Proteomic Analysis Of Spontaneous Regression Proteins In Neuroblastoma Using 2D-DIGE Technology

Background:The spontaneous regression of tumor is a very rare but important scientific event, studies on its pathogenesis is helpful for exploring and understanding the etiology of cancer. Neuroblastoma already becomes an ideal model for cancer self-healing mechanism study due to its frequet incidence of spontaneous regression, for which most of the studies so far is limited to the genome thus it is necessary to screen the proteins associated with spontaneous regression in neuroblastuma tissue. In recent years, the technology of proteomics combined with bioinformatics has been applied extensively. additionally, the two dimensional fluorescent difference gel electrophoresis (2D-DIGE) has more advantages over silver staining and blue dyeing.2D-DIGE is the only proteomic way to apply internal standard so far, which is made from the mixture of the equal amount of proteins from all samples tested. The internal standard will be displayed on every gel, and will support the analysis software as a benchmark to analyze all detected differences of protein expression.Object:In this study, we aim to explore the proteins associated with spontaneous regression in neuroblastoma tissue by use of 2D-DIGE technique.Method:Patient criteria:Newly diagnosed neuroblastoma with stage IVs, or stage IV under 1 year old as control.Primary sites were adrenals.3 patients for each of the group have been employed.Consent forms were signed by their guardians, no previous chemotherapy. During the operations, all the tumor tissues and their suround normal adrenal tissues were removed. After the surgery, all the patients were treated by standard protocol and follow up for 3 years. Whole proteins were extracted from part neuroblastoma tissues and normal adrenal gland tissues, then they were detected by two-dimensional gel electrophoresis(2-DE)after labeled with DIGE fluors Cy3,Cy5 and Cy2. equal amount of protein from all the samples tested were pooled together as internal control. Intensity changes of prolein spots detected with statistical significance were identified by MALDI-TOF MS or MS/MS. To evaluate the reliability and accurate of the proteomic results, some proteins with different expression were further detected by western blot in the 3 kinds of tissue.Results:After 2D-DIGE, there were 973 matched protein spots identified.27 spots were significantly between stage IVs tumor tissue, stage IVs tumor tissue and normal adrenal gland tissue (ratio≥3.5, p≤0.05), among which 17 gene products were identified. In group of stage IVs tumor samples 7 up-regulated gene products and 2 down-regulated gene products were identified compared to stage IV tumor samples. In group of stage IV tumor samples 4 up-regulated gene products and 7 down-regulated gene products were identified compared to normal adrenal gland samples. In group of stage IVs tumor samples 4 up-regulated gene products and 4 down-regulated gene products were identified in stage IVs tumor samples compared to normal adrenal gland samples. The western blot detection revealed that Rho-GDI was up-regulated in stage IVs tumor samples compared to stage IV tumor samples or normal adrenal gland samples.Conclusions:Our results suggest that there are 10 different expressed proteins between stage IVs samples tumor tissue and stage IV tumor tissue; and there are 8 different expressed proteins between stage IVs samples tumor and normal adrenal gland samples. Rho-GDI was identified to be up-regulated in stage IVs tumor samples compared to stage IV tumor samples.