Proteomic Studies On Malignant Glioma

BackgroundGlioma is the most common intracranial tumor arising from glia cell or their precursors.Based on the degrees of malignancy,glioma is classified into different subtypes.In view of its most malignant and invasive property,GBM has an extremely poor prognosis.Even when the MRI-detected tumor is surgically removed and patients are treated with radiat:ion and chemotherapy,the mean survival rate of GBM can only extend to some extent.Highly invasive nature limits surgical options,eventually leading to tumor recurrence.On the contrary,LGA has a low cell proliferation rate and is not prone to invade,leading to a favorable prognosis.Great attempts have been made to explore the pathophysiology of glioma in the past years and some molecules have been proved to exert important impacts.However,due to the complex characteristics,a comprehensive understanding of the mechanism of glioma malignancy is still lacking.Proteomics is a new kind of technology which could identify and analyze all the proteins in samples.Serving as a useful tool in biology,quantitative proteomics allows reproducible identification and accurate quantification of proteins in multiple biological samples.Although genomic and transcriptomic studies have discovered many oncogenes and tumor suppressors,proteomic analysis focuses on translated proteins,which exert direct roles in most biological processes.While a few number of proteins can be studied simultaneously with traditional methods,such as ELISA and western blotting,a typical proteomic study yields information ofthousands ofproteins in a digital form.Comparative proteomics between diseases and normal tissues or among diseases may offer the opportunity to reveal disease specific bio markers,even to elucidate underlying mechanisms.The conventional proteomic techniques have a limited use,owning to several inevitable pitfalls.Recent years have witnessed the development of mass spectrometry with high resolution.In addition,combination of liquid chromatography and tandem mass spectrometry allows us to identify complex protein components with high throughput and resolution.Objective1.To identify potential biomarkers of malignant glioma2.To explore the pathogenesis of malignant gliomaMethods1.De-identified tissue samples of GBM and LGA were collected,verified and characterized by immunohistochemistry with known markers used routinely to diagnose LGA and GBM by pathologists.2.Samples were lysed by RIPA lysis buffer.After dialysis,protein concentrations were quantified by BCA protein assay.3.Total proteins were digested in-solution and trypsinized lysates were desalted by C18Ziptip.4.The desalted peptides were fractionated in a C18 nanocapillary column equipped on high pressure liquid chromatography interfaced with Orbitrap Elite mass spectrometer.The pept:ides were eluted from the column,ionized in a positive-ion mode at 2.2 kV and data was acquired in a data-dependent CID mode.Each sample was run triplicate with the same method.5.For protein identification,the acquired data were searched using Proteome Discoverer 1.4 powered with MASCOT and SEQUEST algorithms.6.For label-free quantification,raw data were analyzed by Progenesis LC-MS software.7.The Search Tool for the Retrieval of Interacting Genes(STRING)was employed for functional enrichments and interaction network analysis on differentially expressed proteins.8.Western blotting and immunohistochemistry were employed to verify the expression of GBM-associated proteins.Whole proteins were separated by SDS-PAGE,tansferred to nitrocellulose/PVDF transfer membrane and probed using primary antibodies against the candidates of interest.Primary antibodies were detected with proper fluorescence-conjugated secondary antibodies.The blots were scanned with Odyssey infrared imaging system.9.SF3Al knockdown was performed using siRNA in U87 and U251 glioma cell line,along with the rescue experiment using SF3Al plasmid which was mutated according to siRNA sequence.Cells were characterized in vitro to assess molecular status,cell proliferation,migration,invasion,cell cycle and apoptosis using Western blotting,qRT-PCR,transwell,cck-8 reagent and flow cytometry.Results1.LC-MS/MS identified a total of 2,661 and 2,848 proteins in LGA and GBM,respectively.Among these proteins,136 proteins were differentially expressed in association with the development of GBM.2.Ontological analysis revealed a close correlation between GBM-associated proteins and RNA processing.3.Interaction network analysis indicated that the GBM-associated proteins in the RNA processing were linked to crucial signaling transduction modulators,including epidermal growth factor receptor(EGFR),signal transducer and activator of transcription 1(STAT1),and mitogen-activated protein kinase 1(MAPK1),which were further connected to the proteins important for neuronal structural integrity,development and functions.4.Western blotting and histoimmunochemistry verified the differential expressions of RPS5,SF3A1,FTH1,TNR and STAT1 in GBM and LGA.6.Knockdown of SF3AI inhibited proliferation,migration,invasion ability of glioma cells.It also inhibited cell cycle progression and triggered apoptosis,which may be due to the abnormality of alternative splicing of MDM4 and ectopic expression ofp53 protein.The rescue experiments confirmed the effect of SF3Al on glioma cells.ConclusionWe revealed a number of differentially expressed proteins in GBM in comparison with LGA.Our data suggested the importance of RNA processing in glioma malignancy and proteins in this network were linked to crucial signaling transduction modulators including EGFR,STAT1 and MAPK1,which were further connected to proteins important for neuronal structural integrity,development and functions.The panel of GBM-associated proteins and the important modulators centered at RNA processing network may become novel biomarkers and help elucidate the underlying mechanism.SF3A1 is differentially expressed in GBM and LGA,whih may influence cell cycle and apoptosis of glioma cells by regulation of splicing of MDM4 and expression of p53 protein.Altogether,SF3A1 may become a novel biomarker and help elucidate the underlying mechanism of glioma malignancy..