(A Preliminary Study On Proteomics Screening Of Diffuse Large B-cell Lymphoma Resistance-associated Protein)

In China, one of the most common malignant tumors is lymphoma, which falls into two categories-Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL). Ninety percent of the lymphoma patients have non-Hodgkin lymphoma, and among them Diffuse large B-cell lymphoma (DLBCL) is the most common subtype, accounting for about30to40percent. At present, chemotherapy is the leading treatment of DLBCL, but the overall5-year survival rate is still less than50percent, mainly due to tumor cell resistance to chemotherapeutic drugs. Therefore, the research on mechanisms of chemotherapy resistance of DLBCL is crucial to improving the treatment efficacy of DLBCL patients.We carry out an differential proteomics analysis on the chemosensitivity of DLBCL tissue with high-throughput screening of apoptosis and resistance-associated protein, and we are expected to find the protein associated with the resistance to chemotherapy and provide new ideas for screening molecular markers of tumor resistance and reversal resistant to targeted therapy.Chapter1Differential proteomics analysis on the DLBCL tissue with different chemosensitivityObjective:In this study using proteomics approach we isolate and identify the differentially expressed proteins of DLBCL organizations in both CHOP chemotherapy high-sensitive group and low-sensitive group, which provides an important basis for determining the sign of the lymphoma chemotherapy sensitivity prediction and its key role in the molecular and chemosensitivity prediction objects.Method:We collect the fresh DLBCL tissue specimens, according to CHOP chemotherapy drug sensitivity tests divide them into low-sensitive group and high-sensitive group, evaluate the difference protein between the two group using2-DE+MALDI-TOF-TOF-MS method, obtain DLBCL chemotherapy resistance-associated protein differential expression spectrum. Then we carry out Bioinformatics analysis of the differentially expressed protein. By Western Blot validation we validate the expression levels of part of the differentially expressed protein and observe the relationship of four protein expression and DLBCL chemosensitivity.Results:By proteomic approach we obtain differential protein expression spectrum of high-sensitive group and low-sensitive group of CHOP chemotherapy of DLBCL tissue, and identify19differentially expressed proteins, including:S100A9, aldehyde dehydrogenase1, Annexin A5, Ezrin, Plecstrin (PLEK),14-3-3ε,61-kD tumor-associated antigen protein, histone H2A.2,14-kDa phosphohistidine phosphatase isoform3, collagen al, ferritin light subunit,6-phosphogluconolactonase, human rab GDP dissociation inhibition, coronin-like protein, heterogeneous nuclear ribonucleoprotein H3isoform a, pre-mRNA-processing factor19,3-hydroxyacyl-CoA dehydrogenase type2isoform1, glutathione S-transferase and heat shock protein beta-1. The function of these proteins involves metabolism enzyme, cell communication, cell cycle, immune system process, stress response, cell structure, molecular chaperones, genetic stability, protein synthesis and transport in the signaling pathway and etc. Part of these proteins may interact to each other by signal transduction network. We apply Western blot validation test to four proteins of the two groups, protein EZR, PLEK, GSTP1and HSPB1, and the result shows that expression EZR and PLEK are higher in the chemotherapy highly sensitive group than those in the low-sensitive group, while GSTP1and HSPB1are lower in high sensitive group of chemotherapy than those in low-sensitive group. Therefore we prove Proteome Research results have better repeatability, and these four proteins are of great significance in terms of chemotherapy sensitivity.Conclusion:1. Using2-DE combined mass spectrometry we obtain differential protein expression spectrum of high-sensitive group and low-sensitive group of CHOP chemotherapy of DLBCL tissue;2, we obtain19differentially expressed proteins related to CHOP chemotherapy resistance of DLBCL tissue, and these proteins’functions are related to metabolism enzyme, cell communication, cell cycle, stress response, cell structure, molecular chaperones, genetic stability, protein synthesis and transport in the signaling pathway and etc;and part of these proteins may interact to each other by signal transduction network;3, EZR, PLEK, GSTP1and HSPB1are related to DLBCL chemosensitivity through Western blot validation.Chapter2, Clinical pathological significance of part of the diffrentially expressed proteins and the effect of HSP27expression on chemosensitivity of diffuse large B-cell lymphomaObjective:In this study we further use immunohistochemistry method to detect the expression of the meaning of these four proteins in clinical samples. Then we further explore the role and mechanism of HSP27expression in chemotherapy sensitivity of diffuse large B-cell lymphoma in in vitro cell and molecular biology level. Methods:By immunohistochemistry validation we further validate the expression levels of part of the differentially expressed protein and observe the relationship of four protein expression and DLBCL chemosensitivity. Further We use shRNA blocking HSP27expression, Western blot verify blocking effect and detect the expression of14-3-3ε; we detect IC50value and resistance index of Farage-LV-HSP27-shRNA cells to doxorubicin, cyclophosphamide, vincristine; we use flow cytometry technique to examine the effect change of doxorubicin, cyclophosphamide, vincristine on tumor cells and promote apoptosis-related role after blocking HSP27expression levels in Farage cells.Results:Further we use immunohistochemistry method to detect the expression of the meaning of these four proteins in clinical samples. The clinical tissue samples are also divided into chemotherapy high-sensitive group and chemotherapy low-sensitive group according to the clinical efficacy of chemotherapy, and the results are agreed with Western blot results. Therefore we furthur prove Proteome Research results have better repeatability, and these four proteins are of great significance in terms of chemotherapy sensitivity. After shRNA blocking HSP27expression, Western blot verify the blocking is effective; IC50of doxorubicin, cyclophosphamide, vincristine decreased significantly in Farage-LV-HSP27-shRNA cells compared with the control cells, indicating that HSP27down regulation is able to increase the sensitivity of diffuse large B-lymphoma cells to chemotherapeutic drugs. Blocking HSP27expression in tumor cells apoptosis rate increase, while in the intensive chemotherapy drug doxorubicin, cyclophosphamide, vincristine increase apoptosis rate more obviously, which further prove that the HSP27downward is able to increase Farage cells sensitivity to doxorubicin, cyclophosphamide, vincristine. In this study we also find that blocking HSP27cause downregulation of14-3-3ε, suggesting that molecular mechanism of HSP27mediated chemotherapy sensitivity is associated with14-3-3εConclusion:1. Blocking Hsp27expression is able to increase sensitivity of DLBCL cells to doxorubicin, cyclophosphamide, vincristine;2, The interaction of14-3-3ε and HSP27is meaningful in diffuse large B-cell lymphoma chemosensitivity.