| Background: Glioma is the most common tumor of the central nervous system(CNS),which is derived from glial cells.Glioma is a diffuse invasive tumor which can affect the surrounding brain tissue.Glioblastoma is the most malignant type,and pilocytic astrocytoma is the lowest type of malignancy.In recent years,the diagnosis and treatment of glioma has made great progress,but the prognosis of patients with glioma is still poor,of which the median survival of glioblastoma is only 14 months.With the amount of further researches,there are many differences in genes.As the research moves along,gliomas were found have many difference in gene expression.The diagnosis and treatment based on these molecular typing will improve the prognosis of patients with glioblastoma.Maitake Polysaccharide(MP)is a kind of active ingredient which extracted from the fruiting body and mycelium of maitake.It has many kinds of biological activity substances such as anti-tumor,enhance immunity and so on.Domestic studies have reported that MP joint Vitamin C(Vitamin C,VC)on the proliferation of tumor cells has significant synergistic inhibition effect,and the molecular mechanisms of anti-tumor,inducing tumor cell apoptosis and tumor cells in the nervous system of combined use of MP and VC research has not yet been reported.Objective:(1)To find out the genes and signaling pathway related with glioma tumorigenesis by analysis of differential gene expression and gene functional annotation.(2)To better understand the tumorigenesis and find out the hub genes,we build the co-expression scale-free network of lower grade gliomas by Weight gene co-expression network analysis(WGCNA)and identified specific gene modules and valuable hub genes associated with gliomagenesis.(3)Combined with the clinical data of the samples,to find clinical factors related to glioma patients prognosis.(4)To study the effects of combination usage of maitake polysaccharide and vitamin C to tumor cell proliferation on glioma M059 K cells,and to further explore the molecular mechanisms of antitumor effect of the combination on glioma M059 K cell.And to provide experimental and the oretical basis of the effective medicine to guide clinical practice and to explore new ideas of glioma clinical treatment in the future.Methods:(1)We used the TCGA-Assembler download level-3 RNASeqV2 gene expression data,miRNA-seq data of samples and clinical information of those patients.We deleted expressed data close to zero,and selected round numbers of all array.Compared the normal group with glioblastoma,we identify the differently expressed genes(DEG)and make the functional annotation.(2)WGCNA was employed to build the co-expression scale-free network and visualized it with Cytoscape software.(3)Combined with the clinical data of the samples,Cox regression analysis was used to identify the factors associated with the prognosis of patients with glioma,and build the survival curve.(4)Then the in vitro experiments to glioma M059 K cells were accomplished,MTT method was used to detect the effect of single and combination drug treatment on glioma M059 K cell proliferation;Flow cytometry and Annexin V-FITC/PI double dyeing method were used to check the cell cycle and cell apoptosis after treatment.Meanwhile,Hoechst33258 staining was used to identify the occurrence of apoptosis by fluorescence microscope.Western blotting(WB)were used to detect the expression of apoptosis related proteins: Bax,Bcl-2,PARP and Caspase-3 after single and combination drug treatment treatment.In addition,the activity of caspase-3,caspase-8 and caspase-9 of cell were detected in order to study the activity changes of apoptosis on the level of molecular biology and to discusses what kind of signal transduction pathways of apoptosis was followed by M059 K cells.Results:(1)Compared with the normal brain tissue,the 3492 differently expressed genes were identified in Glioblastoma,among which 2640 genes were down-regulated and 1302 genes were up-regulated.GO enrichment pathway revealed that the down-regulation of genes involved in cell senescence,metabolism,protein modification etc.GO enrichment pathway results show that the upregulation of genes in glioblastoma involved in cell mitosis,cell cycle regulation,cell division,amino acid modification and so on.Ndc80 complex is located at the core of GO function pathway.(2)The expressed genes in lower grade glioma are clustered into 19 modules by weighted gene co-expression networks analyses(WGCNA).In addition,the red module related in grade of glioma.As is showed in the network,LC12A5,GABRA1,MAL2,PACSIN1,SULT4A1,SYT1,TBR1,DNM1,GPR22,VSNL1,GABRB2,OLFM3,SV2 B,CCT2,CNOT2,ESPN,FRS2,MAP7D2,MDM2,NAPB,PAX7,PTPRB,STX1 A,YEATS4 are on the central of network.(3)Merged the clinical data,we found that histological type,histologic grade,laterality,tumor location,mental status changes,ldh1 mutation,motor movement changes,karnofsky performance score,radiation therapy and age at initial pathologic diagnosis are intensively related with the lifetime.For lower glioma,Bevacizumab(Avastin)combined Temozolomide(Temodar)cannot prolong survival.On the contrary,the combination of Temozolomide(Temodar)with Bevacizumab(Avastin)was associated with a significant reduction of survival for lower glioma patients.(4)Results of Western blot showed that the changes in the expression of apoptosis-related proteins occurred(upregulation of Bax and Caspase-3,down-regulation of Bcl-2,and activation of poly-(ADP-ribose)-polymerase).More over,the activities of caspase-3,caspase-8,and caspase-9 were enhanced in M059 K cells.The inhibiting effect of combined treatment with MP and VC on M059 K cells indicates the mechanism of anticancer activity involved induction of cell apoptosis and the cells apoptosis were mainly follow the mitochondrial signal transduction pathways.Conclusions:(1)By the system biostatistics analysis,we may provide a better understand way to find the mechanisms tumorigenesis of gliomas.Ndc80 complex may play a vital character in gliomagenesis.(2)Clinical prognosis should be multidimensional,the prognosis of malignant tumors not only related with the pathological grade of the tumor,more importantly,with other clinical information and treatment methods.(3)The further experimental results indicated that the combined application of MP and VC have antitumor activity through inducing M059 K cells to apoptosis and the combined application of small doses of Grifola frondosa polysaccharide and vitamin C has significant antitumor activity to M059 K cells. |
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