| BackgroundHodgkin's lymphoma(HL)is one of B cell lymphomas characterized by special clinic and pathology.Clinical pathological type is mainly classified as nodular lymphocyte-predominant Hodgkin lymphoma(NLPHL)and classic Hodgkin lymphoma(cHL).The cHL is characterized by rare Hodgkin and Reed-Stemberg(HRS)tumour cells(about 1%),with the background of predominant reactive inflammatory cells(such as Tissue cells,lymphocytes,plasma cells,eosinophils).Many evidences indicate that the occurrence of tumor is mostly due to the result of the disordered or blocked differentiation of normal cells.Kuppers reported that the majority of HRS cells come from aberrant Germinal center(GC),and happen when germinal center B cells can not differentiate into plasma cells or memory B cells.Therefore our research group has being engaged in the researches of the origin and differentiation of HRS cells.Insulin-like growth factor ? mRNA-binding protein 3(IGF2BP3,IMP3)gene attracts more eyes of the researchers in recent years,not only because expression of IGF2BP3 in adult tissues is extremely low,or even difficult to detect,but also because IGF2BP3 presents high expression in a variety of human tumor cells,closely related to cellular proliferation,invasion,differentiation and metastasis of tumor,and can be used as an important clinical molecular diagnostic and prognostic markers.While reports about IGF2BP3 in lymphoma are very few.King reported IGF2BP3 expression of 26 cases of HL specimens and surprisedly found that the protein has special expression pattern that IGF2BP3 is just expressed in the cytoplasm of HRS cells with strong expression,which makes HRS cells identification easier.The discovery attracts us.And we speculate IGF2BP3 is may be an important marker protein/protein function in HRS cells,and may be a target of HL diagnosis and research.Objective1.To explore expression and location of IGF2BP3 on HL tissues and cell lines.2.To explore effect of IGF2BP3 gene on biological characteristics of HRS cells in HL.3.To explore relationship between IGF2BP3 and abnormal differentiation of HRS cells in HL.4.To explore relationship between IGF2BP3 and miR9-TGF ?R1/smads differentiation axis to reveal the molecular mechanisms of IGF2BP3 in HL.Methods1.Expression and location of IGF2BP3 on HL tissues and cell lines.(1)Collected 81 HL paraffin tissue specimens stored in the department of pathology of Nanfang hospital,Sun yat-sen university affiliated tumor hospital and the first people's hospital of Guangdong province between 2009 and 2012.(2)Detected and compared expression differences of IGF2BP3,CD30,CD15,PAX5 and MUM1 in these paraffin tissue samples by immunohistochemical(IHC).(3)Sorted out B lymphocytes from fresh amygdala tissue by flow cytometry,and extracted the total RNA of the B lymphocytes.(4)Expanded and cultured different kinds of lymphoma cell lines(Raji,Daudi,Ramos,BJAB,Ocl-ly8,Ocl-ly10,8226 andL428),then collectd these cells to partially extract total RNA and protein,and to partially wax cell block.(5)Used real time PCR,Western blot and immunocytochemistry to detect expression of IGF2BP3 in B lymphocytes from tonsil and different kinds of lymphoma cell lines.2.Effect of IGF2BP3 gene on biological characteristics of HRS cells in HL.(1)Designed and selected out sequence of downregulated IGF2BP3 expression,constructed downregulated expression lentiviral vector for IGF2BP3 gene,packaged into lentivirus.Then transfected the lentivirus into HL cell lines(L428)and diffuse large B cell lymphoma cell line(Ocl-ly8),sorted out the GFP positive cells by flow cytometry(L428-shIGF2BP3,Ocl-ly8-shIGF2BP3),and verified the transfected efficiency by real time PCR and Western blot experiments.(2)Designed and constructed overexpression adenovirus vector for IGF2BP3 gene,instantaneously transfected adenovirus into L428-shIGF2BP3 and Ocl-ly8-shIGF2BP3 cells,respectively.Verified whether IGF2BP3 expression recovery or not by real time PCR and Western blot experiments.(3)Observed cell proliferation ability in vitro through CCK8 and cell cycle experiments.(4)Observed cell aggressive ability through improved Transwell method.3.Silencing IGF2BP3 induces HRS cells differentiate toward terminal B cells.(1)Effects of IGF2BP3 gene on RNA levels of differentiation genes MUM1,PRDM1 and CD38 were detected by real time PCR.(2)Effects of IGF2BP3 gene on protein levels of differentiation genes MUM1,PRDM1 and CD38 were detected by Western blot experiment.(3)Effects of IGF2BP3 gene on expression levels of differentiation genes MUM1,PRDM1 and CD38 were detected by confocal microscope.(4)Effects of IGF2BP3 gene on expression levels of relative differentiation antigens were detected by flow cytometry analysis.4.Silencing IGF2BP3 induces HRS cells differentiation by regulating miR9-TGF-?R1/smads pathway.(1)Effects of IGF2BP3 on protein levels of TGF-?R1/smads signal pathway were detected by Western blot experiment.(2)PRDM1 and IGF2BP3 expression were detected by Western blot after L428 cells were transducted with TGF-?R1/Smads agonist TGF-?1 or when L428-shIGF2BP3 cells were transducted with TGF-?R1/Smads blocker SB431542.(3)MiR9 mimics was transfected into L428-miR9-inhibitor cells to restore miR9 expression.Changes of IGF2BP3 and PRDM1 gene expression were compared by real time PCR and Western blotting when downregulated or recovery miR9 in L428 cells.(4)MiR9 expression was detected by real time PCR in L428-shIGF2BP3 cells.Statistical analysisThe experimental results were processed and analyzed by SPSS 20.0 statistical software.Data was represented as mean ± standard deviation(x±SD)of at least three indepent experiments.Differences between variables were assessed by the ?2 test,Fisher's exact test,independent T test and One-way ANOVA.Different groups of cell proliferation ability by CCK8 and cell cycle were analyzed by the use of factorial design analysis of variance.Differences are considered statistically significant at P<0.05.Results1.Expression and location of IGF2BP3 on HL tissues and cell lines.The immunohistochemical staining results showed nearly all(98.8%,80/81)of the HL specimens were positive for IGF2BP3 immunostaining,and the majority(72.8%,59/81)showed strong staining intensity in the cytoplasm of HRS cells.All of the NLPHL specimens(100%,10/10)were also IGF2BP3-positive,with immunostaining detected only in the cytoplasmic compartments of the large multinucleate HRS cells.The non-neoplastic background cells(lymphoid cells,plasma cells,neutrophils,eosinophils,and histocytes)were uniformly nonreactive for IGF2BP3.The majority of cHL specimens were positive for CD30(94.4%,67/71)and CD15(74.6%,53/71),and exhibited a characteristic expression in a membrane pattern with accentuation of dot-like staining in the Golgi region of cytoplasm in the HRS cells.In addition,many of the cHL specimens were positive for PAX5(87.3%,62/71)in the HRS cell nuclear compartments.PAX5 expression was also detected in the nuclei of non-neoplastic bystander B cells,but the staining intensity was significantly stronger than in the HRS cells.MUM1 positive immunostaining occurred in a large portion of the cHL specimens(91.5%,65/71),with expression detected in both the HRS cells and some of the activated B cells.The intensity of positive staining differed among these four markers in the HRS cells.All 10 NLPHL specimens showed uniform negativity for both CD 15 and CD30,while approximately half showed positivity for PAX5(60.0%,6/10)or MUM1(40.0%,4/10)which included one specimen with strong staining intensity for both PAX5 and MUM1.The expressions of IGF2BP3 and several other protein markers on HRS cells were compared by immunohistochemical method.The results showed that positive expression rate of IGF2BP3 on HRS cells was higher than that of other protein markers.What is more,IGF2BP3 presented strong expression in NLPHL where CD 15 and CD30 did not colour.Therefore,we hypothesized that IGF2BP3 was an important marker protein in HRS cells.Real time PCR,Western blot and immunocytochemistry results showed that IGF2BP3 presented differentially expression in these lymphoma cell lines,such as IGF2BP3 expression was relatively low in Burkitt lymphoma cell lines with EBV positive,while higher in diffuse large B cell lymphoma and plasmoma.IGF2BP3 expression level in HL cells was middle with location in cytoplasm.2.Effect of IGF2BP3 gene on biological characteristics of HRS cells in HL.GFP positive cells were separated by flow cytometry after cells were transfected with lentiviral.Results of Real time PCR and Western Blotting experiment showed that compared with the control group,IGF2BP3 expression in GFP positive cells decreased,which suggested cell line model of downregulated IGF2BP3 is successfully constructed.Adenovirus vector for overexpression IGF2BP3 was transfected into L428-shIGF2BP3 and Ocl-ly8-shIGF2BP3 cells.Then IGF2BP3 expression was validated by real time PCR and Western Blot experiments and the results showed that compared with the control group,IGF2BP3 expression was restored in cells transfected with adenovirus.Cell cycle results showed that downregulated IGF2BP3 caused an increase percentage of L428 cells in the G0/G1 phase and decrease in the S phase.The difference was statistically significant(P<0.05).When restored IGF2BP3 gene expression in L428-shIGF2BP3 cells,the percentage of cells in the G0/G1 phase reduced,while in the S phase increased.The difference was statistically significant(P<0.05).Cell cycle results of the diffuse large B cell lymphomal were consistent with that of HL cell line L428.CCK8 results show that downregulated IGF2BP3 could inhibit L428 cell proliferation in vitro.There was statistically significant difference(t=4.565,P=0.010).In addition,there was interactive effect in the cellular level and time level(F = 7.503,P=0.001).Difference between the two cell groups was statistically significant(F=72.829,P=0.000),and difference between each time level was statistically significant(F= 80.035,P=0.000).When restored IGF2BP3 gene expression in L428-shIGF2BP3 cells,cells proliferation ability increased in vitro,and there was statistically significant difference(t=7.571,P=0.002).Among them,there is interactive effect on the cell level and time level(F= 30.824,P=0.000).Difference between the two cell groups was statistically significant(F = 139.477,P =0.001).Difference between each time level was statistically significant(F= 648.819,P=0.001).CCK8 results of diffuse large B cell lymphoma were consistent with that of HL cell line L428.Transwell and CCK8 results show that downregulated IGF2BP3 made less L428 cells through transwell chambers pore,while recovery IGF2BP3 expression,the number of cells through transwell chambers pore increased.OD value detected by CCK8 validated these results and the differences were statistically significant(P<0.05).Transwell and CCK8 results of diffuse large B cell lymphoma were consistent with that of HL cell line L428.3.Silencing IGF2BP3 induces HRS cells differentiate toward terminal B cells.Real time PCR results showed that downregulated IGF2BP3 induced mRNA levels of MUM1,PRDM1 and CD38 increased in L428 cells,and the differences were statistically significant(P<0.05).Western blot results showed that downregulated IGF2BP3 expression caused higher levels of PRDM1,MUM1 and CD38 in L428 cells,while IGF2BP3 was restored in L428-shIGF2BP3 cells,protein levels of PRDM1,MUM1 and CD38 dropped.Confocal microscope results showed that reduced IGF2BP3 in L428-shIGF2BP3 cells led to higher levels of PRDM1 with expression in the nucleus,increased CD38 expression in cytomembrane,upregulated expression of MUM 1 in the cytoplasm and partially nucleus.Flow cytometry results showed that reduced IGF2BP3 in L428-shIGF2BP3 cells led to appearance of CD38 and CD138,and lose of CD30 and CD15 expression,but B cell markers CD 19 expressed no change obviously.4.Silencing IGF2BP3 induces HRS cells differentiation by regulating miR9-TGF-?R1/smads pathway.Western blot results showed that downregulation of IGF2BP3 caused upregulated expression levels of TGF-?R1 and P-Smad2 protein,but dropped level of P-Smad3.Changes of Smad2,Smad3 and Smad4 protein levels were not obvious.When IGF2BP3 gene expression was recovery in L428-shIGF2BP3 cell,levels of TGF-?R1 and P-Smad2 dropped,and P-Smad3 rised.There was also no obviously change in the expression of Smad2,Smad3 and Smad4.Western blot results showed that expressions of TGF-?R1 and PRDM1 appeared when L428 cells were treated with TGF-?R1/smads signal pathway agonist TGF-?1 for 72 hours,but IGF2BP3 protein levels did not change.While L428-shIGF2BP3 cells were treated with TGF-?R1/smads signal pathway blockers SB431542 to inhibit the activity of TGF-?R1/smads signal pathway,expressions of TGF-?R1 and PRDM1 reduced,but there was still no obviously change in IGF2BP3 expression.It was surprising that there is combination between miR9 and IGF2BP3 3 'UTR region by TargetScan database software.Real time PCR,Western blot and immunofluorescence results showed that silencing miR9 in L428 cells led to lower expression of IGF2BP3.When miR9 mimic was transfected into L428-miR9-inhibitor cells to restore miR9 expression,expression of IGF2BP3 increased,while TGF-?R1 and PRDM1 reduced.MiR9 expression was detected in L428-shIGF2BP3 cells by real time PCR.The result showed that downregulated IGF2BP3 expression did not result in change of miR9 expression.Conclusion1.IGF2BP3 may be an important protein marker of HRS cell in HL.2.IGF2BP3 can promote HRS cell growth,proliferation and aggressive ability.3.Silencing IGF2BP3 could induce HRS cell differentiation toward terminal B cell.4.Silencing IGF2BP3 could induce HRS cell differentiation by regulatingmiR9-TGF-?R1/smads pathway.New points1.IGF2BP3 is a new protein marker of HRS cells in HL.2.It is suggested that IGF2BP3 can inhibit growth,proliferation and local infiltration capacity of HRS cells in vitro,which indicates that IGF2BP3 may be play a promoting tumor role in HL.3.Silencing IGF2BP3 could induce HRS cells redifferentiation through regulating miR9-TGF-?R1/smads pathway.4.That provides a new theoretical basis and experimental data on the treatment for HL. |
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