| BackgroudHodgkin’s lymphoma (HL) is one category of malignant lymphomas, with characteristic Hodgkin/Reed-Sternberg (H/RS) tumorous cells. But the proportion of H/RS cells is of little and less than1%of the whole bulk of the tumor, with the remanent predominant T cell background of reactive inflammation. The frequent variation of the tumors further strengthen the diagnostic dilemma. To master a series problems including the intrinsic qulity of the H/RS cells, the mechanism of the origination and development of H/RS cell, the complicated relationship between H/RS cells and the background must be elucidated. To explore these promlems from the root, a suitable animal model with similar characteristic pathologic feature of HL is required. Though a large quantity of human originated H/RS exist, their tumorigenesis of H/RS cell in animal is rarely reported. Research indicates that successful tumorigenesis of H/RS cell in animals restricted to the severe immunodeficient mice like SCID, BNX, NOG. But the more severe the immunodeficiency of the animal, the less the tumorigenesis can reveal the character of the tumor with a background of abundant immunity and inflammation. It is our a long-term vision to built animal model with H/RS cells of mouse.Kiippers et al. revealed that H/RS cell originated from crippled B cells in the germinal center. The research speculated that some cells underwent a favored mutation selection, and accepted by T helper cells and follicular dendritic cells. With a repeating process of proliferation, mutation and selection, the positively selected cells differentiated to plasma cells and memory B cells. The other group, however underwent an unfavorable mutation, like negative mutation, automatic reactive acquisition, and became the so called functionally crippled pro-apoptotic cells, and underwent a programmatic cell death. Some crippled pro-apoptotic cells lost their superficial markers partly or completely, and presented the characteristic antigen marker CD15and CD30, as one of the biological character of H/RS cell. Because of the change of immune phenotype and receptor of H/RS cell, the cell escaped the immune surveillance and cell apoptosis, survived and proliferated in clone.The human CD99is a glycosylated transmembrane protein. Research showed that the down-regulation of CD99was related to the forming of human H/RS. Our research group has previously transfected the CD99gene into the CD99negative HL cell line L428by lipidosome transfection, and screened out the CD99stably expressing cell suline L428-CD99. The preliminary studies indicated that L428-CD99cell subline reappeared with some of the B cell features. We further found that the mouse originated CD99(mouse CD99antigen-like2, mCD99L2) was highly homologous with that of human. Letivirus ShRNA vector was applied the transfect the endogenous mCD99L2gene positive B lymphoma cell line A20, and screened out the interfering vector stably expressing cell subline LV-mCD99L2-A20, and preliminarily proved that some of the LV-mCD99L2-A20cells owned similar character with that of human H/RS cells.Our research group have previously constructed subseries of L428cell line with stable overexpression of CD99gene and A20cell line with low mCD99L2gene. On this basis,2D-DIGE and Mass Spectrometry will be applied to compare the protein expression difference between the two cells lines before and after their treatment. The target protein that interact with human and mouse CD99will be isolated and indentified. From the data obtained from the bioinformatics research and analysis of both the group of human and mouse, we expect to screen out the important proteins that functioning during the cell transformation, analyze and prove their functions and the possible related signal transduction pathway. Our study may provide the role and a theoretical basis for interacting protein with mCD99L2in building H/RS cell model and HL animal model.ObjectiveContents and methods1. Identification of L428-CD99cell subline and LV-mCD99L2-A20cell subline.(1) Identification of L428-CD99cell sublineThe previously constructed L428-CD99clone cell line with stable expressiom of CD99gene was contiously subcultured, and the mRNA and protein expression of CD99was measured by real-time PCR, Western blot and confocal microscope.The effect of CD99low expression on proliferation, celluar size and cytoskeletal proteins was detected by MTT, HE staining and phalloidin staining.The diognosis markers were detected by immunocytochemistry and flow cytometry.(2) Identification of LV-mCD99L2-A20cell sublineThe previously screened out LV-mCD99L2-A20cell line with stably interfering expressiom of mCD99L2was contiously subcultured. The general primers for amplification of the vector gene were applied for PCR detection of the situation of the interfering vector that integrating into LV-mCD99L2-A20cells. The mRNA and protein expression of mCD99L2was measured by real-time PCR, Western blot.The effect of mCD99L2overexpression on morphological changes and proliferation was detected by HE staining and MTT.2.Proteomic analysis of CD99+/mCD99L2-regulating the transformation between H/RS and B lymphoma cellsDifferential proteome analysis of upregulating human CD99or downregulating mouse CD99Like2gene during translation between Hodgkin/Reed-Sternberg and B lymphocyte cell was conducted using two-dimensional differfntial in gel electrophoresis combined with matrix-assisted laser desorption/time of fight (MALDITOF)mass spectrometry. Further, cluster analysis was carried out using the ways included the software Gofact (http://www.hupo.org.cn/gofact), annotation of differentially expressed proteins with the proceeds of the involved biological processes, composition of cell components and molecular function.3. The screening and primary identification of differentially expressed proteins in CD99+/mCD99L2-regulating the transformation between H/RS and B lymphoma cells(1) Screening and analysis of differently expressed proteinsBioinformatics network was employed to predict interactive proteins with CD99and mCD99L2before2-D DIGE and mass spectral analysis to screen the differentially expressed proteins in human CD99+/mice mCD99L2-regulating the transformation between H/RS and B cell lymphoma cells(2) Expression verification of targeted proteins in human cells and tissuesmRNA and expression of targeted proteins in L428-CD99and L428-CTR were tested by RT-PCR, immunocytochemistry, Western blot and Confocal Immunofluorescence Microscopy(3) Expression verification of targeted proteins in mice cells Expression of targeted Proteins in LV-mCD99L2-A20with stable RNA interference of mCD99L2and LV-Gus-A20cells transfected with empty vector were examined by immunocytochemistry and Western blot.4. Primary biofunction verification and signal transduction analysis of differently expressed Septin-2in L428cells and B lymphoma cells(1) The interaction between Septin-2and regulating factors of H/RS cells and B lymphocytesThe interaction between Septin-2and nf-kappaB and c-Myb was examined by Bioinformatics and Western Blot(2) The biofunction verification of Septin-2in H/RS-B cell lymphoma cells transformationMicroscopy and Fluorescent Phalloidin Staining of Septin-2interfered L428cells were performed to test the effect of Septin-2RNA interference on cellular morphology and cytoskeletal protein. CCK-8experiment and flow cytometry was conducted to test the effect of Septin-2RNA interference on cell proliferation and immunophenotype of L428cells.(3) The analysis of the possible role of Septin-2in signal transduction of H/RS-B cell lymphoma cells transformationSignal cascase proteins were tested by Western blot before and after stable RNA interference of CD99and transient interference of Septin-2. Combined with bioinformatics, proteomics and literature data, the role of Septin-2in the signal transduction pathway of CD99regulating H/RS to transform into B cell lymphoma was analyzed.Results1. Identification and analysis of cell fraction of L428-CD99and LV-mCD99L2 -A20cells(1) Iidentification of cell fraction of L428-CD991) RT-PCR, Real-Time Reverse Transcription PCR and Western Blot showed the increase CD99gene and its expression in L428-CD99compared to empty vector2) Significant difference was found (t=7.131, P=0.018) in cell volume of L428-CD99microscopically, phalloidin staining revealed cytoskeleton remodelling in L428-CD99compared with control group.3) MTT Assay showed cells with CD99over expression has a decreased cell proliferation(F=773.374, P=0.000),compared with empty vector4) Flow cytometry (FCM) and immunohistochemistry (IHC) showed that in comparison with control group, cells with CD99over expression significantly reduced the expression of CD30, CD15and MUM1, promoted the expression of CD10、CD19、CD79α、BCL-6、PAX5and CD38, but had no impact on CD20and CD138.(2) Iidentification of cell fraction of LV-mCD99L2-A201) PCR confirmed the stable intergration of interference vector into LV-mCD99L2-A20genomem, RT-PCR and Real-Time RT-PCR showed the mRNA copy numbers of mCD99L2in LV-mCD99L2-A20is reduced with an efficient rate of51%.2) Reverse microscope and HE staining demonstrated that the cell volume of the subcultureed LV-mCD99L2-A20increased significantly, presented with giant cells the resembling human H/RS cells. MTT assays revealed RNA interence of mCD99L2significantly supressed the proliferation of A20.2.Proteomics of CD99+/mCD99L2-regulating the transformation between H/RS and B lymphoma cells.(1)2-D DIGE and mass spectral analysis of human tumor derived cell lines harvested38differently expressed proteins,21of which was positivly correlated with L428-CD99and17had negative correlation. Rho GDP-dissociation inhibitor2(GDIR2) and Septin-2(SEPT2) are related to cytoskeleton organization; DNA mismatch repair protein Msh2(MSH2), Hematopoietic lineage cell-specific protein are related to cell differentiation; Rho GDP-dissociation inhibitor2(GDIR2)、 Prostaglandin E synthase3(TEBP)、Prohibitin (PHB)、Hematopoietic lineage cell-specific protein (HS1)、Sorcin (SORCN) and GEM-interacting protein (GMIP) are related to signal transduction; Prohibitin(PHB)、Heat shock protein beta-1(HSPB1)'Hematopoietic lineage cell-specific protein (HS1) are related to regulation of gene expression(2)2-D DIGE and mass spectral analysis of cell lines derived from murine tumor targeted42proteins,21and20of them were positively and negatively correlated with LV-mCD99L2-A20, respectively. Actin, cytoplasmic1(ACTB)、Septin-2(SEPT2)、 PDZ and LIM domain protein1(PDLI1)、Stathmin (STMN1) are related to cytoskeleton organization; stathmin(STMN1) and ADP-ribosylation factor-like protein6(ARL6)are related to cell differentiation; Ran GTPase-activating protein1(RAGP1)、 ADP-ribosylation factor-like protein6(ARL6) are related to signal transduction; Eukaryotic translation initiation factor4E (IF4E)、Nucleophosmin (NPM)、60kDa heat shock protein, mitochondrial (CH60)、PDZ and LIM domain protein1(PDLI1)、 Hypermethylated in cancer2protein (HIC2)、Poly(U)-binding-splicing factor PUF60(GCP60) are related to regulation of gene expression.3. Screening and identification of the differential expressing proteins in the process of CD99+/mCD99L2regulating the transformation between H/RS and B lymphoma cells(1) Bioinformatics retrieving and differential protomics analysis of humanity and mouse transforming cell lines pinpointed Septin-2and Stathmin as the targeted protein for further investigation.(2) Real time RT-PCR, IHC, ICC, WB and Confocol showed significant difference between L428and L428-CD99in terms of mRNA and proteins expression of Septin-2and Stathmin. Septin-2negatively correlated with CD99expression while Stathmin positively correlated with CD99expression.(3) ICC and WB detection indicated that differential expression of Septin-2and Stathmin existed between mouse originated transforming cell A20andLV-mCD99L2-A20, while they were consistent with that of humanity transformed cells.4. Biofunction verification and signal transduction pathways analysis of Septin-2in L428and B lymphoma cells(1) The Septin-2upstream regulating factors NF-kappaB1, c-Myb and XBP-1were also the regulating factors in promoting H/RS formation and B lymphocyte differentiation. WB showed that NF-kappaB1and c-Myb were involved in H/RS-B cell lymphoma cells transformation. The expression of Septin-2was regulated by nf-kappaB and c-Myb. Septin-2expression was positively correlated with that of nf-kappa B and negatively correlated with that of c-Myb, while the expression nf-kappa and c-Myb were not modulated by Septin-2.(2) Consecutive observation with inverse microscope and confocol assay showed that L428with transcient RNA interference of SEPT2presented with remodeling of cell structure and vanishing of pseudo foot process.(3) CCK8experiment revealed the reducing of cell proliferation of L428-sisept2compared with that of L428-cn (F=204.927, P=0.000).(4) FCM showed partial changes of antigen phenotype in L428after RNAi of Septin-2. The expression of characteristic markers CD30and CD15reduced while the B cell marker CD19increased. The expression of germinal center marker CD10and early stage marker of plasma cell CD38also increased. (5) WB tests showed RNAi of SEPT2and over-expression of CD99in L428was related to the reduction of RhoA expression. Combined with of differential protein expression analysis and bioinformatic retrieving of RhoGDI2, HS1and Stathmin, we suggested that CD99regulating H/RS cell and B lymphoma cell transformation by up-regulating RhoGDI2for the inhibition of Rho GTPases in the Rho Family GTPases signal transtdution pathway. It mainly inhibited the activity of RhoA as the way to inhibit the expression of cell structure GTP binding protein Septin-2, promote the remodeling of cell structure and inhibit cell proliferation. Meanwhile, Septin-2also participated in the B cell receptor pathway by regulating RhoA protein, remodeling the transformed cell structure through co-balancing with HS1, participated in regulating B cell transcript factor and B cell antigen differentiation with the help of Stathmin..Conclusion1. The formerly constructed cell subline L428-CD99and LV-mCD99L2-A20are confirmed by consecutive subculture and assay that CD99+/mCD99L2regulates the transformation between H/RS cell and B cell lymphoma.2.Among the38targeted proteins identified in CD99over-expressed L428,21are up-regulated and17down-regulated. Among the41proteins selected from mCD99L2dow-regulated A20,22are upregulated and20are down-regulated. Some of targeted proteins are involved in the process of cell differentiation, signal transduction and gene transcription regulation.3. Septin-2and Stathmin are differently expressed in both human and mice derived cells. Septin-2negatively correlated with CD99while Stathmin positively correlated with CD99.4. Primary conclusion can be drawn that Septin-2is involved in cellular morphological change by participating structure remodeling in H/RS cells transformation, reducing cell proliferation and plays an important role in antigen differentiation process in H/RS-B lymphocyte cells transformation by Rho Family GTPases signal cascade and B cell receptor signal pathway.Discoveries and innovations1. Based on previously established cell subline with the character that CD99+/mCD99L2-regulates H/RS-B lymphocyte cells transformation with cell models of L428-CD99and LV-mCD99L2-A20, this study further investigate the role of different expression of Septin-2and Stathmin in transfoming cells. Protomics detection reveals the related proteins in cell differentiation, signal transduction,gene transcription regulation and cell transformation.2. This study provided Preliminary verification that in the process CD99+/mCD99L2-regulating the transformation between H/RS and B lymphoma cells, Septin-2may suppress cell proliferation by regulating cell structure remodeling and morphological change, as well as participation in antigen differentiation. While CD99regulates H/RS to lymphocyte transformation, Septin-2participates in ho Family GTPases and BCR signal transduction pathways. |
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