Preliminary Study On The Mechanisms Of Stathmin Involves In The Differentiation Of H/RS Cells Into B Cells

BackgroudHodgkin lymphoma is one of the malignant tumor of lymphoid tissues. The classic Hodgkin lymphoma (cHL) which is the major type of HL, accounts for about95%of the cases. The cHL is a disease of monoclonal leukomonocyte lymphoma and characterized by the rare so-called Hodgkin/Reed-Sternberg (H/RS) tumour cells (<1%) embedded in the background of predominant recative inflammatory cells. About98%of the HRS cells differentiated from mature B cells in germinal center stage, and quite few originated from the peripheral T cells.Our research group have previously constructed subseries of L428cell line with stable overexpression of CD99gene, named "L428-CD99". We have identified38differentially expressed proteins, using two-dimensional gel electrophoresis and mass spectrometry. Finally we choosed one differentially expressed protein---stathmin as our research target.Stathmin is a small molecule phosphoprotein, widely exists in the cytoplasm. It belongs to an microtubule-associated protein, which can adjust the microtubules depolymerization, participate in microtubules assembly, and keep the microtubules dynamic balance. Stathmin also combines with a variety of proteins and associated with cell cycle, proliferation and movement, and many intracellular biological process. Many studies show that Stathmin protein is closely related with tumorigenesis and overexpressing across a broad range of human malignancies.The present studies indicate that stathmin protein also plays a very important role in cell differentiation process. In normal lymph hematopoietic system, stathmin is involved in T cell maturation in thymus gland; suppression of stathmin is biologically important for megakaryocyte maturation and platelet production. In hematopoietic malignancies, the high level of stathmin expression in leukemic cells is necessary for the maintenance of the transformed phenotype. However, there is no report referring to whether and how stathmin takes part in B cell differentiation process.Our object is to detect the expression and localization of stathmin by the methods of immunohistochemistry, real-time PCR, western blot and confocal microscope in L428and L428-CD99cell lines, verify the expression of PRDM1, differentiation-related cell phenotype and biological characteristics after transient interference of stathmin gene, and explore whether stathmin can induce redifferentiation of H/RS cells to terminal B cells. Further, we would investigate the relationship among NF-κB signaling pathway, stathmin and PRDM1and the possibility of induction of redifferentiation of H/RS cells by regulated NF-κB signaling pathway. Our research will provide reference for further elaborating the mechanisms of generation and development of H/RS cells.ObjectiveThis study will be carried out in four parts: 1. The expression of stathmin in B-cell original lymphoma tissues and cell lines.Detecting differences of stathmin expression in RH, B-cell original lymphoma tissues and cell lines and investigating the pattern of stathmin in B-cell differentiation stage.2. The expression level and localization of stathmin in cHL cell lines.Detecting differences of stathmin expression level and localization in L428and L428-CD99cells.3. The regulation on stathmin inducing redifferentiation of H/RS cells.Exploring the interaction between stathmin and B-cell differentiation, and detecting the effect of downregulation of stathmin gene on PRDM1, differentiation-related phenotype and biological characteristics in L428and L428-CD99cells, revealing the role of stathmin gene in B cell differentiation.4. The regulation of NF-κB signal pathway on stathmin inducing redifferentiation of H/RS cells.Exploring the interaction between NF-κB signal pathway and stathmin, and revealing the role of stathmin gene via NF-κB signal pathway in B cell differentiation.Contents and methods1. The expression of stathmin in B-cell original lymphoma tissues and cell lines.The specimens of RH、cHL、GCB-DLBCL、ABC-DLBCL and PCM cases were collected and analyzed by immunohistochemistry of stathmin protein. Stathmin protein expression was further measured by Western blot in five B-cell original lymphoma cell lines.2. The expression level and localization of stathmin in cHL cell lines.The stathmin and acetylation a-tubulin protein expression and co-localization were observed by western blot and confocal microscope. The stathmin and PRDM1expression were measured by real-time PCR, western blot and immunocytochemistry in L428and L428-CD99cell lines.3. The regulation on stathmin inducing redifferentiation of H/RS cells.(1) The effect of transient interference of stathmin downexpression on B cell differentiation in L428and L428-CD99cell lines.Interference efficiency was detected by real-time PCR after transient transfection of stathmin siRNA. The stathmin and PRDM1protein expression were detected by Western blot, and the differentiation-related proteins were detected by flow cytometry after knockdown of stathmin gene.(2) The effect of transient interference of stathmin downexpression on biological characteristics of L428cells.The effect of stathmin downexpression on proliferation、apoptosis and cell cycle were detected by CCK8and flow cytometry.4. The regulation of NF-κB signal pathway on stathmin inducing redifferentiation of H/RS cells.(1) The effect of regulation of NF-κB signal pathway on B cell differentiation. The stathmin and PRDM1protein expression were detected by RT-PCR and Western blot after regulation on NF-κB signal pathway on L428、L428-CD99cells, and the differentiation-related proteins were detected by flow cytometry after regulation on NF-κB signal pathway on L428cells.(2) The effect of regulation of NF-κB signal pathway on biological characteristics of L428cells.The effect of regulation of NF-κB signal pathway on proliferation, apoptosis and cell cycle were detected by CCK8and flow cytometry. Results1. The expression of stathmin in B-cell original lymphoma tissues and cell lines.(1)In reactive lymphoid hyperplasia tissues, the majority of stathmin-positive cells were present in the germinal centres, whereas cells in the mantle zone were essentially negative and the interfollicular areas showed occasional positive cells; In Hodgkin lymphoma, the H/RS cells showed strong or intermediate stathmin expression, and other cell types in background showed variable stathmin expression; DLBCL contained a large fraction of stathmin-expressing cells, and GCB-DLBCL had stronger staining intensity than ABC-DLBCL; PCM showed stronger staining intensity than ABC-DLBCL(2) Stathmin were highly expressed in L428, OCI-Ly8, RPMI-8226and KM3cells, but lowly expressed in OCI-Ly10cells. In RPMI-8226and KM3, the expression of stathmin protein was stronger compared with that of L428, OCI-Ly8.2. The expression level and localization of stathmin in cHL cell lines.(1) CD99-upregulated L428cells showed increased expression of CD99gene and protein expression compared with mock and empty vecter groups detecting by real-time PCR(F=33.311, P=0.013), western blot.(2) Expression of stathmin and acetylation α-tubulin significantly increased in L428-CD99cells detecting by western blot and confocal microscope. The co-localization of stathmin and a-tubuli、stathmin and acetylation a-tubulin were observed in L428and L428-CD99cells with confocal microscope. The stathmin protein was located in cytoplasm and cell membrane, and stathmin protein was mainly located in filopodia and filamentous actin of cell membrane in L428cells, however increased expression of stathmin protein in cytoplasm and decreased expression in cell membrane were observed in L428-CD99cells. Stathmin protein took part in the reorganization of actin cytoskeleton in L428-CD99cells. (3)The expression of stathmin and PRDM1significantly increased in L428-CD99cells compared with control cells detecting by western blot, RT-PCR (F=23.834, P=0.020)(F=205.232, P<0.001) and immunocyte chemistry.3. The regulation on stathmin inducing redifferentiation of H/RS cells.(1) Transient interference of stathmin downexpression in L428cells resulted in decrease of stathmin expression in mRNA level detecting by qRT-PCR(F=47.155, P<0.001), and decrease of stathmin protein and increase of PRDM1protein detecting by western blot. Transient interference of stathmin downexpression in L428-CD99cells resulted in decrease of stathmin expression in mRNA level using qRT-PCR(F=47.770, P<0.001), and decrease of stathmin and PRDM1protein detecting by western blot.(2)Transient interference of stathmin downexpression in L428cells resulted in decrease of diognosis markers of cHL (CD15), and significant increase of plasma cell markers (CD38and CD138).(3) Transient interference of stathmin downexpression in L428cells resulted in decrease of cell proliferation ability detected by CCK8assay (F=122.349, P<0.001; F=1106.644, P<0.001; F=25.100, P<0.001), and increase of cell apoptosis rate by flow cytometry. Stathmin inhibition showed G2/M arrest.4. The regulation of NF-κB signal pathway on stathmin inducing redifferentiation of H/RS cells.(1) The regulation of NF-κB signal pathway in L428cells resulted in decrease of stathmin expression in mRNA level detecting by qRT-PCR(t=30.280, P<0.001), and decrease of stathmin protein and increase of PRDM1protein detecting by western blot. The regulation of NF-κB signal pathway in L428-CD99cells resulted in increase of stathmin expression in mRNA level using qRT-PCR (t=-18.082, P<0.001), and increase of stathmin protein, decrease of PRDM1protein and no significant change of CD99protein using western blot.(2) The regulation of NF-κB signal pathway in L428cells resulted in decrease of diagnostic markers of cHL (CD15), and significant increase of B cell differentiation-related proteins (CD10and CD45) and plasma cell markers (CD38and CD138) detecting by flow cytometry analysis.(3) The regulation of NF-κB signal pathway in L428cells resulted in decrease of cell proliferation ability detecting by CCK8assay ((F=297.382, P<0.001; F=968.735, P<0.001; F=71.293, P<0.001), and increase of cell apoptosis rate detecting by flow cytometry. NF-kB activation inhibition induces G1/S arrest.Conclusion1. The stathmin showed decreasing expression inclination from germinal center stage to post germinal center stage, and increasing expression inclination from post germinal center to plasma cell stage. This phenomenon suggests that the expression of stathmin is related to B-cell differentiation.2. Stathmin protein takes part in reorganization of actin cytoskeleton in the process of H/RS cells towards B cells.3. Stathmin may negatively regulate the expression of PRDM1in L428cells and positively regulate the expression of PRDM1after overexpressed CD99gene in L428cells.4. NF-κB signal pathway may positively regulate stathmin, and negatively regulate PRDM1.5. Both transient interference of stathmin downregulation and activity inhibition of NF-κB signal pathway in L428cells resulted in decrease of diagnostic markers of cHL (CD15), and significant increase of plasma cell markers (CD38and CD138) detected by flow cytometry analysis, trend of H/RS cells towards to terminal B-cells, decrease of cell proliferation ability, and increase of cell apoptosis rate. Stathmin inhibition showed G2/M stage arrest. NF-kB activation inhibition induces G1/S arrest.Discoveries and innovations1. We have preliminarily revealed that the expression rule of stathmin in B-cell differentiation.2. We have preliminarily revealed that NF-κB signal pathway may regulate stathmin to mediate PRDM1expression, which induces H/RS cells differentiation towards terminal B cells. This research provide reference for further elaborating mechanisms of generation and development of H/RS cells.