Comparative Proteomic Study Of Cerebrospinal Fluid In Between Patients With Medulloblastoma And Patients With Ependymoma

Background Both medulloblastoma and ependymoma are neuroepithelial tumors. Medulloblastoma has a higher degree of malignancy than ependymoma. The incidence of medulloblastoma ranked second in central nervous system tumors in childhood, while the incidence of ependymoma ranked third. They have a great bad impact on children's health. Because they are the most common tumors in the fourth ventricle and get close to brain stem, it is difficult to resect the tumors completely. Moreover, they have a tendency to spread through the cerebrospinal fluid (CSF), which results in the low rate of cure with poor prognosis. CSF has the important clinical value because of contact with the tumors and the clinical feasibility of the approach. Therefore, if the tumor biomarkers are found in the CSF, it will be helpful to make the early diagnosis as well as differential diagnosis, to determine prognosis, to monitor tumor recurrence and to observe treatment response.Proteomics study the protein expression profiles in the certain time and surroundings by means of the combination of high-resolution protein separation technique and high-throughput protein identification technique. Comparative proteomics analyze the changes of protein expression level between different samples. Comparative proteomics contain two main kinds of technique, protein separation technique and protein identification technique. Two-dimensional differential in-gel electrophoresis (2D-DIGE), an advanced protein separation technique, can significantly improve the sensitivity of the detection of trace protein. Introducing the concept of internal standard,2D-DIGE is more conducive to detect the differentially expressed proteins accurately. Mass spectrometry (MS) and tandem mass spectrometry (MS/MS) in proteomic research are the major protein identification techniques. MS/MS is more accurate and more sensitive for identification of protein.Objective To provide the clues in search of biomarkers in CSF of medulloblastoma and ependymoma and to offer theoretical and experimental basis for revealing the pathogenesis of medulloblastoma and ependymoma, the differentially expressed proteins in CSF between medulloblastoma, ependymoma and normal control group are isolated and identified via the comparative proteomic techniques. Combined with genechip technology, gene expression profiles of medulloblastoma, ependymoma and cerebellar tissue are established in order to identify differences in the mRNA expression of the genes encoding the differentially expressed proteins. The study of gene expression profile provides some experimental evidences to speculate the sources of differentially expressed proteins in CSF.MethodsThe experiment was divided three groups:medulloblastoma group:nine cases of CSF in patients with medulloblastoma, three cases of medulloblastoma tissue; ependymoma group: nine cases of CSF in patients with ependymoma, three cases of ependymoma tissue; normal control group:six cases of CSF in patients with headache who had no other neurological disorders(normal CSF examination),3 cases of cerebellum tissue from traumatic surgical decompression.(1) Quantitative analysis of comparative proteomics between the three groups:①With 10% TCA/acetone, total proteins in CSF of three groups were precipitated and extracted respectively. Then the proteins were quantified with Bradford method.②Analytical gel electrophoresis:2D-DIGE was used. The internal standard comprised equal amounts of proteins extracted from three groups. Internal standard and proteins from two of three groups were minimally labeled with fluorescent dyes, Cy2,Cy3 and Cy5, then the three samples pooled. Every sample's labelled protein is 50μg. First dimension IEF was performed with 24 cm immobilized pH 3-10 linear gradient dry strip for isoelectric focusing. Then, the proteins were isolated according to the difference of isoelectric point. Second dimension SDS-PAGE was performed with homogeneous 12.5% gels after equilibration twice. Then, the proteins were isolated according to the difference of molecular weight. The fluorescence signals of the three differently CyDye-labeled protein samples were imaged by using a laser scanner and wavelengths of 488 nm (Cy2, blue),532 nm (Cy3, green) and 633 nm (Cy5, red). The detection and quantification of protein spots were performed for the differential in-gel analysis (DIA) module of DeCyder software. Biological variation analysis (BVA) module of DeCyder can provide data on differential protein expression levels beteeen the three groups.③Preparative gel electrophoresis:600μg proteins from every group run 2D-PAGE respectively. Then the gels were stained with Coomassie brilliant blue.④Protein identification:differentially expressed proteins were selectively identified by matrix-assisted laser desorption ionization-time of flight-time of flight-mass spectrometry (MALDI-TOF-TOF-MS), Mainly up-regulated proteins in 2 fold in CSF of medulloblastoma and/or ependymoma. After comparison between preparative gel and analytical gel, the spots in preparative gel that matched differentially expressed spots in analytical gel were excised. After enzymolysis of trypsin, MS and MS/MS were obtained with mass spectrometer. Peptide mass data were searched in data IPI human with GPS software.(2) Quantitative analysis of comparative gene expression profiles between the three groups:①Total RNA of medulloblastoma, ependymoma and cerebellum were extracted respectively. Then quantification, clean up and checking of total RNA were performed.②Synthesis and clean up of cDNA.③Synthesis of biotin-labeled cRNA in vitro transcription.④Fragmenting the cRNA and hybridization with Affymetrix Human Genome U133plus 2.0 genechip.⑤Elution and scan of the genechip.⑥Data analysis with GCOS software.Results(1) 2D-DIGE analysis showed that the levels of 35 protein spots were significantly altered in medulloblastoma CSF compared with control CSF when average ratio of the matched spot was assigned as>2.0 or<-2.0, of which 17 spots were up-regulated and 18 spots were down-regulated.(2) 2D-DIGE analysis revealed that levels of 74 protein spots were significantly changed in ependymoma CSF compared with control CSF when average ratio>2.0 or<-2.0, of which 38 spots were up-regulated and 36 spots were down-regulated.(3) 2D-DIGE analysis demonstrated that levels of 59 protein spots were significantly altered in medulloblastoma CSF compared with ependymoma CSF when average ratio>2.0 or<-2.0, of which 12 spots were up-regulated and 47 spots were down-regulated.(4) 28 up-regulated (average ratio>2.0) spots were selected to be identified with MALDI-TOF/TOF, of which 18 spots were identified successfully, corresponding to 12 unique proteins.(5) Isoform 1 of complement factor H, alpha-2-macroglobulin, serotransferrin, fibrinogen beta chain, immunoglobulin heavy constant alpha 1, haptoglobin isoform 2 preproprotein, highly similar to Haptoglobin and highly similar to actin cytoplasmic 1 (beta-actin) were markedly increased in ependymoma CSF compared with the medulloblastoma CSF and control CSF, which were not significantly altered between the medulloblastoma CSF and control CSF. Isoform 1 of complement factor B was markedly increased in medulloblastoma CSF and ependymoma CSF compared with control CSF, which was not significantly changed between ependymoma CSF and the medulloblastoma CSF. Isoform 1 of alpha-1-antitrypsin and isoform 3 of neuronal cell adhesion molecule was markedly increased in medulloblastoma CSF compared with ependymoma CSF and control CSF, which was not significantly altered between ependymoma CSF and control CSF. Putative uncharacterized protein ALB and putative uncharacterized protein DKFZp686M08189 were markedly increased only in medulloblastoma CSF.(6) Gene expression profiles:the gene encoding complement factor H was detected to express only in medulloblastoma, while the gene encoding haptoglobin was detected to express only in ependymoma. The gene encoding alpha-1-antitrypsin was detected to express in ependymoma and cerebellum, but not in medulloblastoma. The gene encoding alpha-2-macroglobulin, the gene encoding neuronal cell adhesion molecule, the gene encoding serotransferrin and the gene encoding beta-actin was detected to express in medulloblastoma, ependymoma and cerebellum. The gene encoding complement factor B, the gene encoding fibrinogen beta chain and the gene encoding immunoglobulin heavy constant alpha 1 were not detected to express in medulloblastoma, ependymoma and cerebellum.Conclusions There were some significantly differential protein spots in 2D-DIGE of CSF between medulloblastoma, ependymoma and cerebellum.12 differentially expressed proteins in CSF of medulloblastoma and/or ependymoma were identified, which provided meaningful clues for exploring the potential biomarkers of two kinds of tumor in CSF. However, these potential biomarkers needed further validation. The study of gene expression profile was helpful to speculate the the sources of differentially expressed proteins in CSF and pointed out the direction to select the valuable proteins for further study.