Differential Proteomics Analysis Of Oligodendrogliomas With And Without 1p LOH

Glioma is the most common primary brain tumor. The incidence of glioma is high and the treatment efficacy has not been satisfactory. Better prognosis of glioma has been achieved with the improvement of surgical technique and the widespread application of chemotherapy and radiotherapy, however the overall prognosis of most gliomas is still poor. Oligodendroglioma(OG) is one major type of gliomas, which accounts for 2-5% of primary brain neoplasms and 4-15% of gliomas. Because these tumors respond well to chemotherapy and have good prognosis, the reason for this is becoming a hot topic in glioma research. It was reported that response to chemotherapy and good prognosis of OG patients were closely related to the presence of allelic losses on lp(or 1p and 19q) in the resected tumor tissue. Further research revealed that OGs with or without 1p LOH had profound differences, such as tumor location, response to radiotherapy, prognosis. So the two types of OGs undistinguishable under optical microscopy may be tumors with different molecular biological character. The molecular classification of OGs may be helpful to their diagnosis and therapy. Except for the difference of lp/19q LOH, other features, such as expression of genes, proteins, and molecular biological mechanisms, are not clearly defined for the OGs. Further studies are needed to elucidate the molecular signatures, and thus possible mechanisms of OGs with 1p LOH.Modern proteomic technologies allow detection and quantitation of a great number of proteins at the same time. Currently these technologies are being applied in research areas of both basic sciences and clinical medicine. Although genomics and transcriptomics studies have revealed many possible oncogenes and tumor suppressors, proteomics analysis has an advantage of studying proteins, the functional molecule in most of the biological process, directly. In addition to that, post-translational modifications, such as phosphorylation, acetylation, and translocation of the proteins, can only be studied with proteomic technologies. While only a handful of proteins can be studied simultaneously with traditional methods such as western blots, a typical proteomics study would yield information on hundreds or thousands of proteins, which allowed molecular profiling of complex disease such as glioma. Isobaric tags for relative and absolute quantification (iTRAQ) is a new quantitative proteomics technology, which has proved to be efficient, reproducible, and able to simultaneously perform quantitative protein analysis of eight samples. In this study, we use iTRAQ technology combining with two-dimensional liquid chromatography tandem mass spectrometry(2D-LC MS/MS) to identify specific protein markers in OG tissues with 1p LOH.Partâ… :Detection of loss of heterozygosity on chromosome 1p in oligodendrogliomas by fluorescence in situ hybridizationObjective:To explore the incidence of loss of heterozygosity (LOH) on chromosome 1p in tissue samples from Chinese patients with oligodendrogliomas.Methods:Sixteen specimen of oligodendrogliomas and five specimen of normal control cerebral tissues (obtained from the decompression operation of brain trauma) were examined by fluorescence in situ hybridization (FISH).Results:Of the 16 cases of oligodendrogliomas,10 cases (62.5%) had LOH in chromosome 1p; Of the 6 cases of gradeâ…¡oligodendrogliomas,5 cases had LOH in 1p; Of the 10 cases ofâ…¢grade oligodendrogliomas,5 cases had LOH in 1p.Conclusion:LOH in chromosome 1p is a frequent characteristic of oligodendrogliomas in Chinese patients. We can distinguish OGs with or without 1p LOH by FISH method effectively and apply FISH in clinical diagnosis.Partâ…¡:iTRAQ-based differential proteomics analysis of oligodendrogliomasObjective:To perform differential proteomics analysis of oligodendrogliomas with different 1p LOH status; To identify specific proteins for diagnosis and selection of treatment modality.Methods: Differential proteomics analysis of 8 cases of OGs with different 1p LOH status were performed using iTRAQ combining with 2D-LC MS/MS; the data were analyzed with bioinformatics in the database of DAVID and KEGG; marker proteins were obtained using statistical analysis.Results:With stringent criteria, we identified 449 proteins from 8 cases of OGs using proteomics analysis; eleven proteins (HMGB1, MAP2, TMSB4X, LRPAP1, MARCKSL1, RPLP2, CISD1, ATP6V1E1, PSAP, PPIF, and ARPC5L) had a higher expression in the OG group with 1p LOH than in the OG group without 1p LOH, while UBA1 and PRKARIA had a higher expression in the OG group without lp LOH than in the OG group with 1p LOH.Conclusion:There are many differentially expressed proteins between the OGs with and without lp LOH. We obtained a wider coverage of proteins compared to what were found in other studies. Bioinformatics analysis revealed that the actin cytoskeleton pathway may be involved in the molecular biological mechanism of different OGs with and without 1p LOH. HMGB1 and UBA1 may be related to sensitivity of chemotherapy. MAP2 may be related to prognosis.Part III:The expression of HMGB1, MAP2, UBA1, ATP6V1E1 in OGs with different 1p LOH and its significanceObjective: To examine the expression of HMGB1, MAP2, UBA1, ATP6V1E1 in OGs tissues with different 1p LOH.Methods:The expression of some of the marker proteins in OGs with different lp LOH were examined using Western blot; the expression of HMGB1, MAP2, UBA1, ATP6V1E1 were further validated in 39 independent OGs tissue samples with different lp LOH status using IHC method.Results:The result of Western blot confirmed the iTRAQ analysis for the marker proteins; The result of IHC validated that the expression of HMGB1, MAP2 and ATP6V1E1 was higher in OGs with 1p LOH than in OGs without lp LOH, while the expression of UBA1 was higher in OGs without 1p LOH than in OGs with 1p LOH.Conclusion:The results of Western blot and IHC were consistent with the iTRAQ-based proteomics analysis; These specific proteins may be useful in diagnosis, prognosis and treatment selection for OG patients.