The Role Of LTBP1/TGF-β1 In NK/T Cell Lymphoma

NK/T cell lymphoma(NK/T cell lymphoma,NKTCL)is an aggressive lymphoma derived from NK or cytotoxic T cells.It is associated with Epstein-Barr virus(EBV)infection and is characterized by significant necrosis,vascular injury,and a cytotoxic phenotype.NKTCL is prevalent in Asia,Mexico,and Central and South America,and is more common in men than women.NKTCL always presents as extradodal disease,usually involving the upper respiratory tract,such as the nasal cavity,nasopharynx,paranasal sinuses,and palate.Although chemoradiotherapy or L-asparaginase-containing regimens.may improve prognosis,approximately 40%to 50%of patients experience progression/recurrence,the median survival of advanced or recurrent NKTCL is approximately 6-12 months,with a poor prognosis.Latent transforming growth factor β binding protein 1(Latent transforming growth factor β binding protein 1,LTBP1)belongs to the LTBPs family,and LTBPs can form potential complexes with transforming growth factor β(Transforming growth factor TGF-β)and bind to fibrillary proteins.LTBP1 is located on chromosome 2p22.3 and involved in the assembly and secretion of potential TGF-β1.LTBP1 also cross-links to the ion.The TGF-β family consists of three subtypes,TGF-β1,TGF-β2,and TGF-β3.Before TGF-β plays its biological role,it is necessary to activate TGF-β through the release of latency-associated peptide(Latency-associated peptide,LAP)and potential transforming growth factor-βbinding protein(LTBPs)from the complex.Therefore,LTBP1 can affect the synthesis and secretion of TGF-β,which plays an important role in the occurrence and development of many cancers,and it is speculated that LTBP1/TGF-β may play a synergistic regulatory role in some diseases.TGF-β1 can induce apoptosis in B-cell lymphoma Raji cells by inhibiting extracellular regulated protein kinases 1/2(Extracellular regulated protein kinases 1/2,ERK1/2)pathway activity through activation of protein phosphatase 2A(Protein phosphatase 2A,PP2A).However,the effect of LTBβ1/TGF-β1 on the occurrence and progression of NK/T cell lymphoma and its mechanism are still unclear.The purpose of this thesis is to study the role of LTBP1/TGF-β1 in NK/T cell lymphoma.Firstly,the clinical NK/T cell lymphoma tissue morphology was observed by HE assay,and ELISA,qRT PCR and Western blot were performed to meassure LTBP1 and TGF-β1 levels in the serum and organization of clinical NKTCL patients,and the relationship of the LTBP1 expression and clinical data in the NKTCL tissues was analyzed,and the correlation of LTBP1 and TGF-β1 in clinical NKTCL organization was analyzed,and the expression of LTBP1/TGF-β1 in NK/T cell lymphoma and significance were preliminarily investigated.Later,at a cellular level,qRT-PCR and Western blot assays was carried out to detect the LTBP1 and TGF-β1 mRNA and protein expression in different NK/T lymphoma cell line(SNK-6,YTS,HANK1)cells,and then LTBP1 interference vector was constructed,which infected SNK-6 and YTS cells after packaging and titration infection,and CFSE and flow cytometry experiments were implemented to test the influence of LTBP1/TGF-β1 on NK/T cell lymphoma cell proliferation,apoptosis,and cell cycle.In addition,CCK-8 was performed to determin the effect of LTBP1/TGF-β1 on the sensitivity of SNK-6 and YTS cells to chemotherapy drugs adriamycin and geocitabine,and Western blot was carried out to detect the effect of LTBP1/TGF-β1 on the expression of the TGF-β1/Smad,p38MAPK signaling pathway related proteins TGF-β1,Smad2,p-Smad2,Smad3,p-Smad3 and p38MAPK in SNK-6 and YTS cells,and the effect of LTBP1/TGF-β1 on NK/T cell lymphoma cell function was further investigated.Finally in vivo studies,SNK-6 cells was transplanted to construct the NK/T cell lymphoma transplantation tumor model in nude mice,and the influence of LTBP1 and TGF-β1 and their combination on growth of xenograft was discussed,and the LTBP1 expression in tumor tissue was measured by qRT-PCR,and Western blot was carried out to detect the effect of LTBP1/TGF-β1 on the activity of the TGF-β/Smad and p38MAPK signaling pathway,and the effect of LTBP1/TGF-β1 on the growth of NK/T cell lymphoma xenograft was investigated.This study is mainly divided into three parts:Part Ⅰ:The expression of LTBP1/TGF-β1 in NK/T cell lymphoma and significance;Part Ⅱ:The effect of LTBP1/TGF-β1 on NK/T cell lymphoma cell function and its mechanism;Part Ⅲ:The effect of LTBP1/TGF-β1 on the growth of NK/T cell lymphoma xenograft.The main content:Part Ⅰ:The expression of LTBP1/TGF-β1 in NK/T cell lymphoma and significance1.Histological morphology of clinical.NK/T cell lymphoma was observed by HE staining assay2.The levels of LTBP1 and TGF-β1 in clinical NKTCL tissues and serum were detected by ELISA,qRT-PCR and Western blot.3,Statistical analyses of the data were processed using the SPSS21.0 software package.The Chi-square tests were performed for analyzing the relationship between LTBP1 expression in NKTCL tissues and clinical case data.4.Statistical analyses of the data were processed using the SPSS21.0 software package.The correlation between LTBP1 and TGF-β1 levels in clinical NKTCL tissues was analyzed by Pearson correlation analysis.Results1.The growth state of NK/T cell lymphoma was observed by HE staining assay,and it was found that the positive expression rate of TGF-β1 in NK/T cell lymphoma was significantly higher than that in lymph node reactive hyperplasia.2.ELISA,qRT-PCR,and Western blot results showed that both LTBP1 and TGF-β1 mRNA and protein levels were significantly increased in NK/T cell lymphoma tissues and serum compared with lymph node reactive hyperplasia tissues and serum.3.LTBP1 expression in NKTCL tissues was correlated with ANN Arbor staging of clinical patients,and had no significant relationship with age,gender,site of occurrence,symptoms and LDH level of patients.4.There was a positive correlation between LTBP1 and TGF-β1 expression in NK/T cell lymphoma.Part Ⅱ:The effect of LTBP1/TGF-β1 on NK/T cell lymphoma cell function and its mechanismMethods1.The mRNA and protein expressions of LTBP1 and TGF-β1 in different NK/T lymphoma cell lines(SNK-6,YTS and HANKL)were determined by qRT-PCR and Western blot.2.LTBP1 interference lentiviral vector was constructed to infect SNK-6 and YTS cells after packaging and titration.3.CFSE and flow cytometry assay were carried out to detect the effects of LTBP1/TGF-β1 on the proliferation,apoptosis and cell cycle of SNK-6 and YTS cells.4.The effect of LTBP1/TGF-β1 on the sensitivity of SNK-6 and YTS cells to chemotherapeutic drugs doxorubicin and gemcitabine were detected by CCK-8.5.The effect of LTBP1/TGF-β1 on the expression of TGF-β/Smad and p38MAPK signaling pathway related proteins TGF-β1,Smad2,p-Smad2,Smad3,p-Smad3,p-p38MAPK and p38MAPK in SNK-6 and YTS cells were meassured by Western blot.Results1.Compared with NK cells,the mRNA and protein levels of LTBP1 and TGF-β1 were significantly increased in SNK-6,YTS and HANKL cells,especially in SNK-6 and YTS cells.Therefore,SNK-6 and YTS cells were selected for subsequent cell function analysis.2.Interference of LTBP1 lowered the level of TGF-β1 in SNK-6 and YTS cells.3.Interference of LTBP1 could inhibit the proliferation of SNK-6 and YTS cells,arrest the cell cycle in G2 phase,and promote cell apoptosis,while TGF-β1 treatment could reverse this inhibition and promotion effect.4.Interference of LTBP1 increased the sensitivity of SNK-6 and YTS cells to doxorubicin and gemcitabine,while TGF-β1 treatment significantly reduced this effect.5.Interference of LTBP1 could inhibit the expression of TGF-β/Smad signaling pathway and p38MAPK signaling pathway related proteins TGF-β1,p-Smad2,p-Smad3 and p-p38MAPK in SNK-6 and YTS cells,while TGF-β1 treatment significantly reduced this inhibition.Part Ⅲ:The effect of LTBP1/TGF-β1 on the growth of NK/T cell lymphoma xenograftMethods1.SNK-6 xenograft model in nude mice was constructed and divided into three groups:sh-NC,sh-LTBP1 and sh-LTBP1+TGF-β1.The effect of LTBP1/TGF-β1 on tumor size and weight was investgated,and the effect of them on tumor bearing mice survival rate was analyzed by the Kaplan-Meier method and the two-sided Log-rank test.2.The expression of LTBP1 protein in tumor tissues was determined by qRT-PCR and Western blot.3.The effect of LTBP1/TGF-β1 on the expression of TGF-β1/Smad,p38MAPK signaling pathway related proteins TGF-β1,Smad2,p-Smad2,Smad3,p-Smad3,p38MAPK and p-P38MAPK were detected by Western blot.Results1.In NK/T cell lymphoma xenograft model,Interference of LTBP1 inhibited the growth of xenograft tumor and significantly increased the survival rate,while TGF-β1 treatment significantly reduced this inhibition and elevation.2.In the NK/T cell lymphoma xenograft model,the expression of LTBP1 was significantly decreased in the xenograft tissues of sh-LTBP1 group and sh-LTBP1+TGF-β1 group.3.Interference of LTBP1 inhibited TGF-β1-expression in NK/T cell lymphoma xenograft models,while TGF-β1 expression was signifi cantly increased after treatment with TGF-β1.4.In NK/T cell lymphoma xenograft tumor model,interference of LTBP1 inhibited the expression of TGF-β/Smad and p38MAPK signaling pathway related proteins p-Smad2,p-Smad3,and p-p38MAPK.Conclusion1.The mRNA and protein levels of LTBP1 and TGF-β1 were significantly increased in NK/T cell lymphoma tissues compared to those in lymph node reactive hyperplasia tissues.The expression of LTBP1 in NKTCL tissues was correlated with ANN Arbor staging of clinical patients,but not with age,gender,tumor site,symptoms and LDH level.In NK/T cell lymphoma,the expression of LTBP1 and TGF-β1 was positively correlated.2.Interference of LTBP1 could inhibit the proliferation of SNK-6 and YTS cells,arrest the cell cycle in G2 phase,promote cell apoptosis,and increase the sensitivity of NK/T cell lymphoma cells to adriamycin and gemcitabine by lowering the level of TGF-β1 through inhibiting the TGF-β/Smad and p38MAPK signaling pathway.3.In NK/T cell lymphoma xenograft model,interference of LTBP1 could inhibit the growth of xenograft tumor by inhibiting the expression of TGF-β1,thereby inhibiting the activity of TGF-β/Smad and p38MAPK signaling pathway,and thus playing a tumor suppressive role.