Exploration Of Exosomal Biomarkers And Pathogenic Mechanisms In Diffuse Large B-cell Lymphoma

Diffuse large B-cell lymphoma(DLBCL)is the most common type of non-Hodgkin's lymphoma(NHL),with highly heterogeneous characteristics in origin,gene expression profile and prognosis.Currently,R-CHOP remains the standard treatment for all DLBCL subtypes.Although 5-year overall survival rate in DLBCL patients treated with R-CHOP is 60-70%,still 30-40%of patients progress to relapsed/refractory DLBCL.Chimeric antigen receptor(CAR)T cell therapies have been approved for relapsed or refractory DLBCL patients and have achieved 30-40%of lasting remissions.But serious side effects such as cytokine toxicity,cytokine release syndrome,and neurotoxicity remain urgent issues.Currently,there are several approved or investigational targeted therapies for FAD that may be suitable for specific subgroups of patients.For example,ibrutinib was 37%effective in activating B-cell type DLBCL(ABC-DLBCL).Lenalidomide also showed significant efficacy in the ABC-DLBCL subgroup.Therefore,development of new molecular targets and targeted therapies is of great significance to improve the prognosis of patients with DLBCL.Fer,a class of non-receptor tyrosine kinases,is expressed in almost all kinds of cells.FerT is a truncated variant of Fer and is expressed only in tumor cells and spermatogenic cells.Fer/FerT kinase constitutes of mitochondrial electron transport complex-1,which activities in tumor cells and promotes ATP production.Fer is highly expressed in lung cancer,liver cancer,prostate cancer,breast cancer and other malignant tumors,and is associated with poor prognosis of patients in a variety of cancers.Several studies have shown that Fer downregulation inhibit tumor progression by decreasing cell proliferation,migration and invasion,promoting cell apoptosis and reducing chemotherapeutic resistance.Besides,Fer also reported to participate in vesicle transport and mitochondrial reprogramming.In the regulatory mechanism,Fer could be activated by several growth factor receptors,such as epidermal growth factor receptor(EGFR),platelet derived growth factor(PDGF)and insulin-like growth factor-1 receptor(IGF-1R).In the downstream of Fer was reported to interact with STAT3,CTTN,Rac-GTPase regulators,and MAPK,PI3K/AKT/mTOR,Wnt/?-catenin,Notch pathways.In addition,Fer could be activated by KIT or FLT3 to promote cell survival in acute myeloid leukemia(AML).However,the regulatory mechanism of Fer in DLBCL remains unclear.Exosomes is a kind of extracellular vesicles which are 30-150nm in diameter,originate from endosomes and released into the extracellular space.Exosomes are widely involved in the physiological and pathological processes of cells,such as tumor metastasis,cell differentiation,cell migration,immune response,antigen presentation,angiogenesis.Accumulating evidence suggest that exosomes may play an important role as gene messengers in the progression of hematological malignancy,such as bone marrow microenvironment reprogramming,drug resistance,immune response suppression.The structure and function of exosomes make them possess the advantages of stability,immune escape,non-toxicity and so on,which may show the potential value in the clinical application.On the one hand,exosomes are abundant in various biological fluids and can be used as diagnostic markers for DLBCL.On the other hand,exosomes can be used as a drug delivery system to improve the efficiency of targeting therapy.Finally,exosomes exerted as massagers between cells,are considered as targets for DLBCL treatment.This study observed the expression level of Fer in DLBCL and explored its relationship between Fer expression with the clinical characteristics of DLBCL patients.In the treatment study,Fer inhibitor E260 was reported to selectively activate the energy consumption process of hepatoma tumor cells,leading to cancer cell necrosis and autophagy.Thus,we aimed to explore the function and regulatory mechanisms of E260 in the progression of DLBCL.In the last,Fer was observed upregulated in lymphoma tissues of DLBCL patients,but whether Fer would express in plasma exosomes of DLBCL patients remains unclear.Therefore,we decided to detect the expression level of plasma exosome Fer in DLBCL patients and to explore its relationship with disease progression.The expression and prognostic effect of Fer in DLBCL were detected in Oncomine and TCGA databases.The results showed that the mRNA expression of Fer was upregulated in the DLBCL patients,compared with the control group.In addition,Kaplan-Meier survival curve showed that high expression of Fer was associated with shorter overall survival(OS)in DLBCL patients.Fer expression in DLBCL tissue was tested by immunohistochemical assay,the results showed that compared with normal controls,Fer was upregulated in DLBCL tissues.The positive expression of Fer was correlated with clinical pathologic features of DLBCL patients,including higher Ann Arbor stage,LDH,Ki67,?2-M,IPI score and shorter OS(median=7.28,p<0.05).In addition,univariate and multivariate analyses showed that Fer was an independent prognostic factor in DLBCL(p<0.05).The expression level of Fer in DLBCL cell lines was detected by qRT-PCR and Western blot.The results showed that Fer was highly expressed in DLBCL cell lines(p<0.05).CCK-8 assay showed that Fer knocking down significantly reduced the proliferation ability of OCI-LY3 and U2932 cell lines,while Fer overexpression promoted cell proliferation(p<0.05).We further confirmed the growth promoting effect of Fer in xenograft mice.Consistent with the above data,Fer knockdown reduced Ki67 expression and inhibited tumor growth.Annexin V/7AAD assay of apoptosis revealed that Fer knocking down enhanced apoptosis in OCI-LY3 cells(8.8%vs 1.6%)and U2932 cells(7.1%vs 3.3%)compared to control(p<0.001).By Transwell analysis,Fer knocking down significantly antagonized the invasive ability of OCI-LY3 and U2932 cells(p<0.01),and overexpression of Fer promoted the invasive ability of DLBCL cells(p<0.05).CCK-8 assay showed that sh-Fer combined with doxorubicin or ibrutinib significantly inhibited cell proliferation,compared with the monotherapy group(p<0.05).In treating,CCK-8 assay showed that E260 and EXO-E260 inhibited the proliferation of OCI-LY3 and U2932 cells in a dose-dependent and time-dependent manner(p<0.05).In the U2932 cell line,EXO-E260 showed a higher anti-proliferation efficacy than E260 at 48h(p<0.05).Cell apoptosis was detected by Annexin V/7AAD staining and the results showed that Annexin V/7AAD staining indicating cell apoptosis/necrosis was significantly increased in E260 and Exo-E260-treated cells(p<0.01).Western blot further revealed that DLBCL cells treated with E260 did not express cleaved-caspase3 and cleaved-PARP apoptotic markers,suggesting that E260 induced cell necrosis rather than apoptosis in DLBCL cells.The effects of E260 on the invasion ability of DLCBL cells was detected by transwell assay.It was found that E260 and Exo-E260 significantly weakened the invasive ability of OCI-LY3 and U2932 cells(p<0.01).In conclusion,E260 inhibited lymphoma progression,whereas exosome-mediated delivery of E260 showed a better antitumor efficacy(p<0.01).By co-treating DLBCL cells with EXO-E260 and conventional chemotherapeutic agents,the results showed that the combination of EXO-E260 with doxorubicin or ibrutinib significantly reduced cell proliferation,compared with the monotherapy group(p<0.05).To confirm the antitumor activity of EXO-E260 in vivo,OCI-LY3 xenograft tumor mice were intraperitoneally injected with PBS,E260(25 mg/kg)and 100?g EXO-E260.Compared with tumors treated with PBS or E260,Exo-E260 significantly inhibited the tumor growth of OCI-LY3 xenograft in mice(p<0.01).Immunohistochemical staining showed that the expressions of Fer and Ki67 were also decreased in mice treated with EXO-E260.Mechanistically,we first used STRING to predict the interacting proteins of Fer.GO and KEGG enrichment analysis of the interacting proteins showed that the biological processes involved in Fer were mainly related to cell proliferation,apoptosis,cell cycle,cell adhesion and drug reaction.KEGG analysis showed that Fer was significantly correlated with Hippo,JAK-STAT,FOXO,PI3K-AKT and mTOR signaling pathways.The expression of Hippo pathway related interacting proteins(AJUBA,YWHAH,CDH1,CTNNB1)were further tested.However,we found out that only A JUBA was upregulated in DLBCL cells.In addition,AJUBA was reduced in sh-Fer cells and its expression could be reversed by restoring AJUBA expression.The interaction between AJUBA and Fer was clarified by Co-IP analysis.Western blot analysis showed that E260 downregulated AJUBA expression and enhanced the phosphorylation of MOB1 and YAP Ser 397 in DLBCL cells.In addition,E260 can induce cytoplasmic retention of YAP and decrease the expression level of nuclear YAP.On the other hand,overexpression of AJUBA in E260 treated cells reversed p-YAP expression as well as inhibition of cell proliferation and invasion.Plasma exosomes of DLBCL xenograft mice were isolated and the expression of Fer mRNA was detected by qRT-PCR.We found that the expression of Fer mRNA was increased in the plasma exosomes of xenograft mice(p<0.05)and showed positively correlation with the tumor volume(r=0.65,P=0.04).Compared with normal controls,the expression level of Fer in plasma exosomes of DLBCL patients was increased(p<0.001).Furthermore,the expression level of exosomal Fer in DLBCL patients was decreased after R-CHOP treatment(p<0.05).In addition,exosomal AJUBA expression was also upregulated in DLBCL patients(p<0.001)and positively correlated with the expression level of Fer(r=0.76,p<0.0001).ROC curve showed that exosomal Fer could be used as a potential diagnostic indicator for DLBCL,and exosomal Fer combined with LDH had the highest diagnostic efficiency(AUC=0.93,p<0.01).In addition,we analyzed the relationship between exosomal Fer expression and clinicopathological features and found that the high expression of Fer was positively correlated with Ann Arbor stage,LDH,Ki67 and IPI score(p<0.05).Here,we found that Fer overexpression was observed and related to inferior outcomes in DLBCL patients.Fer silencing strongly inhibited cell proliferation,invasion and potentiated chemosensitivity,while Fer overexpression showed opposite effects.E260,a targeted inhibitor of Fer,significantly abrogated cell survival and invasion,but potentiated cell chemosensitivity in DLBCL.Moreover,exosomes were applied to deliver E260 to DLBCL cells,which obviously potentiated the anti-tumor efficacy of E260.E260 reversed the activity of Hippo signaling by inhibiting AJUBA expression,resulting in decreased YAP cytoplasmic sequestration.We further detected the circulating exosomal Fer in DLBCL patients,the results demonstrated that Fer may act as an indicator for the diagnosis and progression of DLBCL.In summary,this study proved that Fer exerted as a non-invasive indicator and therapeutic target in DLBCL.Exosomes delivery of E260(EXO-E260)exhibited exciting anti-tumor effects in DLBCL,which may compensate for the shortcomings of conventional therapy and open up novel avenues for applicating of exosomes in clinical therapy.The diffuse large B-cell lymphoma(DLBCL)is the most common subtype of invasive lymphoma in adults,accounting for about 30%-40%of newly diagnosed non-Hodgkin's lymphoma(non-Hodgkin's lymphoma,NHL).DLBCL is highly heterogeneous,which brings great difficulties to the accurate diagnosis and treatment of DLBCL.With the clinical use of R-CHOP(rituximab,cyclophosphamide,doxorubicin,vincristine,prednisone),the 5-year survival rate for DLBCL patients with conventional chemotherapy increased from 32%to 68%.Although rituximab greatly improved the prognosis of patients,only 50%of patients responded well to rituximab,and the reduction of CD20 expression caused by long-term use of rituximab is an important reason for rituximab resistance.About 40%of patients progress to a relapsed or refractory stage with the 5-year survival rate of only 20%.Therefore,intensively study on the molecular mechanism of the initiation and progression of DLBCL and development of new predictive markers and potential therapeutic targets for DLBCL are crucial to improve the prognosis of patients with DLBCL.MicroRNAs(miRNAs)are a series of small,single-stranded,highly conserved non-coding RNAs with 18-22 nucleotides in length.MiRNA inhibits the expression of target genes at the post-transcriptional level by binding to the 3'-UTR of target mRNA and leading to translation inhibition or degradation.More and more evidence showed that miRNAs may be involved in the regulation of tumor initiation,progression and drug response.In addition,miRNAs are also considered to be useful in the diagnosis,progression and prognosis assessment of diseases.Hundreds of miRNAs have been identified as biomarkers of cancer.Notably,lymphoma-associated miRNAs have also been reported to be detected in the circulation.This may be due to miRNAs being protected from degradation by RNA-binding proteins and/or encapsulation in extracellular vesicles.A growing number of studies have reported that circulating exosomal miRNAs can serve as diagnostic and prognostic biomarkers in a variety of tumors.However,the use of exosomal miRNAs biomarkers in the diagnosis and prognosis have not well being studied in the DLBCL.By analyzing the GEO database,the differential miRNAs related to the progression of DLBCL were obtained.We then detected the expression of differential miRNAs in plasma exosomes and explored the relationship between the expression level of miRNAs and the occurrence and development of DLBCL.Further,we examined the role of miRNAs in the progression of DLBCL,so as to provide new ideas and experimental basis for the diagnosis and treatment of DLBCL.To obtain differential miRNAs(DEMs)involved in the initiation and progression of DLBCL,we downloaded two standardized microarray datasets(GES117063 and GSE29493)from the GEO database.The overlap between the two datasets contained 14 miRNAs.We selected 11 down-regulated miRNAs(let-7c,miR-107,miR-133a,miR-142-3p,miR-210,miR-215,miR-30a-5p,miR-346,miR-375,miR-485-3p,and miR-95)for the further study.The target genes of the above miRNAs were predicted using miRWalk 3.0 and miRDB database.Through the construction of miRNA-mRNA interaction network,it was found that most target genes were associated with miR-107 and miR-30a-5p.The expression of target genes was further verified by GEPIA database,the result showed that NSL1,NFIB,MBNL3,AGO4 and KMT2A were up-regulated in DLBCL.The GO and KEGG enrichment analysis of 484 target genes was performed using DAVID online tool.In the biological process,the target genes were mainly enrichment in transcription,transcription regulation since polymerase ? promoter and the function of signal transduction.In cell components,target genes were mainly expressed in nucleus,nucleoplasm and spindle.Among the molecular functions,the target genes were mainly enriched in the functions of protein binding,ATP binding and zinc ion binding.In KEGG pathway analysis,target genes were mainly enrichment in MAPK signaling pathway,transcriptional misregulation in cancer and cAMP signaling pathways.To verify the expression of DEMs in DLBCL,we collected peripheral blood from 42 patients with DLBCL and 31 healthy volunteers.The expression of miRNAs in plasma exosomes of DLBCL patients was detected by qRT-PCR,we found out that the expression of miR-107 and miR-375-3p was decreased,while the expression of miR-485-3p was increased(P<0.01).ROC curve was drawn to evaluate the diagnostic value of exosomal miRNAs in DLBCL patients.The results revealed that plasma exosomal miR-107,miR-375-3p and miR-485-3p could be used as potential indicators to distinguish DLBCL patients from healthy people.The area under the curve(AUC)of miR-107,miR-375-3p and miR-485-3p were 0.854,0.769 and 0.739,respectively,and the optimal cut-off values were 0.67,0.64 and 0.60,respectively.In addition,the low expression of exosome miR-107 was significantly correlated with higher Ann Arbor stage,ECOG score,LDH,?2-MG and IPI score in the DLBCL patients(P<0.05).In conclusion,the low expression of plasma exosomal miR-107 may performed as an indicator in DLBCL disease progression,and we believe that miR-107 may be involved in the initiation and progression of DLBCL.In order to verify the role of miR-107 in the progression of DLBCL,we first detected the expression of miR-107 in DLBCL cell lines,and then performed gain-and loss-of-function functional analysis.The expression of miR-107 in DLBCL(OCI-LY1,OCI-LY3,OCI-LY8,and OCI-LY10)cell lines was lower than that in normal controls(p<0.01).CCK-8 assay showed that overexpression of miR-107 significantly inhibited the proliferation of OCI-LY3 cells,compared with the Agomir NC group(p<0.01).However,down-regulation of miR-107 significantly enhanced the proliferation ability of OCI-LY8 cells(p<0.01).Flow cytometry assay showed that overexpression of miR-107 promoted apoptosis of DLBCL cells,while knocking down of miR-107 inhibited apoptosis of DLBCL cells(p<0.01).Finally,the effect of miR-107 on the invasion of DLBCL cells was evaluated by transwell assay.The results showed that overexpression of miR-107 significantly weakened the invasive ability of OCI-LY3 cells,while knocking down of miR-107 significantly stimulated the invasive ability of OCI-LY8 cells(p<0.01).To further confirm the antitumor effect of miR-107 in mice,we established a xenograft mouse model using OCI-LY1 cell lines.Compared with the Agomir NC group,mice inoculated with miR-107 overexpressed cells showed significantly lower tumor growth rate(Fig.4I).In general,miR-107 exerted as a tumor suppressor in the progression of DLBCL through inhibiting cell proliferation,invasion and promoting cell apoptosis.To explore the regulatory mechanisms of miR-107 in DLBCL,we first used four databases to predict the target genes of miR-107.We then performed GO and KEGG analyses on these 28 target genes by using the DAVID online tool.The results showed that miR-107 was closely related to the regulation of cell proliferation,cell cycle,angiogenesis,PI3K-AKT signaling pathway,oocyte meiosis related signaling pathway,AMPK signaling pathway and Hippo signaling pathway.MiR-107 is involved in the regulation of apoptosis,cell cycle and PI3K-AKT signaling pathway in the miRNAcancermap database.As shown in the network,we found out that miR-107 may be involved in the regulation of PI3K-AKT,oocyte meiosis and Hippo signaling pathways by targeting YWHAH.Cytoscape was used to map the ceRNA network of lncRNA-miRNA-mRNA,and the differentially expressed lncRNAs were further verified by GSE97336 dataset in the GEO database.We found out that lncRNA CKMT2-AS1 and lncRNA ZNF767P were up-regulated in DLBCL.These results will lay a foundation for the study of lncRNA-miRNA-mRNA regulatory mechanism in DLBCL.In order to screen out the downstream target genes of miR-107,we first searched the expression of these 28 target genes in DLBCL from GEPIA database,and found that only 6 genes including 14-3-3?,CDK6,RRAGC,PP2R5C,OGT,and KIF23 were upregulated in DLBCL(p<0.05).Next,we detected the expression of these candidate genes in DLBCL cell lines by qRT-PCR and found that 14-3-3?(YWHAH)was increased in OCI-LY3 and OCI-LY8 cell lines(p<0.05).Furthermore,14-3-3? expression was decreased in miR-107 overexpressing cells and increased in miR-107 decreasing cells(p<0.05).To investigate the expression of 14-3-3? in DLBCL patients,qRT-PCR and confocal detection were performed.Compared with healthy controls,14-3-3? was upregulated in PBMCs of DLBCL patients.Similarly,the expression of 14-3-3? in the lymphoma tissue of the DLBCL patients was detected by immunofluorescence staining.Compared with the control group,the expression of 14-3-3? was increased in the lymphoma tissues of the DLBCL patients,and 14-3-3? was mainly expressed in the cytoplasm.In conclusion,14-3-3? expression was upregulated in DLBCL and negatively regulated by miR-107.Further,we used dual luciferase reporter activity assay to detect whether there was a direct binding between miR-107 and 14-3-3?.The luciferase reporter plasmid was transfected into miR-107 overexpressed cells,and the luciferase activity was significantly decreased in cells transfected with WT plasmid,while the luciferase activity was not inhibited in cells transfected with MUT plasmid.The results showed that miR107 specifically binds to the 3'-UTR region of 14-3-3?.In this study,11 down-regulated miRNAs were detected from GEO,decreased exosomal miR-107 and miR-375-3p and increased exosomal miR-485-3p were observed in the plasma of DLBCL patients(p<0.05).The diagnostic value of plasma exosomal miRNAs in DLBCL patients was analyzed by ROC curve,the AUC were 0.854,0.769 and 0.739,respectively.Notably,upregulation of miR-107 in DLBCL cells was significantly associated with advanced Ann Arbor stage,IPI score,high LDH,and?2-MG in DLBCL patients(all p<0.05).This suggested that miR-107 may play a role in the progression of DLBCL.Thus,upregulation of miR-107 could inhibit cell proliferation and invasion and induce apoptosis of DLBCL cells.Similarly,miR-107 upregulation significantly inhibited tumor growth in OCI-LY8 xenograft mice.In contrast,downregulation of miR-107 by miR-107 Antagomir resulted in upregulation of cell proliferation,apoptotic resistance and cell invasion.Therefore,we speculated that miR-107 acted as a tumor suppressor in the progression of DLBCL by inhibiting cell proliferation,invasion and promoting cell apoptosis.In addition,we found that 14-3-3? expression was up-regulated in DLBCL and negatively regulated by miR-107.Therefore,we suggested that miR-107 regulated the progression of DLBCL by targeting 14-3-3?.This study not only revealed the feasibility of exosomal miRNA as a potential biomarker,but also provided a new target for the treatment of DLBCL.