The Mechanism Of Exosomal MiRNAs And Predictive Biomarker For CAR-T Cell-mediated Neurotoxicity In Diffuse Large B Cell Lymphoma

Part ?:The effect of exosomal miR-665 and its underlying mechanism in diffuse large B cell lymphoma(DLBCL)Background:Diffuse large B cell lymphoma(DLBCL)is the most common type of non-Hodgkin lymphoma worldwide and manifested as rapidly enlarging lymphadenopathy and constitutional symptoms.Morphologically,DLBCL is characterized by diffuse infiltration of medium to large cells with large nucleoli and abundant cytoplasm,which effaces the normal nodal or extranodal architecture.DLBCL tumor cells grow faster,have a high degree of malignancy,and have strong invasive ability,which can cause infiltration and metastasis.Although the latest research shows that the first-line treatment such as R-CHOP of DLBCL has significantly improved the clinical effect,due to the high degree of heterogeneity of DLBCL patients,the response to treatment and prognosis vary greatly,and up to date,about one-third of patients will experience relapse and chemotherapy resistance after treatment.Therefore,It has important clinical significance to find biomarkers that can effectively evaluate the prognosis of DLBCL and improve the overall survival rate of DLBCL patients.Exosomes are nanoscale(30-150 nm diameter)extracellular lipid bilayer vesicles secreted by most cells and extensively existed in serum,saliva,urine and other body fluids.In the exosome membrane and lumen,there are various lipids,proteins,and nucleic acids,including DNA and RNA.Exosomes are taken up by other cells,and these molecules are transferred into recipient cells.As the essential mediators of cell-cell communication,numerous studies have recognized that tumor-derived exosomal miRNAs might serve as effective markers for early diagnosis and predict indicators for metastasis potential and prognosis of various malignancies.Has-miR-665 is a miRNA encoded by 72 deoxyribonucleotide sequences on chromosome 14q32.2 and produced after processing.A large number of studies have found that miR-665 is related to the proliferation,differentiation,apoptosis,invasion,migration and chemotherapy resistance of various tumors such as acute lymphoblastic leukemia,hepatocellular carcinoma,gastric cancer,and ovarian cancer.For example,miR-665 can inhibit cell proliferation,migration and invasion in gastric cancer and ovarian cancer cells.In breast cancer cells,miR-665 can promote tumor formation and lung metastasis in the cells.Current studies have confirmed that the expression level of miR-665 in the serum of DLBCL patients is significantly down-regulated,but its expression level in DLBCL exosomes has not been reported yet.Therefore,in-depth study of the role of exosomal miR-665 in DLBCL and its potential molecular mechanisms will help identify new biomarkers and improve the prognosis of DLBCL patients.Objective:This study intends to compare the expression level of miR-665 in serum-derived exosomes from DLBCL patients and healthy volunteers;and analyze the correlation between the expression level of miR-665 and the clinical characteristics of patients,then elucidate its clinical value in prediction DLBCL progression and outcomes;At last,we explore the role of miR-665 on the occurrence and development of DLBCL and its potential molecular mechanism through in vivo and in vitro experiments.Methods:1.Isolation and identification of serum exosomes of DLBCL patients and healthy volunteers:Serum-derived exosomes were isolated using ultracentrifugation and precipitation methods.And then the morphology and structure of obtained exosomes were characterize using Transmission Electron Microscope.Exosome-specific markers(CD63 and CD81)were identified using western blotting analysis.2.Total RNAs in exosomes were isolated using an Exosome RNA Purification Kit,and miR-665 expression level in the serum exosomes of DLBCL patients(n=30)with that of healthy volunteers(n=30)was detected using Real-time Reverse transcription quantitative PCR(RT-qPCR).Correlations between the expression of miR-665 and clinicopathological features(gender,age,subtype,Ann Arbor stage,LDH level,B symptoms,and IPI score)of DLBCL patients(n=30)were analyzed using ?2 test.3.Cell transfection:miR-665 mimics/inhibitor,and LASP1 siRNA were introduced into DLBCL cells to regulate miR-665 and LASP1 expression levels which is detected by RT-qPCR and Western Blotting in the regulatory cells;4.Cell viability in each group was examined using CCK-8 assay.5.Flow cytometry analysis quantified population of apoptotic cells following Annexin V-FITC staining.6.Transwell invasion assay evaluated the invasive capability of DLBCL cells.7.Western Blotting detects the expression levels of apoptosis and invasion-related proteins in the DLBCL cell lines after modulation;8.The potential target genes of miR-665 were predicted using Bioinformatics method.9.Dual-Luciferase Reporter Assay System was performed to confirm the target relationship between miR-665 and LASP1.The wild and mutant luciferase reporter vector in the 3'UTR region of LASP1 gene was constructed to detect the change of luciferase activity after miR-665 was up-regulated.10.Hematoxylin?eosin(H?E)staining,Immunohistochemistry(IHC),and TdT mediated dUTP Nick End Labeling(TUNEL)assay were used to evaluate histopathological changes of tumor tissues,expression level of Ki-67 and cell apoptosis,respectively after the mouse is subcutaneously bearing tumor.Results:1.We identified exosomes using electron microscopy and Western Blotting analysis.The typical"cup-shaped" vesicles of exosomes were observed under electron microscopy.Besides,the exosome-specific markers CD63 and CD81 were also detected in the exosomes.2.The results of RT-qPCR suggested that miR-665 expression was significantly down-regulated in exosomes of DLBCL patients.According to the median expression value of miR-665 in the exosomes of 60 DLBCL patients,we divided these patients into two groups:the low expression group(n=30)and the high expression group(n=30).As shown in Table 1,there was no significant association between miR-665 expression and gender,age,subtype,and B symptoms.However,low miR-665 expression was closely associated with the higher Ann Arbor stage,lower LDH,and higher IPI score.3.SU-DHL-4 cells transfected with miR-665 mimics displayed reduced mRNA and protein levels of LASP1,and suppression of miR-665 in FARAGE cells elevated LASP1 mRNA and protein expression(P<0.01).SU-DHL-4 and FARAGE cells were transfected with two siRNAs against LASP1.The results showed that these two siRNAs knocked down LASP1 successfully both at mRNA and protein levels in DLBCL cells.We chose the more effective siLASP1 to transfect FARAGE cells with miR-665 inhibitor.4.CCK-8 assay and Transwell invasion assay showed that miR-665 knockdown remarkedly reduced the proliferation and invasion of SU-DHL-4 cells,while miR-665 overexpression enhanced cell growth and invasion of FARAGE cells(P<0.05);5.Flow cytometry assay revealed that miR-665 up-regulation in SU-DHL-4 cells elevated the percentage of apoptotic cells and miR-665 knockdown alleviated apoptosis of FARAGE cells(P<0.01);6.Western blotting analysis showed that overexpression of miR-665 inhibited MMP-2 and MMP-9 levels,while miR-665 knockdown promoted the expression of MMP-2 and MMP-9.Moreover,miR-665 overexpression markedly increased the protein levels of pro-apoptotic factors cleaved caspase-3 and Bax,while reduced anti-apoptotic factor Bcl-2(P<0.01).7.Luciferase reporter assay confirmed that 293T cells co-transfected with miR-665 mimics and LASP1-wide type showed significant decrease in luciferase activity,while the co-transfection of miR-665 and LASP1-mutant type had little influence on the luciferase activity(P<0.01).8.miR-665 suppressed DLBCL cell growth,invasion,and apoptosis through down-regualting LASP1.9.We verified the effects of miR-665 on DLBCL growth and apoptosis in vivo.SU-DHL-4 cells were inoculated into the flank of nude mice followed by orthotopic injection with miR-665 agomir.The expression of miR-665 was up-regulated,and LASP1 protein expression was inhibited in tumors by miR-665 agomir.The volume and weight of tumors separated from nude mice injected with miR-665 agomir were decreased compared with that from NC agomir mice.H?E staining was performed to observe the pathological change in tumors.The images showed that tumor cells were significantly reduced in miR-665 agomir group.Immunohistochemical staining also indicated that Ki-67 expression in tumors was reduced in miR-665 agomir group compared with NC agomir group.Moreover,more TUNEL-positive cells in tumors were observed in miR-665 agomir group than in NC agomir group.These findings revealed that miR-665 overexpression suppressed DLBCL growth in vivo.Conclusion:1.miR-665 was significantly reduced in DLBCL and played anti-tumor role in DLBCL progression and development.2.Overexpression of miR-665 effectively suppressed cell viability,invasion and induced cell apoptosis through targeting LASP1.Part ?:The effect of bone marrow mesenchymal stem cell-derived exosomes' miRNAs in diffuse large B-cell lymphoma(DLBCL)Background:Diffuse large B-cell lymphoma(DLBCL)is the most common non-Hodgkin's lymphoma.The etiology of DLBCL is very complex,except for the currently proven risk factors such as genetic characteristics,immune system disorders,Virus infection,occupational environmental exposure,etc.The role of tumor microenvironment is also very important.Bone marrow mesenchymal stem cells(BMSCs)are pluripotent stem cells derived from bone marrow,which have the potential for self-renewal,rapid proliferation,and multi-directional differentiation.In the field of tumors,it has also been found that BMSCs are related to the formation of tumor microenvironment,and can interact with tumor cells to affect their growth and metastasis potential.In recent years,BMSCs have also been used as one of the potential targets for tumor therapy.BMSCs can secrete exosomal vesicles,but the role of BMSCs-derived exosomes in DLBCL is still unclear.This study aims to identify and discover miRNAs from the BMSCs-derived exosomes that are related to the occurrence and development of DLBCL.Objective:This study intends to screen out exosomes derived from bone marrow mesenchymal stem cells(BMSCs)that are closely related to the occurrence and progression of diffuse large B-cell lymphoma;and to evaluate the clinical value of exosomes derived from bone marrow mesenchymal stem cells;Preliminary exploration of the role of exosomal miRNAs derived from bone marrow mesenchymal stem cells in diffuse large B-cell lymphoma.Methods:1.BMSCs-derived exosomes isolation and identification:BMSCs-derived exosomes were isolated through ultracentrifugation and precipitation methods.And then the isolated exosomes were identified using Transmission Electron Microscope(TEM),nanoparticle tracking analysis(NAT),and western blotting analysis.2.Differentially expressed miRNAs in BMSCs-derived exosomes were identified by TaqMan Advanced miRNA assay.3.RT-qPCR was employed to determine the expression levels of BMSCs-derived exosomes miRNAs in DLBCL patients.4.Cell viability and apoptosis were examined using CCK-8 assay and Annexin V/PI after co-culture of DLBCL cell line with exosomes derived from bone marrow mesenchymal stem cells(BMSCs);.5.Kaplan-Meier analyzed the correlation between exosomal miRNAs and the overall survival of DLBCL patients.Results:1.The typical "cup-shaped" vesicles of exosomes were observed under electron microscopy.Besides,the exosome-specific markers CD63 and CD81 were also detected in the exosomes(100 nm),indicating the successful isolation of exosomes from BMSCs.2.BMSCs exosomes could promote DLBCL cell proliferation and reduce cell apoptosis.3.A total of 83 differentially expressed miRNAs were identified in BMSCs-derived exosomes from DLBCL patients compared with healthy patients,includes 82 down-regulated miRNAs and 1 up-regulated miRNA.4.miR-126-3p,miR-22-3p,and miR-223-3p were associated with poor prognosis in DLBCL patients.Conclusion:1.BMSCs-derived exosomes facilitated DLBCL cell proliferation and inhibited apoptosis;2.miR-126-3p,miR-22-3p,and miR-223-3p might serve as potential biomarkers for DLBCL prognosis.Part ?:Identification of predictive biomarker associated with CAR-T Cell-Mediated Neurotoxicity in DLBCL based on Bioinformatics analysisBackground:DLBCL is a highly heterogeneous disease,and its pathogenesis is complicated.Although DLBCL has a good overall therapeutic effect,more than one-third of patients will still relapse and be refractory to treatment.Rescue chemotherapy or accept Hematopoietic stem cell transplantation is the first-line treatment for relapsed/refractory DLBCL,but in fact only half of the patients can successfully receive the transplant,and PFS is only 53%after 3 years.In recent years,the emergence of Chimeric Antigen Receptor T-Cell Immunotherapy(CAR-T)has brought hope to patients with relapsed/refractory DLBCL,even though CAR-T therapy is used in the treatment of hematological tumors.It has achieved surprising curative effects,but its severe side effects have greatly restricted its wide use.The clinical side effects of CAR-T cell therapy mainly include cytokine release syndrome(CRS)and CAR-T cell-associated encephalopathy syndrome(CRES).Early identification of patients with the highest risk of neurotoxicity may be a problem.Early intervention before severe neurotoxicity provides an opportunity.This study used the gene chip data in the GEO database to screen the differentially expressed genes related to DLBCL CAR-T treatment neurotoxicity,and through further GO enrichment analysis,pathway analysis,and KEGG pathway analysis,And combined with prognostic analysis to further screen predictive biomarkers for predicting CAR-T neurotoxicity.Objective:To screen predictive bio-markers for the severity neurotoxicity associated with Chimeric Antigen Receptor T Cells in DLBCL.Methods:The mRNA expression profile data of GSE153438 were downloaded from the Gene Expression Omnibus(GEO)database(http://www.ncbi.nlm.nih.gov/geo).Limma R tool was utilized to identify differential expression genes(DEG)in non-severe neurotoxicity(grade 0-2)and severe(grade 3 or higher)neurotoxicity patients.DEGs was screened according to:fold change more than 2 and P value less than 0.05.Gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment assessment were carried out.We screened the hub gene by protein-protein interaction(PPI)network analysis and Cytoscape software.Gene set enrichment analysis(GSEA)was also analyzed with the GSEA software.Moreover,the predictive value of the hub gene for severe neurotoxicity was evaluated via receiver operating characteristic(ROC)curve analysis.Results:1.Total 51 DEGs were identified in the GSE153438 between non-severe and severe neurotoxicity tissues,includes 25 up-regulated and 26 down-regulated genes.2.GO analysis showed that these DEGs were mainly enriched in T cell activation,leukocyte cell-cell adhesion,and positive regulation of cell adhesion.3.KEGG analysis revealed that DEGs were significantly enriched in T cell receptor signaling pathway,cell adhesion molecules,and Epstein-Barr virus infection.4.GSEA revealed that the glycolysis pathway was significantly associated with severe neurotoxicity.The top centrality hub gene GZMB was identified from the PPI network.5.ROC curve analysis showed that GZMB had a potential predictive value for severe neurotoxicity.Conclusions:1.T cell activation and glycolysis pathways significantly associated with CAR-T cell-mediated severe neurotoxicity in DLBCL microenvironment before CAR-T cell infusion.2.GZMB might be used as a predictive and therapeutic molecular marker for neurotoxicity associated with CAR-T cell therapy in DLBCL.