| PART I.The effect of hypoxia on the gene expression and biological metabolism in glioblastoma cells and its biological significanceAs the most common primary tumors in brain, malignant gliomas showed characters of short lifetime, easy relapse and difficult treatment. In recent years a large number of cellular, animal experiments and clinical studies have revealed abundant findings for the molecular mechanism of occurrence and development of malignant gliomas, and on this basis the therapy of surgery combined with radiotherapy, chemotherapy, gene therapy, targeted therapy, immunotherapy, etc. comprehensive treatment method was widely developed and applied in patients with malignant glioma. However, the mean survival period of gliomas still showed less than 1 year, and most of them showed strong resistance to radiotherapy and chemotherapy. Currently, the key problem for glioma is lack ofspecific markers for early diagnosis and therapeutic targets for intervention. Therefore, investigation diagnosis biomarkers and therapeutic targets for glioblastoma will greatly improve the prognosis for patients and bring out widespread clinical values. In recent years, the development of high-throughput sequencing technology provided further technical support and opportunity to reveal the molecular mechanism of carcinogenesis and the discovery of specific biomarkers and therapeutic targets for tumor. And these methods have been widely applied during cancer research. Furthermore, besides tumor self, the microenvironment, such as hypoxia, was closely associated with the property of unlimited proliferation, invasion, resistance and high recurrence and so on. However, the effect and biological significance of hypoxia on gene transcription and substance metabolism of glioblastoma remains to be illustrated. Thus, in this study, with trancriptome and metabolome methods, we evaluated the transcriptive and metabolic signature of glioblastoma in normoxia and hypoxia and revealed activated processes and changed signal pathways and genes and metabolites of glioblastoma by bioinformatics. Furthermore, with checking in CCLE database and validation in clinical tissues and serum samples, this study not only provides new findings for clarifying the initiation and development mechanismof malignant gliomas, but also brings out experimental basis for discovering biomarkers and targets for glioblastoma diagnosis and therapy, more importantly, provides opportunity for individualized treatment for patients with malignant glioma. Therefore, this work showed important theory significance and potential applicative value for glioblastoma therapy.MethodsWe have utilized human brain HEB cell line as non-neoplastic brain cell and U87-MG cell line as a human GBM cell model cultured in 21% and 1% O2 for RNA sequencing. After the filtering of raw reads with standard procedure, we mapped clean reads to reference genome(hg19). Differentially expressed genes were screened by Nioseq package of R software, and the |Fold Change| ≥2 and Probability ≥ 0.8 were chosen as cut-off criterion. According to the gene expression values in U87-MG cells, the differentially up-regulated genes were divided into high, intermediate and low three categories by>=1000, >=10 and <1000, <10. The genes in high category were chosen as particular expressed genes in U87-MG cells and its biological function was analyzed with protein-protein interaction databases Int Act, MINT, Matrix DB, DIP, I2 D and Innate DB. Then the plasma concentration of inflammatory response related genes IL1 B, CSF3 and TIMP1 belonging to high category were detected in GBM patients with ELISA assay. GO and KEGG pathway enrichment analysis were performed for target genes using Enrichr and REVIGO. Moreover, we explored the expression of genes in glioma cell lines which related to Toll-like receptor pathway and particular expressed in U87-MG cells with CCLE database.Based on the gene expression in HEB and U87-MG cells cultured in normoxia, we screened the differentially expressed genes of HEB and U87-MG cells in hypoxia. Combining the gene expression profiling of various glioma cell lines in CCLE database, we found the hypoxia sensitive genes consecutively expressed in U87-MG cells. And we detected the expression of protein STC1, HMOX1 and TGFB1 with western blotting in cells and IHC in GBM patients.For the study of metabolomics, HEB and U87-MG cells culturedin 21% and 1% O2 environment for 24 h respectively were collected. On the basis of the quality control, the chromatographic data were obtained by the liquid chromatography-mass spectrometry analysis. After extraction chromatographic peak by R software, following by standardization with internal reference and cell number, we processed multivariate statistical data analysis(with SIMCA-P12.0), and univariate statistical analysis(with SPSS19.0), to seek the different metabolites of between U87-MG and HEB cells and between U87-MG cells or HEB cells in normoxia and hypoxia. After validation in HMDB and METLIN database, we enriched the different metabolic pathway with Metabo Analyst 3.0 and made the metabolic schematic graft with the KEGG metabolic pathways as reference. Combining changes of metabolic enzymes, we claryfied the metabolic signature and metabolsm alteration in hypoxic of glioblastoma.Results1. A total of 5215 differentially expressed genes were identified in U87-MG cells, including 2759 up- and 2456 down-regulated genes. The up-regulated genes were significantly enriched in inflammatory response, angiogenesis and cell migration. Moreover, the interaction of cytokines and Toll-like receptor pathways which were core signal pathways in inflammation were activated in U87-MG cells.2. Twelve genes particularly expressed in U87-MG cells were involved in inflammatory response, angiogenesis and cell migration. The plasma concentrations of IL1 B, CSF3 and TIMP1 in GBM patients were significantly higher than healthy individuals.3. Under hypoxia condition, the biological processes of cell division, DNA replication, tricarboxylic acid cycle, mitochondrion transport and ATP metabolism were suppressed; however, positive regulation of homeostatic process and cellular response to lipid were activated.4. Hypoxia sensitive genes STC1, HMOX1 and TGFB1 consecutively expressed in U87-MG cells with a higher expression. Moreover, the brain tissues of GBM patients presented a higher expression than normal brain tissues.5. 119 different metabolites were found between U87-MG and HEB cells, of which 59 upregulated, 60 down-regulated. These metabolites were mainly enriched in the citric acid cycle, lipid metabolism, glycolysis, amino acid and nucleic acid metabolism pathway.6. With 1% O2 environment culture, U87-MG cells showed increased amino acid metabolism and glucose metabolism. Different from U87-MG cells, lipid synthesis and metabolism was enhanced in HEB cells.ConclusionCompared with HEB cells, U87-MG cells showed a significant different gene expressing pattern, with substantial activation of inflammation, angiogenesis and cell mobility. Under further analysis, we found that the inflammation associated Toll like receptor pathway and the signal pathway mediated interactive roles of cytokines showed high activities in U87-MG cells. Moreover, different from HEB cells, U87-MG cells showed a distinguished response model in hypoxia environment, with suppression of cell division, DNA replication, tricarboxylic acid cycle, mitochondrion transport and ATP metabolism and up regulation of homeostatic process and response to lipid. And the transcriptome study and experiments from clinical samples predicted that inflammatory associated genes IL1 B, CSF3 and TIMP1 and hypoxia sensitive genes STC1, HMOX1 and TGFB1 could be regarded as the biomarkers for early diagnosis and pathological diagnosis and prognosis evaluation for GBM patients. Moreover, compared with HEB cells, U87-MG cells showed enhanced glycolysis and metabolism of amino acid and lipid and decrease of aerobic oxidation and nucleic metabolism. And the metabolism of glucose and amino acid of U87-MG cells showed more important roles in hypoxia environment. PART II. The clinicopathological significance of IF1 in non-small cell lung cancerLung cancer continues to be the most common causes of cancer-related deaths worldwide. Patients with non-small cell lung cancer(NSCLC) accounts for more than 85% of lung cancer. Most patients were diagnosed in advanced stages and therefore missed the best opportunity to be cured. ATPase inhibitory factor 1(IF1) was overexpressed in several human cancers and has the unique capacity to regulate tumor metabolism reprogramming. We demonstrated that elevated expression of IF1 may serve as an independent prognostic factor in NSCLC patientsMethodsAimed to evaluate the expression levels of IF1 in NSCLC and the prognostic value for the patients. IF1 protein expression levels were detected in 149 cases of NSCLC by using immunohistochemistry.Results1. The z-scores were significantly higher in NSCLC than that in the normal tissues(P=0.034). The expression levels of IF1 were significantly increased in patients with advanced TNM stages(stage I/II)compared with early TNM stages(stage III/IV)(P=0.033).2. IHC staining showed the expression levels of IF1 were significantly increased in patients with advanced TNM stages(P=0.032) or lymphatic metastasis(P=0.043) as compared with those with early TNM stages or without lymphatic metastasis, respectively.3. Multivariable Cox-regression analysis revealed that elevated expression of IF1 could be an independent prognostic factor(HR=1.667, P=0.034) in NSCLC. Kaplan-Meier analysis showed that the IF1 high group suffered significant worse outcome than the IF1 low group(P=0.042).4. The recurrence rate in patients with IF1 low expression was much lower than those with IF1 high expression(P=0.003). |
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