Serum Proteomic And Bioinformatic Analysis In Children With Hematologic Malignancies

Part One Discovery and identification of serum potential biomarkers in children with hematologic malignancies using iTRAQ-coupled 2D LC-MS/MSBackground and objective Hematologic malignancies are the most common malignant cancers of childhood.Currently,bone marrow aspiration and lymph node biopsy are used for diagnosis.However,because of great damage,most of patients show different degrees of mental stress towards this examine.In recent years,with the intensified chemotherapy,the prognosis of hematologic malignancies has been greatly improved but part of patients are difficult to achieve complete remission or struggle with relapse,and traditional chemotherapy drugs cause toxic side effects,which may lead to death.Noninvasive and specific biomarkers for the early diagnosis and therapeutic targets of pediatric hematologic malignancies are urgently needed.Using Isobaric tags for relative and absolute quantification(iTRAQ)quantitative proteomic technique combined with two-dimensional liquid chromatography tandem mass spectrometry(2DLC-MS/MS)and IPA method we screen the differences in protein expression of hematologic malignancies,to investigate the diagnosis and therapeutic target and the pathogenesis of hematologic malignancies.Methods 1.Serum were collected assigned into the normal group,acute B-cell acute lymphoblastic leukemia(B-ALL)group,T-cell acute lymphoblastic leukemia(T-ALL)group,acute myeloid leukemia with maturation(M2)group,acute promyelocytic leukemia(M3)group,acute monocytic leukemia(M5)group,B-cell non-Hodgkin's lymphoma(B-NHL)group and T-cell non-Hodgkin's lymphoma(T-NHL)group with 20 cases for each group.2.Equal volumes of serum from patients group and control group were pooled,respectively.Samples from the 8 groups were depleted of 14 high abundance proteins labeled with iTRAQ tags and then analyzed by 2DLC-MS/MS.We take reverse database to estimate its false positive rate.The iTRAQ experiment is replicated by 3 times.3.According to identificating proteins iTRAQ relative quantitative values to ensure differential proteins for groups.At last,the differences in protein were further analyzed by IPA database and get partial signaling pathways and networks.4.The proteomic results were validated by enzyme-linked immunosorbent assay.Results 1.The final result shows 534 non-redundant proteins,with 1.2 and 0.8 as the up-regulation and down-regulation cutoff point for differential proteins,comparing the experimental groups(hematological malignancies)and control group,respectively.2.Ingenuity pathway analysis of differential proteins in 7 experimental groups:(1)There are 81 differentially expressed proteins in B-ALL group as oppose to control group,among them,20 proteins are up-regulation and 61 down-regulation.These differentially expressed proteins are related to the function of Cell-To-Cell Signaling and Interaction?Tissue Development?Cellular Movement?Immune Cell Trafficking?Cardiovascular Disease and Hematological System Development and Function.There are 6 key changing pathways,include Acute Phase Response Signaling?LXR/RXR Activation?Coagulation System?Intrinsic Prothrombin Activation Pathway?Atherosclerosis Signaling?Extrinsic Prothrombin Activation Pathway and there are 4 protein-protein interaction networks.(2)There are 85 differentially expressed proteins in T-ALL group as oppose to control group,among them,43 proteins are up-regulation and 42 down-regulation.These differentially expressed proteins are related to the function of Cell-To-Cell Signaling and Interaction?Tissue Development?Cellular Movement?Immune Cell Trafficking?Cardiovascular Disease and Hematological System Development and Function.There are 7 key changing pathways,include Acute Phase Response Signaling ? LXR/RXR Activation ? Atherosclerosis Signaling?Clathrin-mediated Endocytosis Signaling?Production of Nitric Oxide and Reactive Oxygen Species in Macrophages?IL-12 Signaling and Production in Macrophages ? Agranulocyte Adhesion and Diapedesis and there are 6 protein-protein interaction networks.(3)There are 91 differentially expressed proteins in M2 group as oppose to control group,among them,27 proteins are up-regulation and 64 down-regulation.These differentially expressed proteins are related to the function of Cellular Movement?Immune Cell Trafficking?Cardiovascular Disease?Cell-To-Cell Signaling and Interaction ? Tissue Development ? Organismal Injury and Abnormalities ? Hematological System Development and Function ?Hematological Disease.There are 8 key changing pathways,include LXR/RXR Activation ? Acute Phase Response Signaling ? Coagulation System ?Atherosclerosis Signaling ? Intrinsic Prothrombin Activation Pathway ?Clathrin-mediated Endocytosis Signaling?IL-12 Signaling and Production in Macrophages?Production of Nitric Oxide and Reactive Oxygen Species in Macrophages and there are 5 protein-protein interaction networks.(4)There are 83 differentially expressed proteins in M3 group as oppose to control group,among them,33 proteins are up-regulation and 50 down-regulation.These differentially expressed proteins are related to the function of Cellular Movement?Immune Cell Trafficking?Hematological System Development and Function ? Cell-To-Cell Signaling and Interaction ? Tissue Development ?Cardiovascular Disease ? Inflammatory Response.There are 6 key changing pathways,include LXR/RXR Activation?Acute Phase Response Signaling?Atherosclerosis Signaling?Complement System?Production of Nitric Oxide and Reactive Oxygen Species in Macrophages?IL-12 Signaling and Production in Macrophages and there are 5 protein-protein interaction networks.(5)There are 84 differentially expressed proteins in M5 group as oppose to control group,among them,26 proteins are up-regulation and 58 down-regulation.These differentially expressed proteins are related to the function of Cardiovascular Disease?Cell-To-Cell Signaling and Interaction?Tissue Development?Metabolic Disease ? Hematological Disease ? Hematological System Development and Function?Organismal Functions?Immune Cell Trafficking.There are 7 key changing pathways,include Acute Phase Response Signaling ? LXR/RXR Activation ? Complement System ? Atherosclerosis Signaling ? Coagulation System?IL-12 Signaling and Production in Macrophages?Production of Nitric Oxide and Reactive Oxygen Species in Macrophages and there are 5 protein-protein interaction networks.(6)There are 79 differentially expressed proteins in B-NHL group as oppose to control group,among them,34 proteins are up-regulation and 45 down-regulation.These differentially expressed proteins are related to the function of Cell-To-Cell Signaling and Interaction ? Tissue Development ?Metabolic Disease?Cellular Movement?Immune Cell Trafficking?Hematological System Development and Function.There are 6 key changing pathways,include Acute Phase Response Signaling?LXR/RXR Activation?Intrinsic Prothrombin Activation Pathway?Coagulation System?Extrinsic Prothrombin Activation Pathway?Atherosclerosis Signaling and there are 4 protein-protein interaction networks.(7)There are 73 differentially expressed proteins in T-NHL group as oppose to control group,among them,45 proteins are up-regulation and 28 down-regulation.These differentially expressed proteins are related to the function of Organismal Injury and Abnormalities ? Metabolic Disease ?Cardiovascular Disease ? Cell-To-Cell Signaling and Interaction ? Tissue Development?Cellular Movement?Hematological System Development and Function?Immune Cell Trafficking.There are 6 key changing pathways,include Acute Phase Response Signaling ? LXR/RXR Activation ? Atherosclerosis Signaling ? Production of Nitric Oxide and Reactive Oxygen Species in Macrophages?Clathrin-mediated Endocytosis Signaling?IL-12 Signaling and Production in Macrophages and there are 4 protein-protein interaction networks.3.ELISA results indicated that LRG1 and S100A8 were significantly up-regulated in serum of childhood hematologic malignancies.SPARC was markedly down-regulated in serum of childhood acute leukemia compared to control group respectively.There were no difference between non-Hodgkin's lymphoma group and control group.Conclusion 1.The iTRAQ-2DLC-MS/MS technology,with the characteristics of fast and high throughput of protein changes,good repeatability,easy operation,has been widely used to tag peptides for multiplexed protein quantification and can be applied to look for different proteins associated with childhood hematologic malignancies.2.IPA analysis can further looking for different proteins related with hematologic malignancies and the interaction networks of signaling pathways.It may be helpful to early diagnosis and therapeutic targets for children with hematologic malignancies,and it may provide experimental basis for molecular mechanisms of childhood hematologic malignancies.3.The expression of LRG1 and S100A8 related to the pathogenesis of childhood hematologic malignancies,and the expression of SPARC related to the pathogenesis of childhood acute leukemia.These differential proteins may be serve as new diagnostic biomarker and potential therapeutic targets.Part Two Identification of B-cell acute lymphoblastic leukemia Associated Antigens and Autoantibodies by Serological Proteome AnalysisBackground and objective Acute lymphoblastic leukemia(ALL)is the most common malignancy of childhood,accounting for 30% of cancers,with the most common type of B-ALL.Currently,bone marrow aspiration is used for diagnosis.However,because of great damage,most of patients show different degrees of mental stress towards this examine.In recent years,with the intensified chemotherapy,the prognosis of ALL has been greatly improved but about 20% of patients with acute lymphoblastic leukemia struggle with relapse,despite intensive chemotherapy,and traditional chemotherapy drugs cause toxic side effects,which may lead to death.Therefore,noninvasive and specific biomarkers for the therapeutic targets of pediatric ALL are urgently warranted.Cancer onset and progression produce mutated or aberrantly expressed proteins that are able to act as antigens and evoke an immune response,a process which results in the production of autoantibodies.These autoantibodies can be detected months or years before the clinical diagnosis of cancer;therefore,tumor-associated antigens(TAAs)and their corresponding autoantibodies could be used as biomarkers for the early diagnosis?therapeutic targets and prognosis of cancer.Serological proteomics analysis(SERPA)is a new technology combing proteomics with immunology.The purpose of this study was to identify B-ALL related antigens and autoantibodies in sera from children with B-ALL using SERPA.Methods 1.We separated total proteins extracted from the three B-ALL cell lines(NALM-6?REH and BALL-1)by 2-DE.One of the three parallel 2-DE gels went for commassie blue staining while the others transferred onto PVDF membrances and underwent immunoblot.Mixed serum samples from 20 B-ALL children were used as the primary antibodies of the immunoblot.We detected 6 antigenic proteins spots that reacted to B-ALL sera but not to normal controls.Positive dots of immunoblot were identified by MALDI-TOF/TOF-MS/MS and PMF matching.The SERPA experiment is replicated by 3 times.2.The autoantibodies of?-enolase(ENO1)and voltage-dependent anion-selective channel protein 1(VDAC1)were further validated respectively by Indirect ELISA.3.The candidate antigens ENO1 and VDAC1 were further validated respectively in bone marrow by immunohistochemistry.4.The candidate antigens ENO1 and VDAC1 protein expression level in B-ALL cell lines were contrastly studied by Western Blot.Results 1.Six dots were identified with MALDI-TOF/TOF and PMF as Aconitase?apoptosis-inducing factor(AIF)?dihydrolipoamide dehydrogenase(DLD)?ENO1?medium-chain acyl-Co A dehydrogenase(MCAD)?VDAC1.2.The frequency of autoantibodies against ENO1 and VDAC1 with B-ALL were 27% and 23% respectively,which were significantly higher than that normal controls(4% and 0),and had no significant difference compared to other cancers(23% and 17%)in children.3.The result of immunohistochemistry showed significantly increased expression of ENO1 and VDAC1 were observed in bone marrow of children B-ALL compared with the normal bone marrow of children(P<0.05).4.Western Blot showed that there was no significant difference in the 3 B-ALL cell lines on expression of ENO1 and VDAC1(P>0.05).Conclusion 1.ENO1 and VDAC1 might be the candidate immunogenic antigens of children B-ALL.2.The autoantibody against VDAC1 may be a potential serological marker of children B-ALL.