategy for glioma patients.The Overall Survival(OS)of glioma patients is short due to the strong invasiveness,high recurrence rate,and resistance to radiotherapy and chemotherapy of glioma.Although some progress has been made in glioma research in recent years,no breakthrough has been achieved in improving the prognosis of glioma patients.Histopathological results are important references for the comprehensive treatment of glioma.With the deepening of the research on glioma,the guiding significance of molecular typing in the treatment process has gradually attracted attention.It is an important direction in glioma research to find the genes related to the survival of glioma patients and to study whether they can be used as potential targets for treatment.The development of bioinformatics has greatly promoted the progress of life science.And its contributions in the field of medical research are also increasing.With its help,we can accurately and effectively find and analyze the potential glioma-related genes,understand the occurrence,development,and biological characteristics of glioma from the gene level,find new molecular classification and therapeutic targets,and improve the accuracy of clinical diagnosis and treatment of glioma.Pyroptosis is a form of programmed cell death marked by cell swelling,cell lysis,and the release of proinflammatory factors.Recent studies have shown that pyroptosis plays a crucial role in inhibiting tumor cell proliferation and tumor growth,such as colon cancer,non-small cell lung cancer,and hepatocellular carcinoma.For gliomas,many small molecules have been proved to exert anti-tumor effects through pyroptosis.Therefore,it is significant to investigate the role of pyroptosis-related differentially expressed genes(PRDEGs)in glioma progression.We screened the differentially expressed PRDEGs in glioma from different databases by bioinformatics analysis.Among the PRDEGs,Caspase-6(CASP6)was the only gene that was overexpressed in all databases.This study aims to explore the relationship between CASP6 and the prognosis and tumor immune microenvironment of glioma patients,as well as its role and mechanism in the progression of glioma,so as to provide relevant theoretical basis for the early diagnosis and precise individualized treatment of gliomaPart One Bioinformatics analysis of pyroptosis-related geneCASP6 in gliomaObjective: To investigate the value of CASP6 as a biomarker for predicting the prognosis of glioma patients.Functional enrichment analysis of CASP6 was performed.To evaluate the relationship between the expression of CASP6 in glioma and the sensitivity of chemotherapy and immunotherapy,as well as the correlation with tumor immune infiltration.Methods:1.The Cancer Genome Atlas(TCGA),Genotype-Tissue Expression(GTEx),Gene Expression Omnibus(GEO)and Rembrandt database were used to screen the overexpressed PRDEGs in gliomas.2.The genomic and clinical information of glioma patients in the Chinese Glioma Genome Atlas(CGGA),TCGA and REMBRANDT databases were used to evaluate the value of CASP6 as a biomarker in predicting the prognosis of glioma patients.3.The Gene Ontology(GO),Kyoto Encyclopedia of Genes and Genomes(KEGG)and Gene set enrichment analysis(GSEA)were performed to analyze the related functional enrichment of CASP6.4.CGGA database and Genomics of Drug Sensitivity in Cancer 2(GDSC2)database were used to analyze the correlation between CASP6 expression and the chemotherapy sensitivity in glioma.The Tumor Immune Dysfunction and Exclusion(TIDE)algorithm in Python was used to evaluate the sensitivity of glioma patients with different CASP6 expression levels to immune checkpoint blockade(ICB)blockers.5.Estimation of STromal and Immune cells in Malignant Tumors using Expression data(ESTIMATE),CIBERSORT,single sample gene set enrichment analysis(ss GSEA)and Tumor Immune Estimation Resource(TIMER)algorithm were used to calculate the immune infiltration of glioma tissues in the high and low expression groups of CASP6.Results:1.In the GSE4290 glioma dataset of the GEO database,a total of 11pyroptosis-related differentially expressed genes(PRDEGs)were identified,of which 10 were overexpressed and 1 was under-expressed.In the TCGA-GTEx dataset,57 over-expressed PRDEGs and 1 low-expressed PRDEGs were screened.11 PRDEGs were identified in the REMBRANDT dataset,of which3 were overexpressed and 8 were lowly expressed.By analyzing the PRDEGs in these datasets,we found that CASP6 was the only PRDEG that was coupregulated in all three datasets,while there were no co-downregulated PRDEG.2.We stratified glioma samples in the CGGA database based on the median expression level of CASP6.Kaplan-Meier survival curve showed that patients with lower levels of CASP6 had longer OS(P< 0.0001).ROC curve was used to evaluate the accuracy of CASP6 expression in predicting the 3-year and 5-year OS of glioma patients.The AUC values of 3-year and 5-year OS were 0.733 and 0.759,respectively.Data analysis of TCGA and REMBRANDT cohorts also showed that glioma patients with lower CASP6 expression had longer overall survival(OS)consistently(P<0.0001).Univariate and multivariate Cox analysis showed that CASP6 was a prognostic factor of glioma patients.The CGGA dataset was stratified by age,sex,chemotherapy,radiotherapy,WHO grade,IDH mutation,1p19 q co-deletion,and MGMT methylation status.Stratified survival analysis confirmed that patients in the low CASP6 expression group had longer survival in each subgroup.3.GO analysis showed that CASP6 was mainly involved in processes including “activation of immune response”,“adaptive immune response”,“aging”,“ameboidal cell migration”,and “antigen processing and presentation”.Furthermore,the annotations of the KEGG pathway revealed an enrichment of CASP6 in pathways including “antigen processing and presentation”,“cell cycle”,“complement and coagulation cascades”,“receptor interaction”,and“focal adhesion”.GSEA analysis showed that higher expression of CASP6 was associated with hallmarks of tumorigenesis including “apoptosis”,“allograft rejection”,“coagulation”,“complement”,and “E2F targets” correlated markedly.4.Notably,TMZ presented a better therapeutic response in patients with glioma with high CASP6 expression.Meanwhile,the TIDE results further predicted that patients with high expression of CASP6 would achieve a poorer response to immunotherapy than those with low CASP6 expression.5.In both CGGA and TCGA datasets,glioma sample group with increased levels of CASP6 expression exhibited a higher immune score than the group with decreased CASP6 expression.The proportion of naive T cells,activated NK cells,and M0 macrophages significantly decreased in glioma samples with higher levels of CASP6 expression,while the proportion of gamma delta T cells,monocytes,M2 macrophages,activated dendritic cells,and neutrophils increased significantly in this group compared to glioma samples with lower CASP6 expression levels.Conclusions:1.In the results of bioinformatics analysis,the pyroptosis-related gene CASP6 was overexpressed in glioma tissues compared with normal brain tissues.2.CASP6 is associated with the prognosis of glioma patients and is a potential biomarker for predicting the prognosis of glioma patients.3.CASP6 is involved in a variety of biological processes and plays a certain role in the occurrence and development of tumors.4.CASP6 expression can predict the sensitivity of glioma patients to TMZ and immune checkpoint blockers.Glioma patients with high CASP6 expression are more sensitive to TMZ treatment,while glioma patients with low expression of CASP6 are more sensitive to immunotherapy.5.CASP6 is related to the tumor immune microenvironment of glioma,and the expression level of CASP6 can affect the changes of immune cell components in the glioma microenvironment.Part Two Verification of CASP6 expression in glioma and itscorrelation with clinical survival timeObjective: To investigate the expression of CASP6 in glioma,and to explore the correlation between CASP6 expression and the clinical survival time of glioma patients.Methods.1.Fresh glioma tissue samples and inactivated brain tissue samples from non-functional areas resected by decompression due to trauma were collected from glioma patients and brain injury patients who were hospitalized and treated in the Department of Neurosurgery of the Second Affiliated Hospital of Hebei Medical University from December 2020 to June 2021.Immunohistochemistry,RT-PCR,and Western Blot were used to detect the expression of CASP6 in different grades of glioma.2.RT-PCR and Western Blot were used to detect the expression level of CASP6 in human astrocytes(HA)and different glioma cell lines.3.The clinical information and pathological tissue sections of 104 glioma patients who underwent surgical treatment at Department of Neurosurgery,the Second Affiliated Hospital of Hebei Medical University from June 2013 to June2017 were collected.The postoperative pathological diagnosis of all glioma patients was confirmed.Kaplan-Meier curve was used to analyze the correlation between the expression level of CASP6 in glioma and the survival time of patients.Results:1.The expression levels of CASP6 m RNA and protein in glioma tissues were higher than those in control brain tissues(P < 0.05),and the expression levels of CASP6 m RNA and protein increased with the increase of glioma grade(P < 0.05).2.The m RNA and protein expressions of CASP6 in glioma cell lines T98 G,U138,and U251 were higher than those in control human astrocytes(P < 0.05).3.Kaplan-Meier survival curves showed that patients with low CASP6 expression had longer overall survival(OS)compared with patients with high CASP6 expression.Conclusion:1.According to the results of our clinical glioma samples,the m RNA and protein expression levels of CASP6 in glioma tissues were higher than those in control brain tissues.The expression level of CASP6 was positively correlated with tumor grade.This result is consistent with the results of bioinformatics analysis in Part One.2.The m RNA and protein expression levels of CASP6 in glioma cell lines were also higher than those in control human astrocytes.3.The expression level of CASP6 is closely related to the survival time of glioma patients,and it is a risk factor for the survival of glioma patients.The overall survival of the CASP6 low expression group was significantly longer than that of the high expression group.CASP6 is a potential target for individualized precision treatment of glioma.Part Three CASP6 regulates the biological behavior of gliomacells through pyroptosis pathwayObjective: To investigate the effect of CASP6 on the biological behavior of glioma and its preliminary regulatory mechanism.Methods:1.Immunofluorescence was used to detect the subcellular localization of CASP6.2.Small interfering RNA(si RNA)was used to knock down CASP6 in glioma cell lines,and the sequence with the highest knockdown efficiency was selected to prepare lentivirus,and CASP6 low expression stably transfected glioma cell line was constructed by lentivirus.3.The stable transfected T98 G,U138,and U251 glioma cell lines were used for CCK-8 cell proliferation assay to detect the effect of CASP6 on glioma cell proliferation.A plate cloning assay was also performed to detect the effect of CASP6 on the proliferation of glioma cells.4.Cell scratching assay and Transwell assay were used to detect the effect of CASP6 on the migration and invasion of glioma cells using stable transfected T98 G,U138 and U251 glioma cell lines.5.TUNEL assay and apoptosis assay were used to detect the apoptosis of T98 G,U138 and U251 glioma cells after stable transfection,and the effect of CASP6 on apoptosis of glioma cells was detected.6.To detect the protein changes in the pyroptosis pathway of T98 G,U138,and U251 glioma cell lines after stable transfection and explore the role of CASP6 in the pyroptosis pathway of glioma cells.7.Subcutaneous tumor formation experiment was performed in nude mice to observe the changes in the tumorigenic ability of U251 glioma cells after knocking down CASP6 in nude miceResults:1.CASP6 is localized in the cytoplasm.2.The expression of CASP6 in glioma cell lines can be stably knocked down after lentivirus transfection.3.In T98 G,U138 and U251 glioma cell lines,compared with the knockdown control group(sh NC),the cell proliferation ability of glioma cells in the knockdown group(sh CASP6)was significantly decreased,and the difference was statistically significant(P < 0.05).4.In T98 G,U138 and U251 glioma cell lines,compared with the knockdown control group(sh NC),the cell migration and invasion ability of glioma cells in the knockdown group(sh CASP6)was significantly decreased,and the difference was statistically significant(P < 0.05).5.In T98 G,U138 and U251 glioma cell lines,the incidence of apoptosis in the CASP6 knockdown group(sh CASP6)was significantly increased compared with the knockdown control group(sh NC),and the difference was statistically significant(P < 0.05).6.In T98 G,U138 and U251 glioma cell lines,after knocking down CASP6,the pyroptosis-related gene NLRP3 was significantly increased(P < 0.05),and GSDMD was significantly increased(P < 0.05).CASP6 can regulate the key genes in the pyroptosis pathway.7.Animal experiments showed that knockdown of CASP6 significantly inhibited the tumorigenic ability of U251 glioma cells in nude mice,and the difference was statistically significant(P < 0.01).Conclusion:1.Knockdown of CASP6 can inhibit the proliferation,migration,and invasion of T98 G,U138 and U251 glioma cells,and increase the incidence of apoptosis.Knockdown of CASP6 can inhibit the tumorigenic ability of U251 glioma cells in vivo.CASP6 may play an oncogene role in the occurrence and development of glioma.2.CASP6 may affect the progression of glioma through the pyroptosis pathway.