| Background and ObjectiveGlioma is the most common primary central nervous system tumor in human tumor.It accounts for 30%~40% of the primary central nervous system tumor,and is one of the lowest survival time and the worst prognosis.With the development of society,tumor has become a more common disease of mankind,and the mortality of tumor is second only to cardiovascular disease,which is one of the most important diseases currently threatening human health.With the rapid development of surgical,chemotherapy,radiation,molecular targeting,and so on,the results of the treatment of the glioma have improved significantly.However,the survival and prognosis of patients with brain glioma are still not optimistic,and the molecular markers of brain glioma are still very limited.In recent years,with the increasing understanding of molecular biology,molecular targeting has gradually become a new therapeutic measure for the treatment of tumors,which has become an important part of modern medical oncology treatment.The human mitochondrial transcription termination factor 3 is a negative regulation of mitochondrial gene expression and protein oxidative phosphorylation activity.It has been reported that the gene copy number amplification and overexpression of hMTERF3 plays an important role in the occurrence,progression and prognosis of hepatocellular carcinoma,breast cancer,non-small cell lung cancer and pancreatic cancer,but the expression and prognosis of hMTERF3 in glioma have not yet been reported.The project is based on the data mining and analysis of the TCGA and Oncomine database and analyzing the relationship between the expression of the hMTERF3 and the clinical pathology of theglioma,and the correlation between the level of hMTERF3 and the prognosis of the gliomas.By immunohistochemical technique to observe hMTERF3 protein in brain gliomas and the expression of tumor tissue.Building and identification of recombinant eukaryotic expression vector p3×FLAG-CMV-hMTERF3,and by using bioinformatics methods to predict hMTERF3 protein structure and function.To further study the effects of transfection recombinant eukaryotic expression vector p3×FLAG-CMV-hMTERF3 on the mitochondrial DNA copy number,mitochondrial protein expression level and cell proliferation,cell cycle and other biological behaviors of U251 cells in brain glioma.This study to clarify hMTERF3 in glioma experimental basis of the role of the occurrence and development,also for the diagnosis and treatment of cerebral glioma provide potential molecular targets.MethodsUsing Oncomine database to retrieve information about hMTERF3 gene,and the hMTERF3 meta-analysis gene expression in glioma;Using Bioconductor/TCGA biolinks to download clinical data from the TCGA database,using R3.3.0 software to analyze the clinical pathology parameters of 526 cases of glioma in the TCGA database and the correlation analysis of the levels of hMTERF3.Using Graphpad 5.0software to express hMTERF3 TCGA data set high and low expression hMTERF3 brain glial the prognosis of patients with survival for survival analysis.At the same time,collected in 2008 ~ 2012 in Dali prefecture's first people's hospital of 20 cases of glioma tissues and 16 cases of immunohistochemical tumor tissue samples.Among the 20 patients with glioma,13 were male and 7 were female,with age ranging from14 to 62 years old,all of whom were confirmed for the first time,without chemotherapy or radiotherapy before surgery.The expression of hMTERF3 protein was detected by immunohistochemistry method,and the expression level was compared.The human MTERF3 gene was amplified by Polymerase Chain Reaction(PCR).The recombinant products of the p3×FLAG-CMV-hMTERF3 were gained by T4 DNA ligase connecting MTERF3 gene fragment and eukaryotic expression vector p3×FLAG-CMV-14 with restriction enzymes Hind ? and BamH?.The recombinant pasmid p3×FLAG-CMV-hMTERF3 were identified by colony PCR,doublerestriction enzyme digest and DNA sequencing.Using semi-quantitative PCR and Western blot hMTERF3 tested expression of glioma U251 cells mitochondrial DNA copy,mitochondria,coding protein expression level of quantity change.The multifactorial analysis shows that the age,the original body parts of the tumor are the independent prognosticative factors that affect the prognosis of brain gliomas.A specific band of 1254 bp was detected form recombinant plasmid p3×FLAG-CMV-hMTERF3 by digestion of Hind ? and BamH?.Sequencing and identification showed that homology between this squence and the human MTERF3 gene sequence form GenBank was 100%,and the size and the direction of the inserted gene were right.ResultsUsing Oncomine database analysis,it was found that hMTERF3 was highly expressed in glioma tissues.The clinical pathological data of 526 cases of glioma patients were found in TCGA database,and the expression and age of hMTERF3(P=0.010),primary tumor site(P=0.048),screen location(P=0.041),grade(P<0.001)and pathological type(P<0.001)were significantly correlated,However,it was not related to gender,site of disease,history of headache,and sample type.To further use the Kaplan-Meier Plotter database to analyze the impact of hMTERF3 on the survival time of a glioblastoma patients,the OS of hMTERF3 high expression group(P=0.001,HR=2.03)and DFS(P=0.004,HR=1.54)were all lower than the hMTERF3 low expression group,and the difference between the two groups was statistically significant.Immunohistochemical analysis showed that MTERF3 protein was highly expressed in glioma tissues,while low expression in non-neoplastic brain tissue.,the expression of hMTERF3 protein in glioma tissues was mainly localized in cytoplasm,with yellowish-yellow or brown-yellow staining.The positive expression of hMTERF3 in glioblastoma tissue is strong,and the dye is deep,and non-oncology brain tissue can also observe the expression of the hMTERF3 protein,but the dye is lighter.Using different bioinformatics software to systematically study hMTERF3 protein,it was found that the secondary structure of hMTERF3 protein was mainly helix and random coil,including 6 MTERF motifs,and the prediction results of thetertiary structure were consistent with the second-order structure prediction results.Subcellular location analysis shows that the protein is located in human mitochondria;Multiple sequence comparison and evolutionary analysis of homologous proteins showed that hMTERF3 protein was highly homologous with the MTERF3 protein in mammals such as rats and mice,and was clustered in the phylogenetic tree.After transfection glioma U251 cells by semi-quantitative PCR detection of mitochondrial DNA copy found that overexpression of hMTERF3 have significant inhibitory effect on mitochondrial DNA replication;Western blot detection showed that the overexpression of hMTERF3 could reduce the ND1 protein level of mitochondrial encoding.The MTT test results show that the expression hMTERF3 is promoting the proliferation of U251 cells;Flow cytometry analysis of cell cycle found that hMTERF3 overexpression can promote cell proliferation and lead to smooth passage of cells through G1/S control point,and cell cycle is not blocked and apoptosis.ConclusionsThe study shows that based on the information from the TCGA and Oncomine database to get the information from the hMTERF3 in the tissue of the brain glioma,to explore the role of hMTERF3 in the development of the brain glioma.The expression of hMTERF3 protein in human glioma is increased,suggesting that hMTERF3 may be involved in the development of human glioma,and may be closely related to the malignancy of the tumor.The eukaryotic expression vector of human MTERF3 was constructed successfully,and may provide the foundation for further research of the interaction mechanism between behavior of MTERF3 in tumor cells and it's development.The overexpression of hMTERF3 in glioma U251 cells significantly inhibited the replication level of mitochondrial DNA,and affected the expression level of the mitochondrial encoding ND1 protein.At the same time,the overexpression of hMTERF3 can significantly promote the proliferation of U251 cells,promote the transformation process of the cell cycle G0/G1 phase to S phase,and it do not cause apoptosis. |
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