| BackgroundAn increasing incidence of non-Hodgkin lymphoma(NHL)has been reported in the worldwide,with 300,000 new cases a year.Diffuse large B-cell lymphoma(DLBCL)is the most common type of NHL,accounting for 30%to 40%of all newly diagnosed cases.DLBCL is regarded as an aggressive behaving and heterogeneous group of lymphomas in terms of morphologic,immunologic,and cytogenetic features.According to the 2008 WHO classification of tumors of haematopoietic and lymphoid tissues,DLBCL can be divided into the following four subtypes:Diffuse large B cell lymphoma,not otherwise specified(DLBCL,NOS);Diffuse large B cell lymphoma subtypes;Other lymphomas of large B cells and the borderline cases.DLBCL,not otherwise specified(NOS)is the most common subtype.Molecular subgroups allow subdivision of DLBCL,NOS into Germinal centre B-cell-like(GCB)and Activated B-cell-like(ABC).The most commonly used initial therapy for DLBCL is R-CHOP(rituximab,cyclophosphamide,doxorubicin,vincristine,and prednisone)or plus radiation therapy.Application of rituximab make early stage of DLBCL,GCB patients reached 80%-90%of the remission rate and disease-free survival.Literature shows that relapse cases of GCB(10%-20%)and refractory cases of ABC accounted for 50-60%of the total cases of DLBCL,NOS,and the majority of patients who fail R-CHOP will ultimately died from their disease.Thus,optimization of front-line therapy,as well as the development of more effective salvage strategies,remains an important objective.Inducing differentiation therapy is known as the redifferentiation of tumor cells,means that inducing agents can help tumor cells differentiation to normal cells by changing morphology,biological behavior and protein markers of the cells.It will become a new direction of tumor therapy.During the earlier stage of experiment,our team found that L428-CD99(over-expression CD99 gene in L428 cells)appeared B cell phenotype,which could open the switch protein(PRDMI)of plasma cell differentiation.CD99 over-expression could induce L428 cell differentiation towards terminal B cells.Next,we carried out two-dimensional electrophoresis(2-D DIGE)analysis L428-CD99 and L428-Con cells,and applied high flux peptide mass fingerprint spectrometry to identify differences protein,and found Hematopoietic cell-specific Lyn substrate I(HS1)was one of the over-expression genes.We further researched HS1 function based on the GO,by using GO fact open software online clustering.This result indicated that HS1 may participate in the differentiation of B cell.Thus,we determinate HS1 as our research object.Hematopoietic cell-specific Lyn substrate 1(HS1,namely as HCLS1,CTTNL,p75,IckBP1)mainly express in normal bone marrow,lymph node,tonsil,spleen,thymus,peripheral blood cells.Interactions among HS1,HAX1 and LEF-1 proteins are essential for G-CSF-triggered granulopoiesis.HS1 interacts with Lyn and is critical for erythropoietin-induced differentiation of erythroid cells.HS1 protein is a major substrate of protein-tyrosine kinases upon B-cell antigen receptor-mediated signaling pathway,regulates B lymphocyte proliferation and antigen receptor induced apoptosis.However,is HS1gene expressed in B cells at various stages of differentiation and plasma cells?Is HS1 expression involved in the differentiation of B cells into plasma cells?Up to now,it has not been reported,and need to be further studied.Objective1.Detecting expression profiles of HS1 in human B cell subpopulations,lymphoma cell lines and lymphoma tissues.Analysis significance of HS1 expression in clinical diagnose between classical Hodgkin lymphoma and T cell/histiocyte-rich large B-cell lymphoma.2.Constructing sub-cell lines of DLBCL with HS1 was knocked down.3.Detecting characteristics of plasma cell differentiation in the cell lines of DLBCL with HS1 knockdown.Exploring the possibility and potential mechanism that HS1 knockdown induces DLBCL cell lines differentiation.4.To explore the relationship between HS1 was knocked down and the activation of NF-?B signal pathway was inhibited in cell redifferentiation of ABC type DLBCL cell5.Detecting cell morphology and cytoskeleton protein in the cell lines of DLBCL with HS1 knockdown.Exploring the influence on cell apoptosis after HS1 was knocked down in DLBCL cell lines.Method1.Detecting expression profiles of HS1 in human B cell subpopulations,lymphoma cell lines and lymphoma tissues.Expression of HS1 was detected in classical Hodgkin lymphoma and T cell/histiocyte-rich large B-cell lymphoma tissues.HS1 protein was detected in tonsils with chronic tonsillitis and lymph node with reactive hyperplasia by using immunohistochemistry.And FACS analysis was used to isolation B cell subpopulation according to the B cell surface markers were differ from different B cell subsets by the use of mAbs of B cell surface marker.HS1 mRNA was detected in B cells,Naive B cells,centroblast cells,centrocyte cells,Memory B cells,plasma cells and lymphoma cell lines by RT-PCR.HS1 protein was detected in normal B cells and lymphoma cell lines by Western Blot,was detected in B cell lymphomas and Hodgkin lymphoma tissues by using immunohistochemistry.Finally,discover the significance of HS1 protein expression in clinical diagnosis.2.Constructing sub-cell lines of DLBCL with HS1 was knocked down,and analysis their biological characteristicsLentiviral vector for shHS 1 and control Lentiviral vector in this experiment were used,both of which were provided by Suzhou Gene Pharm.DLBCL cell lines(GCB:OCI-Ly1,OCI-Ly8;ABC:OCI-ly3 and OCI-Ly10)were transfected with containing recombinant lentiviral vector virus supernatant(including experimental group and control group),and named as OCI-Lyl-shHSl/NC,OCI-Ly8-shHS1/NC,OCI-Ly3-shHSl/NC and OCI-Ly10-shHSl/NC.Both experimental group and control group of the lentivirus with puromycin and GFP(Gluc-GFP)double label.The transfected cells were purified by using puromycin treatment,and the efficiency of transfection and purification was observed under the fluorescence microscope.RT-PCR and Western Blot were used to verify the mRNA and protein levels respectively.Cell apoptosis and cell cycles were detected by flow cytometry,and cell proliferation was detected by CCK8 assays3.HS1 knockdown can induce cell lines of DLBCL,ABC towards plasma cell differentiationDetection expression of plasma cell markers CD38 and CD 13 8,and B cell marker CD 19 in sub-cell lines of DLBCL by using flow cytometry.Observing cell morphology with HE(haematoxylin-eosin)staining under the microscope.Detection immunophenotype(such as CD38?CD138?CD20?BCL6?Mum-1)related to plasma cell differentiation by using immunocytochemistry in the sub-cell lines.Analysis of transcription factors(including PRDM1,Mum-1,BCL6 and Pax5)related to plasma cell differentiation by using RT-PCR and Western Blot assay in the sub-cell lines.4.To explore the relationship between HS1 was knocked down and the activation of NF-?B signal pathway was inhibited in cell redifferentiation of ABC type DLBCL cellAnalysis of protein expression of HS1,NF-?B signal pathway components and transcription factors(including PRDM1,Mum-1,BCL6 and Pax5)related to plasma cell differentiation by using Western Blot assay in the cell lines,which was conducted with HS1 knockdown and/or added NF-?B signal pathway inhibitor.5.Observe cell morphology,distribution of F-actin and DBNL under the laser scanning confocal microscope in the sub-cell lines,and analysis the impact on cell apoptosis.Identification of HS1 interaction with DBNL in normal B cells by CO-IP.Observe cell morphology,distribution of F-actin(Rhodamine tag ghost cyclic peptide)and DBNL(cell immunofluorescence)under the laser scanning confocal microscope in the sub-cell lines.Analysis of cytoskeleton protein(DBNL)after HS1 was knocked down by using Western Blot.Statistical analysisThe SPSS software]7.0 was used for the statistical analysis.Data were represented as the mean ± Standard Error of Mean(S.E.M).All experiments were representative of three independent experiments.Differences were considered statistically significant at P<0.05,analyzed for statistical significance using one-way analysis of variance(ANOVA)for assays with RT-PCR,and two independent samples nonparametric test for CCK8 assays and apoptosis assays.Result1.Detecting expression profiles of HS1 in human B cell subpopulations,lymphoma cell lines and lymphoma tissues.Analysis significance of HS1 expression in clinical diagnose between classical Hodgkin lymphoma and T cell/histiocyte-rich large B-cell lymphoma.(1)HS1 protein was detected in tonsils with chronic tonsillitis and lymph node with reactive hyperplasia by using immunohistochemistry(IHC).To identify HS1 protein expression in normal lymphoid tissue,we carried out IHC experiments on tonsil and lymph node tissues,and found that both tissues were strong staining of HS1.This phenomenon noted that HS1 was ubiquitously expressed in follicular,perifollicular area,interfollicular area,marginal zones,and even in epithelia cells of tonsil.We also found that HS1 expression in germinal center B cells were bright staining than marginal and mantle zones.Somewhat surprisingly,image showed that HS1 protein expression in plasma cell was negative.(2)Isolation of the B cell subpopulation according to the B cell surface markers.In our study,B cells(CD19+)and plasma cells(CD19-CD38+CD138+)were purified from other sorts of cells,B cells(CD19+CD38-)were further sorted into Naive B(IgD+)and Memory B(IgD-)cells,and B cells(CD19+CD38+IgD-)were further sorted into centrocyte(CD77+)and centroblast(CD77-)cells.(3)Relative HS1 mRNA level in B cell subpopulation.Up sorted B cells subpopulation mRNA level of HS1 were tested,and shown that HS1 mRNA was different expressed at various stages of B cell differentiation,highest in naive B cells,moderate in centrocyte,centroblast and mature B cells,lowest in plasma cells.We find that HS1 mRNA level is slide down follow with the B cell from immature to mature.Finally,plasma cell does not express HS1 mRNA.(4)HS1 protein and mRNA expression in B cell lymphoma cell linesHS1 over-expression of protein and mRNA were existed in lymphoma cell lines(Nalm6,OCI-ly1,OCI-ly3,OCI-ly8,OCI-1y10,Raji,Daudi,Ramous,BJAB,L428-99),lower expression in cell lines(RPMI-8226),but not in cell lines(IM9 and L428),in contrast with the normal B cells,by using RT-PCR and Western blot analysis.(5)HS1 protein expression in B cell neoplasm tissueswe examined HS1 expression in 214 cases of lymphoid neoplasm,and found that all of the DLBCL,THRLBCL and FL specimens demonstrated the highest percentage of positive cases,was 53/53(100%),12/12(100%)and 14/14(100%),respectively,and the tumor cells are often with bright staining of HS1.Meanwhile,MCL and NMZL also have the highest percentage of positive cases 100%(13/13 and 11/11,respectively)with weak to moderate staining for HS1.But in other B lymphoma subtypes,HS1 positive staining is differ from the former subtypes,21/35(60%)in B lymphoblastic lymphomas,14/27(51.85%)in Burkitt's lymphoma and 9/28(32.14%)in plasma cell myeloma.And the percent of HS1 positive-staining in plasma cell myeloma is the lowest in all type of non-Hodgkin lymphoma.Classical Hodgkin lymphoma is a unique lymphoid neoplasm in HS1 staining of IHC.And our data showed that nearly all(95.24%,20/21)of the Hodgkin lymphoma specimens were negative for HS1 staining in H/RS cells,and only one cases(4.76%,1/21)were shown weakly staining intensity.(6)The gene expression of HS1 located at the stage of B cell differentiationAccording to our research results,an expression profile of HS1 gene was drawn in B cell differentiation.The gene expression of HS1 located at the stage of B cell is from pre-B to mature B cells,fewer in plasma cells,and not in H/RS cells which derive from crippled pre-apoptotic germinal centre B cells.(7)Combined HS1 with CD30,CD 15 and CD20 in the diagnosis of classical Hodgkin's lymphoma and large B cell lymphoma with T/tissue cellsThe majority of CHL specimens were positive for CD30(17/21,80.95%)and CD 15(13/21,61.9%),but parts of the CHL specimens were positive for CD20(2/21,9.52%)in the H/RS cells.In all THRLBCL specimens,100%(12/12)were positive staining for CD20,one case was positive for CD30(8.33%,1/12),no specimen(0%,0/12)was detected positive staining for CD 15.One THRLBCL specimen show double-positivity for HS1 and CD30(n=1),but CHL specimens of CD20-positive(n=2)or CD30-negative(n=4)did not show positivity for HS1(n=6).Therefore,when immunostaining was performed for HS1,the two groups of tumors were identified by their respective diagnosis.2.Constructing sub-cell lines of DLBCL with HS1 was knocked down,and analysis their biological characteristics(1)Design and synthesis of Lentiviral vector for HS1 knockdownLentiviral vector for HS1 knockdown(HCLS1-Homo-1431,HCLS1-Homo-716,HCLS1-Homo-147 and HCLS1-Homo-272)and control Lentiviral vector(LV3-NC)in this experiment were used,which were provided by Suzhou Gene Pharm.Both experimental group and control group of the lentivirus with puromycin and GFP(Gluc-GFP)double label.(2)Constructing sub-cell lines of DLBCL with HS1 was knocked down.DLBCL cell lines(OCI-Ly8 and OCI-Ly10)were transfected with containing recombinant lentiviral vector virus supernatant(including HCLS1-Homo-1431,HCLS1-Homo-716,HCLS1-Homo-147,HCLS1-Homo-272 and LV3-NC).The transfected cells were purified by using puromycin treatment,and the efficiency of transfection and purification was observed under the fluorescence microscope.RT-PCR and Western Blot were used to verify the mRNA and protein levels respectively.The standard of screening stable knockdown HS1 expression in cell lines:mRNA level and protein expression were decreased to 50%,while the control group had no significant changes.HCLS1-Homo-1431 was screened out to use in the future experiments.DLBCL cell lines(OCI-lyl,OCI-ly3,OCI-Ly8 and OCI-Ly10)were transfected with containing recombinant lentiviral vector virus supernatant(HCLS1-Homo-1431 and LV3-NC).(3)Detection of proliferation and apoptosis in various sub-cell linesSub-cell lines(OCI-lyl-shHSl/NC,OCI-ly3-shHS1/NC,OCI-ly8-shHS1/NC cells and OCI-Iy10-shHSl/NC)were detected in our study.Detection cell proliferation ability changes in cells between experimental group and control group by using CCK8 assays.The results showed that,compared with the control group,OCI-lyl-shHS1,OCI-ly8-shHS1,OCI-ly3-shHS1,OCI-ly10-shHS1 proliferation ability clearly slowed down.Flow cytometry was used to detect cell cycles and cell apoptosis in sub-cell lines,and results showed that:?cell cycles were blocked at G1 phase in cells of OCI-lyl-shHSl(p=0.000,t=-30.174),OCI-ly8-shHS1(p=0.009,t=-4.783),OCI-ly3_shHS1(p=0.005,t=-5.582)and OCI-ly10-shHS1(p=0.026,t=-3.470);?cell apoptosis of OCI-ly3-shHS1(p=0.025,t=-3.492)and OCI-ly10-shHS1(p=0.009,t=-4.763)was increased.Other groups showed no obvious change.3.HS1 knockdown induces cell lines of DLBCL,ABC towards plasma cell differentiation(1)Detection expression of plasma cell markers CD38 and CD 138,and B cell marker CD19 in sub-cell lines of DLBCL by using flow cytometry.These results are as folio:1)Expression of CD138 markedly enhanced in cell lines of OCI-ly3 and OCI-Ly10 when HS1 gene expression was knocked down than that in the control group.The positive rates are,72.76%and 40.82%in shHS1 group,respectively,and 1.12%and 0.52%in NC group,respectively.Expression of CD38 and CD 19 in shHS1 group and NC group had no significant change.2)Expression of CD138,CD38 and CD19 had no significant change in cell lines of DLBCL,GCB(OCI-Lyl and OCI-Ly8)when HS1 gene expression was knocked down than that in the control group.(2)Results of HE staining and immunocytochemistry staining show as follow:1)Contrast with the NC group,OCI-ly3-shHS1 cells show that,the size of cells were smaller,more cells appearing mature plasma cell morphology,i.e.cells were oval,eccentric nucleus,paraventricular nucleus appear light staining area,cytoplasmic basophilic.Results of immunocytochemistry analysis showed that:expression of CD38,CD 138 and Mum-1 protein in OCI-ly3-shHSl were significantly higher than that in control group,while BCL-6 and CD20 protein expression was not significantly changed.2)Contrast with the NC group,OCI-ly10-shHS1 cells show that,the size of cells were smaller,a small part of cells appearing mature plasma cell morphology,i,e.cells were oval,eccentric nucleus,paraventricular nucleus appear light staining area,cytoplasmic basophilic.Results of immunocytochemistry analysis showed that:expression of CD38,CD 138 and Mum-1 protein in OCI-ly10-shHS 1 were significantly higher than that in control group,while BCL-6 and CD20 protein expression was not significantly changed.3)Contrast with the NC group,OCI-ly1-shHSl cells show that,,the size of cells were smaller,a small part of cells appearing mature small lymphocyte morphology,i.e.cells were small,round,nucleus deeply staining with hematoxylin,little cytoplasm.Results of immunocytochemistry analysis showed that:expression of CD38,CD138,Mum-1 and BCL-6 protein in OCI-lyl-shHSl had not changed anymore than that in control group,while CD20 protein expression was significantly decreased than that in control group.4)Contrast with the NC group,the cell morphology of OCI-ly8-shHSl cells had not changed anymore.Results of immunocytochemistry analysis showed that,expression of CD38 and Mum-1 protein in OCI-ly8-shHSl had down-regulation than that in control group.No obvious alterations were observed in OCI-ly8-shHS1 about CD 138,BCL-6 and CD20 protein expression compared with controls.(3)Analysis of transcription factor expression related to plasma cell differentiation in the sub-cell lines.Analysis expression of mRNA and protein of transcription factor(PRDM1,Mum-1,BCL6 and Pax5)related to plasma cell differentiation by using RT-PCR and Western Blot assays in the sub-cell lines.1)RT-PCR and Western blot results showed that,when HS1 was knocked down in OCI-ly3 cell line,Mum-1(p=0.000)up-regulation,PRDM1,BCL-6 and Pax5(p=0.003,p=0.000,p=0.000,respectively)down-regulation both in mRNA and protein level,contrast with the NC group.2)RT-PCR and Western blot results showed that,when HS1 was knocked down in OCI-ly10 cell line,Mum-1 and PRDM1(p=0.002,p=0.000,respectively)up-regulation,BCL-6 and Pax5(p=0.034,p=0.005,respectively)down-regulation both in mRNA and protein level,contrast with the NC group.3)RT-PCR and Western blot results showed that,when HS1 was knocked down in OCI-lyl cell line,Mum-1,PRDM1(p=0.004,p=0.015,respectively)down-regulation both in mRNA and protein level,contrast with the NC group.BCL6 and Pax5 had no alteration in mRNA level expression,but Pax5 protein was detected down-regulation.Last,BCL6 was not be detected in protein expression level.4)RT-PCR and Western blot results showed that,when HS1 was knocked down in OCI-ly8 cell line,PRDM1(p=0.004)down-regulation,Mum-1,BCL6and Pax5 had no alteration in mRNA expression(p>0.05,p>0.05,p>0.05,respectively),contrast with the NC group.PRDM1 and Mum-1 down-regulation,BCL-6 and Pax5 had no alteration in protein expression.4.HS1 knockdown induces ABC type DLBCL cell redifferentiation by the activation of NF-?B signal pathway was inhibited.(1)Western blot results showed that,when HS1 was knocked down both in OCI-ly3 and OCI-ly 10 cell lines,P105,cRel down-regulation,P100 up-regulation,RelA and RelB had no alteration in protein expression,contrast with the NC group.(2)NF-?B signal pathway inhibitor(BAY-11-7082)impacted the expression of HS1 in OCI-Ly3 and OCI-Ly3-NC cells.Western blot results showed that,when added BAY-11-7082(1?M,5?M and 10?M),HS1 protein was down-regulated in OCI-Ly3 cells,but only added the BAY-11-7082(10?M),HS1 expression was down-regulated in OCI-ly3-NC cells.(3)NF-?B signal pathway inhibitor(BAY-11-7082,10?M)impacted the expression of NF-?B signal pathway components in OCI-Ly3 and OCI-Ly10 cells.Western blot results showed that,p105,p100,RelB protein was down-regulated,cRel up-regulated,and RelA had no alteration in OCI-Ly3 cells,contrast with the blank group.p105,RelA protein was down-regulated,cRel and RelB up-regulated,and p100 had not detected in OCI-Ly10 cells.(4)Alteration of protein expression of transcription factor(PRDM1,Mum-1,BCL6 and Pax5)related to plasma cell differentiation were seen both in OCI-Ly3 and OCI-Ly10 cells,when cells were conducted with added NF-?B signal pathway inhibitor(BAY-11-7082,10?M)and HS1 was knocked out.Western blot results showed that,when OCI-Ly3 cells were conducted with BAY-11-7082(10?M)and HS1 was knocked out,HS1,PAX5 and BCL6 down-regulation,Mum-1 up-regulation,PRDM1 up-regulation in the former,and down-regulation in the later,contrast with the blank group.When OCI-Ly10 cells were conducted with added BAY-11-7082(10?M)and HS1 was knocked out,HS1,PAX5 and BCL6 down-regulation,Mum-1 up-regulation,PRDM1 down-regulation in the former,and no alternation in the later,contrast with the blank group.5.Observe distribution of F-actin and DBNL under the laser scanning confocal microscope in the sub-cell lines,and analysis the impact on cell apoptosis.1)Identification of HS1 interaction with DBNL by using CO-IP assay.CO-IP assay was carried out by using mono-antibody to HS1 as a 'bait' to pull down DBNL,conversely,using the mono-antibody DBNL to as a 'bait' to pull down HS1.The result showed that HS1 interaction with DBNL.2)F-actin cytoskeleton visualized with Rhodamine tag phalloidin.F-actin arrangement images under the confocal laser scanning microscope were shown in the cells of OCI-ly3 and OCI-Ly10 after HS1 was knocked down:red-silk fluorescent count increased significantly,assembly structure is complex,the local microfilament poly integrated nodular,microfilament which located in the cytoplasm and perinuclear migrate toward the cell membrane.3)Detected expression of DBNL protein by immunofluorescenceExpression of DBNL images under the confocal laser scanning microscope were shown in the cells of OCI-ly3 and OCI-Ly10 after HS1 was knocked down:red-silk fluorescent count increased significantly,assembly structure is complex,local poly integrated nodular,DBNL protein located in the cytoplasm and perinuclear migrate toward the cell membrane.4)Using Western blot to detect expression of DBNL proteinWestern blot results show that DBNL up-mediate expression related to HS1 knockdown,indicate that HS1 is negative mediate DBNL expression.Conclusion1.Expression profile of HS1 gene at the stage of B cell differentiation.Namely,HS1 protein expression located at the stage of pre-B cells to mature B cells(including normal B cells and corresponding B cell lymphoid tumor),low expressed at the terminal stage of B cells(namely,plasma cells and plasma cell myeloma).The H/RS cells arise from crippled pre-apoptotic germinal centre B cells are not express HS1 protein.2.Considering H/RS cells are not expressed HS1 protein,combination CD30,CD 15 and CD20 expression may do good for diagnose classical Hodgkin lymphoma and T cell/histiocyte-rich large B-cell lymphoma.3.HS1 knockdown can induce cell lines of DLBCL,ABC differentiation towards plasma cell.4.HS1 knockdown induces ABC type DLBCL cell redifferentiation by the activation of NF-?B signal pathway was inhibited.5.F-actin rearrangement and DBNL protein expression increasing can induced cell apoptosis in DLBCL cell lines with HSlwas knocked down.Innovation points1.Reveals expression rule of HS1 in human B cell subpopulation and this pattern will do goods for clinical diagnosis.2.HS1 knockdown can prompt cell lines of DLBCL,ABC differentiation towards plasma cell.And the mechanism is the activation of NF-?B signal pathway was inhibited,leads Mum-land PRDM1 expression up-mediate,BCL6 and PAX5 expression down-mediate.This result provides a new target for the differentiation treatment of DLBCL. |
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