| Objective: Glioma is the most common malignant tumor of the central nervous system.Due to its high degree of malignancy and rapid progression,the morbidity and mortality rates remain high.Especially for glioblastoma multiforme,the median survival time of patients is only about 14 months.At present,surgical resection combined with postoperative radiotherapy and chemotherapy is the main treatment method in clinical practice.Even with the emergence of new treatment modalities,the improvement of the prognosis of glioma patients is very limited.In recent years,more and more studies have found that the poor prognosis and resistance to treatment of glioma are closely related to the metabolic state of the tumor microenvironment.As an important component of the tumor microenvironment,macrophages participate in the shaping of the microenvironment and affect the occurrence,progression and prognosis of tumors.At the same time,under the stimulation of different conditions,the macrophages in the tumor microenvironment can be polarized to the M1 phenotype that suppresses the tumor or the M2 phenotype that promotes tumor progression,and the polarization direction of macrophages determines the tumor to a certain extent.ending.The metabolic state of macrophages with different phenotypes also showed differences in many stimulating factors.How to regulate the phenotype of macrophages in the tumor microenvironment through energy substance metabolism and transport has gradually become one of the research hotspots in the field of glioma treatment.In this study,we found that the solute transport protein SLC7A7 is related to the malignant phenotype and the metabolic characteristics of poor prognosis of glioma through analysis in the public database.It was confirmed by experiments that it is specifically expressed in macrophages and regulates macrophages in the glioma microenvironment.Polarization of cells involved in malignant progression of glioma.And elucidated the specific mechanism of SLC7A7 inducing the polarization of macrophages to the M2 phenotype by combining with ACSL1 to enhance the protein stability of the latter to promote fatty acid oxidation.Methods:(1)Glioma sequencing data: Bulk sequencing data and corresponding clinical information for bioinformatics analysis were obtained from the TCGA(The Cancer Genome Atlas)glioma database.Glioma single cell sequencing data were obtained from the GSM3828672 and GSM4119536 datasets in the GEO(Gene Expression Omnibus data base)database.(2)Clinical glioma tissue samples: All glioma tissue samples in this study were obtained during surgery at the First Hospital of China Medical University.Informed consent was obtained from patients and their families for all sample extraction and scientific applications,and the relevant experiments were approved by the Hospital Ethics Committee.(3)Cell lines and cell models: H4,LN229 human-derived glioma cells and THP1 human monocytic leukemia cells were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences;GSC21 cells were the primary glioma cells of patient origin constructed by the experimental team;tumor tissues were obtained from the First Affiliated Hospital of China Medical University during surgery,and the process was approved by the Hospital Ethics Committee..(4)Molecular biology experiments: Use RT-qPCR experiments and Western Blot experiments to verify the expression levels of different phenotype markers in macrophages to evaluate the polarization state of macrophages,and detect the m RNA and protein expression levels of target genes such as SLC7A7 and ACSL1;Flow cytometry analysis detects the expression of different phenotypic markers in macrophages to evaluate the polarization state of macrophages;Use protein co-immunoprecipitation experiments and liquid chromatography-mass spectrometry to find and verify the interaction between protein molecules;The proliferation ability of glioma cells after co-culture with macrophages treated with different conditions was assessed by cell proliferation assay(MTS);The migration ability of glioma cells after co-culture with macrophages treated with different conditions was assessed by cell migration assay(Transwell);And the oxidation level of fatty acids by macrophages was assessed by extracellular oxygen consumption rate assay(OCR).(5)An in vivo macrophage-glioma cell interaction model was constructed based on an intracranial in situ tumorigenesis model in nude mice to assess the effect of interfering with macrophage SLC7A7 expression on glioma progression in experimental animals.(6)Bioinformatics and statistical analysis: Rstudio,Graph Pad Prism,Image J,GSEA and other software were used to perform bioinformatics and statistical analysis on the data.Differences between data groups were verified by two-tailed t-test and one-way ANOVA and chi-square test,respectively;Pearson test was applied for correlation analysis.p-value less than 0.05 was considered as statistical difference.Results:Part Ⅰ: SLC7A7 is associated with malignant phenotype and poor prognosis of gliomaAfter reviewing the literature and analyzing and screening,we obtained a gene set closely related to the metabolism and transport of substances in the tumor microenvironment,and performed unsupervised clustering in the TCGA glioma database based on m RNA expression levels.In the results,it was found that a cluster(cluster I)tumors showed significant poor prognosis and enrichment of malignant phenotype.It shows that the malignant progression of glioma has a characteristic metabolic state.Through differential gene analysis,we found the SLC7A7 gene among the significantly upregulated genes in cluster I,and confirmed that the high expression of SLC7A7 was significantly correlated with the malignant phenotype of glioma,and was an independent risk factor for the poor prognosis of glioma.In the differential gene enrichment analysis based on the expression level of SLC7A7,we found that immune-related biological processes were significantly enriched in the SLC7A7 high-expression group,and the Estimate immune score analysis found that the immune score and matrix score were significantly higher in SLC7A7 high-expression tumors increased,while the tumor purity was significantly lower than other tumors.It shows that SLC7A7 may affect the malignant progression of glioma by participating in the formation of an inhibitory immune microenvironment during the progression of glioma.Next,we analyzed the glioma single-cell sequencing databases GSM3828672(Grade Ⅳ)and GSM4119536(Grade II)and found that the expression of SLC7A7 in the glioma microenvironment was highly consistent with the expression of macrophage markers CD14 and CD45.The results of immunofluorescence staining also suggested that SLC7A7 was co-localized with macrophage marker IBA-1.Then we confirmed by Western Blot that the expression of SLC7A7 in macrophages was significantly higher than that in glioma cells.Therefore,we believe that SLC7A7 is specifically expressed in macrophages in the glioma microenvironment,and has the potential to become an immunotherapy target related to macrophages.Part Ⅱ: SLC7A7 can regulate macrophage polarization and affect the prognosis of gliomaThrough the first part of the research,we found that the SLC7A7 molecule was specifically expressed in macrophages in the glioma microenvironment,and its high expression was significantly related to the malignant phenotype and immune infiltration of glioma,and it was a poor prognosis of glioma independent risk factors.In this phase of the study,we further explored the specific reasons why SLC7A7 affects the glioma microenvironment and produces poor prognosis.First,we performed GSEA analysis on immune-related biological processes based on the expression level of SLC7A7 in the TCGA glioma database.In the results,we found that biological processes such as chemotaxis,migration,activation and polarization of macrophages were significantly enriched in the SLC7A7 high expression group.Due to the important role of the polarization of tumor-associated macrophages in the formation of the tumor immune microenvironment,and its impact on the malignant progression and treatment resistance of glioma,we conducted further experiments in different phenotypes of macrophages and analysis.In the tumor-associated macrophage model established by using THP-1 cells,we found that the expression of SLC7A7 was significantly decreased in M1 macrophages,while the expression of SLC7A7 was significantly increased in M2 macrophages.In order to verify whether the differential expression of SLC7A7 in macrophages with different phenotypes can regulate the process of macrophage polarization,we constructed tumor-associated macrophage models with knockdown and overexpression of SLC7A7 by using lentivirus and plasmid respectively,and Verify the polarization.It was found that the expression of M1 phenotype markers was significantly increased in SLC7A7-knockdown macrophages,and the expression of M2 phenotype markers was significantly decreased.However,the opposite trend was observed in SLC7A7 overexpressed macrophages.The results of flow cytometry analysis showed that the proportion of M1 macrophages was significantly increased after knocking down SLC7A7,while the overexpression of SLC7A7 led to an increase in the proportion of M2 macrophages.Therefore,we believe that the expression of SLC7A7 can regulate the phenotypic polarization of macrophages.In order to test whether the regulatory effect of SLC7A7 on macrophage phenotype can affect the malignant progression of glioma,we first constructed a co-culture model of glioma cells and macrophages in vitro.It was found that the proliferation and migration of glioma cells co-cultured with SLC7A7-knockdown macrophages were significantly inhibited.However,the proliferation and migration ability of glioma cells co-cultured with SLC7A7-overexpressing macrophages was significantly increased.Finally,we verified it in the glioma-macrophage interaction model in vivo based on the nude mouse intracranial orthotopic tumorigenesis model.When SLC7A7 was knocked down in macrophages,tumor proliferation and survival of nude mice were significantly improved.It shows that SLC7A7 can affect the malignant progression of glioma by regulating the phenotype polarization of macrophages.Part Ⅲ: SLC7A7 binding to ACSL1 affects fatty acid oxidation in macrophagesIn the previous part of the study,we found that the expression of SLC7A7 can regulate the polarization process of macrophages in the glioma microenvironment.In this step,we deeply studied the specific mechanism of SLC7A7 regulation of macrophage polarization.In view of the specific expression of SLC7A7 in glioma-associated macrophages,we first screened the macrophage sequencing data in the glioma single-cell sequencing database GSM3828672(Grade Ⅳ)and GSM4119536(Grade II),and based on the The expression level of SLC7A7 in the cells was analyzed by GSEA,and it was found that the fatty acid oxidation process was significantly enriched in the SLC7A7 high expression group.Next,we conducted a correlation analysis between SLC7A7 and genes involved in fatty acid oxidation,and found that SLC7A7 was positively correlated with CPT2 and ACADM.We also found in the macrophage model that CPT2 and ACADM also showed a consistent trend of change after knockdown and overexpression of SLC7A7.To confirm the effect of SLC7A7 on fatty acid metabolism in macrophages,we evaluated the oxidation level of fatty acids by macrophages by measuring the extracellular oxygen consumption rate with fatty acids as substrate.The results showed that the level of oxygen consumption of macrophages after knocking down SLC7A7 was similar to that of adding fatty acid oxidation inhibitors,indicating that knocking down SLC7A7 could significantly inhibit the process of mitochondrial fatty acid oxidation in macrophages.In order to explore the molecular mechanism of SLC7A7 affecting fatty acid oxidation in macrophages,we examined the molecules that interact with SLC7A7 in the glioma microenvironment by co-immunoprecipitation and mass spectrometry.In the results we found the ACSL1 molecule,whose role is mainly to catalyze long-chain fatty acids into their active forms to enter mitochondria for oxidative breakdown.We confirmed the significant association of SLC7A7 with ACSL1 in the glioma database,and verified the reciprocal binding of SLC7A7 and ACSL1 by co-immunoprecipitation.Protein stability experiments confirmed that the combination of SLC7A7 and ACSL1 can enhance the protein stability of the latter in the glioma microenvironment.In order to verify the effect of ACSL1 on fatty acid oxidation in macrophages,we first constructed a macrophage model with knockdown of ACSL1,and confirmed that the fatty acid oxidation process of macrophages was significantly inhibited after knockdown of ACSL1 by measuring the extracellular oxygen consumption rate.At the same time,we also verified the phenotype of macrophages knocking down ACSL1,and found that macrophages showed a significant M1 phenotype polarization trend after knocking down ACSL1.Finally,we knocked down ACSL1 in a SLC7A7-overexpressing macrophage model and examined the phenotype.It was found that the M2 phenotype polarization of macrophages caused by the overexpression of SLC7A7 was restored by knocking down ACSL1,the expression of markers in M1 macrophages was significantly increased,and the expression of markers in M2 macrophages was significantly decreased.The results of flow cytometry also showed that after ACSL1 was knocked down in SLC7A7-overexpressing macrophages,the polarization trend of M2 phenotype was significantly inhibited.It shows that SLC7A7 regulates the polarization of macrophages by combining with ACSL1.Conclusions:Part Ⅰ: The characteristic substance metabolism and transport state in the glioma microenvironment are related to the malignant phenotype and poor prognosis of glioma;the high expression level of SLC7A7 has predictive value for the malignant phenotype and poor prognosis of glioma;It is specifically expressed in macrophages in the tumor microenvironment.Part Ⅱ: Differential expression of SLC7A7 among different phenotypes of macrophages;SLC7A7 expression level can regulate the phenotypic polarization of macrophages;Knockdown of SLC7A7 can induce the polarization of macrophages to M1 phenotype and inhibit the malignancy of glioma progress.Part Ⅲ: SLC7A7 affects macrophage fatty acid metabolism through ACSL1;ACSL1enhances its protein stability after combining with SLC7A7;knockdown of ACSL1 can inhibit the polarization process of macrophage M2 phenotype induced by overexpression of SLC7A7. |
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