Human Bone Marrow-Derived Mesenchymal Stem Cell-Secreted Exosomes Overexpressing Microrna-124-3p Inhibit DLBCL Progression By Downregulating NFATc1

Background: Exosomes play important roles in intercellular communication by delivering microRNAs (miRNAs) that mediate tumor initiation and development, including those in diffuse large B cell lymphoma (DLBCL). To date, however, limited studies on the inhibitory effect of exosomes derived from human bone marrow-derived mesenchymal stem cells (hBMSCs) on DLBCL progression have been reported. Therefore, this study aimed to investigate the role of hBMSC-secreted exosomes carrying microRNA-124-3p in the development of DLBCL. Methods: Microarray-based expression analysis was adopted to identify differentially expressed genes and regulatory miRNAs, which revealed the candidate NFATc1. Next, the binding anity between miR-124-3p and NFATc1 was using luciferase activity assays. The mechanism underlying NFATc1 regulation was investigated using lentiviral transfections. Subsequently, DLBCL cells were cocultured with exosomes derived from hBMSCs transfected with a miR-124-3p mimic or control. Proliferation and apoptosis were measured in vitro. Finally, the effects of hBMSC-derived miR-124-3p on tumor growth were investigated in vivo. Results: MiR-124-3p was downregulated while NFATc1 was upregulated in DLBCL cells. MiR-124-3p specically targeted and negatively regulated the expression of NFATc1 in DLBCL cells, upregulated miR-124-3p-inhibited DLBCL cell proliferation and promoted apoptosis. In addition, we found that hBMSC-derived exosomes carrying miR-124-3p repressed DLBCL cell proliferation both in vitro and in vivo. Conclusion: hBMSC-derived exosomal miR-124-3p represses the development of DLBCL through the downregulation of NFATc1. found that DLBCL cells the b prognostic in We obtained miR-124-3p-targeted genes in DLBCL. c GO functional enrichment analysis of the 237 miR-124-3p-targeted genes. The results revealed that biological regulation, metabolic process, and response to stimulus were the main biological processes. These genes are related to various cellular components, including membrane, nucleus, and membrane-enclosed lumen. Concerning molecular function, the major activities of these genes include protein binding, ion binding, and nucleic acid binding. d The 237 miR-124-3p-related genes are displayed. e Protein–protein interaction (PPI) network analysis showed that NFATc1 was one of the hub genes that exhibited the greatest number of interactions analyzed using the STRING database and cytoHubba software. f Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis suggested that the 237 miR-124-3p-related genes were mainly concentrated in the signaling pathways "blood vessel development", "regulation of cell adhesion" and "positive regulation of cell cycle". b The morphology of hBMSCs. A relatively large number of puried cells a shuttle shape and swirling arrangement were observed. c-d The ability to induce adipogenic differentiation and osteogenic differentiation. A large number of lipid droplets appeared in the cells, and oil red O staining conrmed that the hBMSCs exhibited adipogenic differentiation (c). Many red calcium nodules were observed at the cell center using alizarin red staining (d). e Transmission electron microscopy (TEM) was used to identify BMSC-derived exosomes. The shape of the exosomes was globular or oval, with a complete lipid membrane. f Nanoparticle tracking analysis (NTA). The Zeta View nanoparticle tracking analyzer revealed that the exosome particles were predominately approximately 100 nm in size. g Western blot analysis. The exosome surface marker proteins CD63, TSG 101, and Hsp70 were expressed in BMSC-derived exosomes, whereas calnexin was not. h The miR-124-3p level in miR-124-3p-transfected hBMSCs and the exosomes derived from miR-124-3p mimic-treated hBMSCs was signicantly higher than that in control cells.

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Background
Diffuse large B cell lymphoma (DLBCL) accounts for 30-40% of all lymphoma cases and has the characteristics of high malignancy, fast in ltrating growth, and poor prognosis [1].The 5-year survival rate of late-stage DLBCL patients is less than 30%.Although approximately half of all DLBCL patients can be cured by the current standard chemotherapeutic regimen of rituximab combined with cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP), more than 40% of patients still experience relapse/refractory issues [2,3].Therefore, effective new treatment approaches that can be applied to improve patient survival or delay the recurrence of DLBCL are urgently needed.
Human mesenchymal stem cells (hMSCs) from adult bone marrow can be induced to differentiate into multiple mesenchymal tissues and then expanded and genetically modi ed in vitro, representing a potential therapeutic strategy for cancer patients (4).hBMSCs can migrate to tumor tissues, but the role of hBMSCs in tumorigenesis and cancer development is not well de ned.Additional emerging evidence indicates that hBMSCs may be crucial for DLBCL development [5,6].Therefore, better knowledge of these functions of hBMSCs in DLBCL could facilitate improved prognosis and suppress malignancy.Exosomes secreted by hBMSCs are often used experimentally as a potential therapeutic target for malignant tumors [7].Exosomes are small, membrane-enclosed vesicles (30-150 nm) that are secreted by various cells and deliver intracellular contents, such as proteins, messenger RNAs (mRNAs), and microRNAs (miRNAs), from the originating cells to the recipient cells [8].Exosomes, as a kind of natural carrier with targeting effects and drug delivery capability, have the characteristics of good histocompatibility, few side effects and high e ciency of internal delivery [9].Therefore, exosomes have great potential in clinical applications.
As tumor suppressors or oncogenes, miRNAs are small noncoding RNA molecules that can regulate gene expression at the posttranscriptional level [10].MiR-124-3p has been shown to act as a tumor suppressor in several tumors, including breast cancer [11], cervical cancer [12] and colorectal cancer [13].According to a previous study, miR-124-3p is poorly expressed in DLBCL and is capable of inhibiting cellular proliferation by binding to NF-κB [14].In the present study, we found that miR-124-3p negatively regulates nuclear factor of activated T cells c1 (NFATc1).As a potential oncogene, NFATc1 has been reported to be involved in the development of DLBCL.Moreover, it was found that NFATc1 can mediate the transcription of cMYC, which is associated with proliferation and apoptosis in DLBCL [15].
Based on the above information, we aimed to explore the therapeutic effect of miR-124-3p transferred by hBMSC-derived exosomes on DLBCL by regulating NFATc1 to provide novel potential therapeutic strategies.

Tissue sample collection
Primary DLBCL tissues (n = 36) were obtained from patients who underwent surgical resection at the Lymphoma and Myeloma Diagnosis and Treatment Center, The Second A liated Hospital of Dalian Medical University between 2016 and 2019.The tissue samples were immediately washed with phosphate buffered saline (PBS) after surgery and then cut into small blocks and preserved in liquid nitrogen in a cryopreservation tube.All DLBCL cases were con rmed through a histopathological examination.None of the patients had received cancer treatment before surgery.Complete patient clinicopathological data were collected and followed up until May 31, 2020.Patients who died from non-DLBCL-related diseases were excluded from this study.

Cell cultures
Human SU-DHL-6 and SU-DHL-10 cells were a kind gift from Professors Jing Wei and Fang Wang of the Biology Laboratory of Sichuan University (Sichuan, China).The SU-DHL-6 cell line (Cobioer Biosciences Co., Ltd.; January 2018) and the SU-DHL-10 cell line (Chinese Academy of Sciences; July 2019) were authenticated using short tandem repeat authentication.The cells were cultured in Roswell Park Memorial Institute 1640 medium (HyClone, Logan, UT, USA).Cells were supplemented with 10% fetal bovine serum (FBS; HyClone) (SU-DHL-10) and 15% FBS (SU-DHL-6) and maintained in a humidi ed atmosphere at 37°C with 5% CO 2 .The medium was supplemented with 100 units/mL penicillin and 100 µg/mL streptomycin.BMSCs were extracted and puri ed from the bone marrow of healthy adults and then cultured in Dulbecco's modi ed Eagle's medium (DMEM) hBMSC isolation and culture [20] Under sterile conditions, 10 mL bone marrow was collected from the femoral shaft fracture end with a 20-mL syringe (containing 2,000 IU heparin), after which the bone marrow cells were mixed quickly with heparin.Next, the samples were transferred into a 15 mL sterile centrifuge tube and centrifuged at 258×g for 10 min.The remaining cells were washed with DMEM 3 times and resuspended in 15 mL medium after the upper adipose tissues were removed.The same volume of Ficoll-Paque™ PLUS lymphocyte isolate (density 1.077 g/ml) was added to the tube after which the suspension was centrifuged at 715×g for 20 min.Nucleated cells were noted to be located predominately in the boundary and upper liquids, while most of the erythrocytes had precipitated to the bottom.The nucleated cells were retrieved from the interface with a pipette and centrifuged at 178×g for 8 min.Next, 5 mL cell culture medium was added to resuspend the nuclear cells.The cell suspension (10 µL) was mixed with 490 µL PBS.Then, 10 µL of the mixture was obtained for cell counting.Subsequently, the cells were incubated in a culture bottle (1×10 5 cells/bottle) with 5 mL DMEM-F12 culture medium at 37 °C with 5% CO2.After 24 h, hBMSCs began to adhere to the wall, and half of the medium was discarded to remove nonadherent cells.After 4-7 days, when the adherent hBMSCs reached 80-90% con uence, the cells were subcultured again.Then, the hBMSCs were passaged every 2-3 days after puri cation and ampli cation.hBMSCs of the third to seventh passages were used for further experiments.hBMSCs were subjected to osteogenic and adipogenic differentiation in OriCell™ medium (Cyagen Biosciences Inc., Guangzhou, China) and then stained with alizarin red and oil red O.A ow cytometer (BD Biosciences Pharmingen, San Jose, CA, USA) was adopted to detect the hBMSC surface markers CD105, CD90, CD31, CD34, CD45, CD166, CD29, CD11b, HLA-DR, and CD73 (antibodies were purchased from BD Biosciences, USA).

Exosome isolation
Serum exosomes: Whole blood (10 mL) was collected from DLBCL patients and controls, and the serum was separated.Subsequently, exosomes were isolated from the serum using an ExoQuick Kit (EXOQ20A-1, System Biosciences, Palo Alto, CA, USA) in strict accordance with the instructions provided by the manufacturer.
Exosomes of DLBCL cell culture: When BMSC con uence reached approximately 80%, the medium was changed to 10% exosome-free FBS at 37°C in a CO2 incubator for 48 h.The cell supernatant (100 mL) was collected and subsequently centrifuged at 300×g for 10 min, 2000×g for 10 min, and 10000×g for 30 min at 4 °C.The supernatant was collected and ltered through a 0.22 μm lter (Beyotime Institute of Biotechnology, Shanghai, China) and then ultracentrifuged at 100000 ×g for 2 hours.The sediments containing the exosomes were resuspended in 50 to 100 μL PBS and stored at -80°C.
Observation of exosomes under an electron microscope and NTA Transmission electron microscopy was used to identify exosomes.The exosome suspension was xed with 2% paraformaldehyde and then adsorbed onto carbon-coated copper grids.The copper grids containing exosomes were incubated with 1% glutaraldehyde for 5 minutes, ddH2O for 2 minutes, uranyl acetate (pH=7) for 5 minutes, and methylcellulose for 10 minutes.Finally, the samples were examined under an electron microscope (HITACHI, Tokyo, Japan) after drying at an accelerating voltage of 80 kV.
Exosomes were dissolved in 1 mL PBS and vortexed for 1 min to ensure uniform distribution.A Zeta View PMX110 nanoparticle tracking analyzer (Particle Metrix, Germany) was employed to measure the size distribution of exosomes.

Internalization of exosomes by DLBCL cells
DiO is used to track exosomes, as it can stably bind to the lipid region of the exosome lipid membrane.All experimental steps were performed following the instructions of the DIO-Membrane EVS Labeling and Puri cation Kit (Rengen Biosciences Co., Ltd., Liaoning, China).Brie y, 5 µL DiO was incubated with 50 µl reaction buffer solution.Next, the system was cultured with 50 µl exosomes at a concentration of 5×10 9 /mL for 30 min at room temperature and then centrifuged at 50×g for 90 seconds on a spin column.One hundred microliters of the exosome labeling preparation was carefully applied to the top of the column.The column was placed in a 1.5 ml lightproof microcentrifuge tube and then centrifuged for 90 seconds at 50 × g.The eluate was collected as puri ed labeled exosomes.The labeled exosomes were cocultured with DLBCL cells in serum-free medium.DLBCL cells were subsequently xed with 4% paraformaldehyde, and nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI).Finally, the capture of exosomes by DLBCL cells was observed under a uorescence microscope (FV3000, Olympus, Tokyo, Japan).
The speci c inhibitors GW4869 (Sigma-Aldrich, St. Louis, MO, USA) and DMA (Santa Cruz, Paso Robles, CA, USA) were used to block exosome secretion.DMSO was used as a negative control.Then, 10 nM GW4869 or 15 nM DMA was added to hBMSCs transfected with miR-124-3p at 48 hours prior to the end of coculture.

Reverse transcription quantitative polymerase chain reaction (RT-qPCR)
Total RNA was extracted using a TRIzol Kit (Invitrogen, Carlsbad, California, USA), and diethylpyrocarbonate (DEPC)-treated ultrapure water was used to dissolve RNA.RNA concentration and purity were measured on a Nanodrop 2000 ultraviolet spectrophotometer (Tiangen, Beijing, China) at wavelengths of 260 nm and 280 nm.Next, the extracted RNA was reverse transcribed into complementary DNA (cDNA) in accordance with the instructions of the All-in-One miRNA RT-qPCR Kit (Takara, Shiga, Japan).Next, the reaction was performed according to the instructions of the kit (Fermentas Inc., Hanover, MD, USA).The primers were designed and synthesized by TaKaRa (Tokyo, Japan) (shown in Table 1).Real-time quantitative PCR (RT-qPCR) was conducted using a quantitative PCR instrument (Bio-Rad iQ5, Bio-Rad, Richmond, CA, USA).U6 was used as the internal reference for miR-124-3p, while glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal reference for NFATc1.The 2-ΔΔCt method was employed to determine relative gene expression.Each experiment was conducted 3 times.

Western blot analysis
Total protein was extracted and quanti ed using a bicinchoninic acid (BCA) kit (Thermo Fisher Scienti c, Rockford, IL, USA).Protein samples (30 μg) were used for sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and subsequently transferred to a polyvinylidene uoride (PVDF) membrane (Amersham plc, GE Healthcare, Chicago, Illinois, USA).Next, the proteins were blocked in bovine serum albumin (BSA) at room temperature for 1 h and incubated with primary antibodies against CD63 (1:1000, Abcam, UK), Hsp70 (1:1000, Abcam, UK), calnexin (1:1000, Abcam, UK), TSG 101 (1:1000, Abcam, UK), cMYC (1:1000, Cell Signaling Technology Danvers, MA, USA), and GAPDH (1:5000, Abcam, UK) and a mouse antibody against NFATc1 (1:1000, Santa Cruz, CA, USA) at 4 °C overnight.The membranes were washed with TBST (TBS containing 0.1% Tween-20) three times followed by incubation with a horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature.The samples were then washed with TBST 3 times and visualized by electrogenerated chemiluminescence detection, and the densitometric values were determined using a gel image analysis system (Bio-Rad Laboratories).Western blotting results were quanti ed using a Gel-Pro analyzer.The following formula was used to calculate the relative expression of the target protein: relative expression = gray value of the target protein band/ gray value of the internal reference band of the same sample.

Apoptosis assay
Apoptosis was assessed by annexin V-FITC and/or propidium iodide (PI) double standard staining analysis using ow cytometry.According to the manufacturer's instructions, cells (1 × 10 6 cells/mL) were collected and washed three times with PBS.The cells were subsequently resuspended in 195 µL binding buffer and gently mixed with 5 µL annexin V-FITC and 10 µl PI at room temperature for 30 min in the dark.Cell apoptosis was analyzed using a BD FACSCanto II ow cytometer (BD Biosciences, San Jose, CA, USA) at 488 nm.Data were analyzed with FlowJo software.
Cell proliferation assay DLBCL cells were seeded at a density of 2 × 10 5 cells/mL and incubated in 96-well plates for 24 and 48 h.At the end of the incubation period, 10 μL CCK-8 solution was added to each well and incubated at 37°C for an additional 2 h at 37°C.The absorbance at 540 nm was recorded on a microplate reader (Bio-Rad Laboratories, Hercules, CA, USA).

Pulldown assay
The biotin-labeled double-stranded oligonucleotide probe targeting the cMYC promoter sequence was synthesized by PCR using biotin-labeled primers from Sangon Biotech Co. (sense, 5'-GCCCTA-TCAGAACAATGAAT-3' and its complementary strand).Nuclear proteins (400 μg) were mixed with the double-stranded biotinylated cMYC promoter probe (4 µg) and streptavidin agarose beads (50 ml) in 500 ml PBSI buffer containing 0.5 mM PMSF, 10 mM NaF, 25 mM and β-glycerophosphate and rotated overnight at 4 °C.The beads were centrifuged and washed with PBSI buffer two times, and then the beads were resuspended in loading buffer and boiled at 100 °C for 10 min.The supernatant was analyzed by western blotting.

Tumor xenografts in nude mice
A total of 12 male NOD/SCID mice (4-6 weeks old, weighing 18-22 g) were obtained from Huafukang Biotechnology Co., Ltd.(Beijing, China).One week after acclimation, SU-DHL-10 cells (1 × 10 7 ) were suspended in 100 μL serum-free culture medium and subcutaneously injected into the upper ank region of NOD/SCID mice.Tumor volumes were measured every 2 days according to the formula V = 1/2 * (short diameter) 2 x (longest diameter).After the tumor reached an approximate volume of 100 mm 3 , the mice were randomly allocated to three groups (MSC-derived miR-124-3p, MSC-derived miR-NC and PBS; four animals per group).hBMSC stable cell lines transfected with miR-124-3p or miR-NC were injected into NOD/SCID mice via the tail vein once every three days (5×10 5 cells/mouse).Subsequently, the tumor size was then measured.After seven injections, the mice were euthanized by means of anesthesia.Thereafter, tumors were collected and weighed.Finally, the tumor samples were frozen in liquid nitrogen or embedded in para n for immunohistochemistry analysis after imaging.

Immunohistochemistry
Immunohistochemical staining was performed using the streptavidin-peroxidase method.Formalin-xed para n-embedded tissues were cut into 4 µm thick sections that were dewaxed and dehydrated with gradient ethanol.Sections were pretreated with 10 mM Tris-Na citrate (pH 6) for 20 min at 95°C and washed.Then, sections were incubated for 10 min in 3% H 2 O 2 in PBS to inhibit endogenous peroxidase.
Next, the sections were subjected to antigen retrieval and blocking.The sections were subsequently incubated with primary antibodies against NFATc1 (1:50) and cMYC (1:200) diluted in 1% BSA overnight at 4°C under agitation.After washing with PBS, the biotinylated secondary antibody-HRP conjugate (1:500) was incubated with normal goat serum (C-0005, Shanghai Haoran Bio Technology Co., Ltd., Shanghai, China) for 30 or 60 min at room temperature.The sections were developed with 3,3'diaminobenzidine and counterstained with hematoxylin (PT001, Shanghai Bogoo Biotechnology, Co., Ltd., Shanghai, China).After dehydration with gradient ethanol and sealing with neutral balsam, histological samples were quanti ed using light microscopy.

Statistical analysis
Statistical analyses were performed with SPSS 21.0 statistical software (SPSS, Inc., Chicago, IL, USA) and GraphPad Prism 6 software (GraphPad Software Inc., San Diego, CA, USA).Figures were generated with GraphPad Prism 6 software.Survival curves were drawn with the Kaplan-Meier method and analyzed by the log-rank test.Measurement data are presented as the mean ± standard deviation (SD) of the experimental groups.Comparisons between two groups were performed with an independent samples t-test; comparisons between multiple groups were performed with one-way analysis of variance (ANOVA).p < 0.05 indicated a statistically signi cant difference.

Results
The expression of miR-124-3p is low in DLBCL tissues and cells A large number of differentially expressed microRNAs were obtained from the exosomes of DLBCL patients and healthy controls (Fig. 1b).Among these microRNAs, miR-124-3p drew our attention.The DLBCL-related microarray (GSE29493 and GSE2399) indicated that the expression of miR-124-3p was lower in DLBCL cells than in normal B cells (Fig. 1a and c).Furthermore, we observed that low miR-124-3p expression levels in DLBCL cells indicated a poorer prognosis and shorter overall survival (OS) than high miR-124-3p expression levels (GSE40239) (Fig. 1d).These results indicate that the expression level of miR-124-3p is relatively low in DLBCL.

Overexpression of miR-124-3p inhibits proliferation and promotes apoptosis in DLBCL cells
To investigate the biological function of miR-124-3p, DLBCL cells were treated with a mimic and inhibitor of miR-124-3p.Compared with the mimic-negative control (NC) group, the apoptotic rate was elevated in the miR-124-3p mimic group (p < 0.05) (Fig. 1e and f).When miR-124-3p was inhibited, cell proliferation was signi cantly increased (Fig. 1g).Thus, we demonstrated that upregulated miR-124-3p could inhibit DLBCL cell proliferation and facilitate apoptosis.
MiR-124-3p may regulate the NFATc1 gene in DLBCL To further predict the target genes regulated by miR-124-3p, three miRNA-mRNA relation prediction databases (TargetScan, miRTarBase, and mirRDB) were used.A total of 1027 genes were identi ed as miR-124-3p-related genes (Fig. 2a).After intersection with the differentially expressed genes (GEPIA website) and prognostic genes (GSE10846), 237 genes were obtained (Fig. 2b).These genes are displayed in Fig. 2d.The genes were then subjected to GO, KEGG pathway, and PPI network analyses.GO functional enrichment analysis revealed that biological regulation, metabolic process, and response to stimulus were the main biological processes.
Concerning cellular components, the major components of these genes include membrane, nucleus, and membrane-enclosed lumen.These genes are related to various molecular function including protein binding, ion binding, and nucleic acid binding (Fig. 2c).PPI analysis using the STRING database and cytoHubba software showed that NFATc1 was one of the hub genes that exhibited the greatest number of interactions (Fig. 2e).Further KEGG enrichment analysis showed that these genes were mainly concentrated in the signaling pathways "blood vessel development", "regulation of cell adhesion" and "positive regulation of cell cycle" (Fig. 2f).Among these miR-124-3p-targeted genes, it was noted that NFATc1 is involved in multiple tumors, including DLBCL [21].

MiR-124-3p targets and downregulates NFATc1
The targeted binding site between NFATc1 and miR-124-3p was predicted through the microRNA database (http://www.microrna.org/)(Fig. 3a) and TargetScan (Fig. 3b).To verify that NFATc1 is the direct target gene of miR-124-3p, a dual luciferase reporter gene assay was performed.The results showed that compared to the NC group, the luciferase activity of' the NFATc1 wild-type (WT) 3'untranslated region (UTR) group was signi cantly inhibited by miR-124-3p (p < 0.05), while no difference was observed in the NFATc1 3'-UTR MUT group (p > 0.05) (Fig. 3c).To further verify the effect of miR-124-3p on NFATc1, qRT-PCR (Fig. 3e) and western blot analysis (Fig. 3d and f) were then employed to determine the mRNA and protein expression of NFATc1.The expression of the NFATc1 gene was decreased when miR-124-3p was overexpressed, whereas this trend was reversed when miR-124-3p was inhibited.These ndings indicate that NFATc1 is a target gene of miR-205 and that miR-205 negatively regulates NFATc1.

Silencing NFATc1 inhibits proliferation and promotes apoptosis in DLBCL cells
NFATc1 is highly expressed in DLBCL (TCGA database) (Fig. 4a).The Oncomine dataset showed that NFATc1 is differentially expressed (Fig. 4b) and associated with a poor prognosis (Fig. 4c).To verify the effect of NFATc1 on DLBCL cells, the NFATc1 gene was silenced.The silencing e ciency of sh-NFATc1 was evaluated by RT-PCR analysis (Fig. 4d).Silencing NFATc1 obviously suppressed cell proliferation (Fig. 4e) and promoted cell apoptosis (Fig. 4f).cMYC was one of the NFATc1-regulated genes predicted through the hTFtarget website, and the binding sites are displayed in Fig. 4g.There was a signi cant decline in c-MYC and NFATc1 expression in NFATc1-silenced cells (Fig. 4h).A pulldown assay was performed to analyze the binding of the NFATc1 protein to the cMYC promoter (Fig. 4h).NFATc1 knockdown suppressed proliferation and promoted apoptosis in DLBCL cells.

Overexpression of NFATc1 rescues the biological function of miR-124-3p
To investigate the relevance of miR-124-3p and NFATc1 in human DLBCL samples, we performed realtime PCR to determine the expression of miR-124-3p and NFATc1 in 36 DLBCL samples.There was a signi cant correlation of the miR-124-3p level with stage, IPI score, and extranodal invasion.The NFATc1 level was also correlated with stage and IPI score but not with other clinical features (Table 2).The Kaplan-Meier survival curve showed that increased miR-124-3p expression and low NFATc1 expression were related to good survival (Fig. 5a and b).A negative correlation between miR-124-3p and NFATc1 was observed in the 36 DLBCL tissues (r = − 0.707, p = 0.001, Fig. 5c).Furthermore, the miR-124-3p mimic was cotransfected with the NFATc1 overexpression plasmid.Exogenous overexpression of NFATc1 rescued the effects of miR-124-3p on DLBCL cells (Fig. 5d and e).NFATc1 and cMYC protein expression was inhibited by miR-124-3p overexpression and reversed by NFATc1 cotransfection (Fig. 5f).These results indicate that the inhibitory role of miR-124-3p in DLBCL cells is mediated by the downregulation of NFATc1.

Isolation and identi cation of hBMSCs
After three to four passages, we observed a relatively large number of puri ed hBMSCs with a shuttle shape and swirling arrangement (Fig. 6b).Antibodies against CD105, CD90, CD31, CD34, CD45, CD166, CD29, CD11b, HLA-DR, and CD73 were used to identify surface antigens by ow cytometry.Generally, CD29, CD90, CD73, CD105, and CD166 are considered markers of hBMSCs [22].CD34 and CD45 are regarded as hematopoietic stem cell markers; CD31 is expressed ubiquitously within the vascular compartment and is located mainly at junctions between adjacent cells [23].CD11b is expressed on the surface of many leukocytes, including monocytes, neutrophils, natural killer cells, granulocytes and macrophages, as well as on 8% of spleen cells and 44% of bone marrow cells.Functionally, CD11b regulates leukocyte adhesion and migration to mediate the in ammatory response [24].HLA-DR is an MHC (major histocompatibility complex) class II cell surface receptor encoded by the human leukocyte antigen complex and is predominately expressed in lymphocytes and macrophages [25].We found that CD29 (99.74%),CD90 (99.81%),CD105 (99.32%),CD166 (96.33%), and CD73 (99.73%) were expressed, while CD34, CD31, CD45, CD11b, and HLA-DR were not.These results support that the cultured cells were hBMSCs (Fig. 6a).Next, the ability of hBMSCs to induce differentiation was assessed.We found that a large number of lipid droplets appeared in the cells after 2 weeks of adipogenic differentiation.Oil red O staining con rmed that the hBMSCs exhibited adipogenic differentiation (Fig. 6c).After 4 weeks of osteogenic differentiation, many red calcium nodules were observed at the cell center using alizarin red staining (Fig. 6d).

Exosomes are successfully extracted from hBMSCs
Transmission electron microscopy (TEM) was used to identify BMSC-derived exosomes.The shape of exosomes was globular or oval, with a complete lipid membrane (Fig. 6e).The Zeta View nanoparticle tracking analyzer revealed that the exosome particles were predominately approximately 100 nm in size (Fig. 6f).Western blot analysis showed that the exosome surface marker proteins CD63, TSG 101, and Hsp70 were expressed in BMSC-derived exosomes, whereas calnexin was not (Fig. 6g).We found that the miR-124-3p level in miR-124-3p-transfected hBMSCs and the exosomes derived from miR-124-3p mimictreated hBMSCs was signi cantly higher than that in control cells (Fig. 6h).These results suggest that hBMSCs can effectively release exosomes containing miR-124-3p.hBMSCs deliver miR-124-3p to DLBCL cells by secreting exosomes Next, to observe whether exosomes derived from hBMSCs can be absorbed by DLBCL cells, we cocultured hBMSC-isolated exosomes labeled with DIO with DLBCL cells (Fig. 7a).As shown in Fig. 7b, slight green uorescence, indicating exosomes absorbed by DLBCL cells, could be observed cells under a confocal uorescence microscope.To determine whether miR-124-3p transferred from the exosomes of hBMSCs could modulate the biological function of DLBCL cells, proliferation and apoptosis experiments were conducted.The results showed that the apoptosis rates were increased (Fig. 7c) and the proliferation of DLBCL cells was inhibited (Fig. 7d) after hBMSC-derived exosomes containing miR-124-3p were added.
To further investigate the roles of hBMSC exosomes in DLBCL cells, the miR-124-3p-transfected hBMSCs were cocultured with DLBCL cells (Fig. 8a).Furthermore, we employed GW4869 and DMA, an exosome inhibitor, to reduce exosome secretion to determine the function of exosomes in this process.The results demonstrated that miR-124-3p-transfected hBMSCs inhibited proliferation (Fig. 8b) and induced apoptosis (Fig. 8c and d) in DLBCL cells by delivering miR-124-3p via the release of exosomes and that the biological function caused by hBMSC-derived exosomal miR-124-3p could be rescued by GW4869 or DMA.We also found that coculture with GW4869 and DMA resulted in a signi cant decrease in miR-124-3p (Fig. 8e) and increase in NFATc1 (Fig. 8f).Thus, GW4869 and DMA could effectively inhibit the production of exosomes from hBMSCs and then reduce the transfer of miR-124-3p in hBMSCs to DLBCL cells, indicating that hBMSCs could affect the biological functions of DLBCL cells though exosomes.

MiR-124-3p secreted from hBMSCs inhibits tumor growth in vivo
To further evaluate the effect of hBMSC-derived miR-124-3p on DLBCL tumor growth in vivo, tumorbearing mice were injected with hBMSC-derived miR-124-3p, hBMSC-derived miR-NC or PBS via the tail vein.Tumor volume (Fig. 9a and b) and tumor weight (Fig. 9c) were measured, and the tumor tissues were subjected to immunohistochemical staining.The tumor volumes and tumor weights in the hBMSCderived miR-124-3p group were signi cantly lower than those in the hBMSC-derived miR-124-3p NC and PBS groups.These ndings support the inhibitory effect of miR-124-3p on tumor growth in vivo.The immunohistochemical staining results showed that hBMSC-derived miR-124-3p signi cantly decreased the NFATc1 level in DLBCL tissues (p < 0.05) (Fig. 9d) compared with hBMSC-derived miR-NC and PBS.
Taken together, these results indicate that hBMSC-derived miR-124-3p suppresses tumor growth both in vitro and in vivo by downregulating NFATc1 (Fig. 10).

Discussion
DLBCL is one of the most aggressive lymphoid malignancies in humans.Although chemotherapy e cacy can be further improved by the application of rituximab, patients who experience relapse/refractory issues usually have a median survival duration of less than 2 years [3].In recent years, MSC-derived exosomes transfected with miRNAs have been shown to play essential roles in tumorigenesis and can serve as mediators in human cancers.Several studies have reported that exosomes released from bone marrow mesenchymal stem cells (MSCs) can carry microRNAs (miRNAs), which are involved in cancer cell proliferation, differentiation, and apoptosis [26][27][28].However, the effect of human BMSC (hBMSC)-derived exosomes on the occurrence and progression of DLBCL remains poorly understood.In our study, we explored the role of hBMSC-derived exosomes delivering miR-124-3p in DLBCL and found that miR-124-3p transferred by hBMSC-secreted exosomes could inhibit the proliferation of DLBCL cells by attenuating NFATc1 expression.
Our initial ndings showed that miR-124-3p was downregulated while NFATc1 was upregulated in primary DLBCL tissues and DLBCL-derived cell lines.MiR-124-3p is a tumor suppressor microRNA with low expression in various hematological tumor types, including multiple myeloma (MM), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), and B-cell lymphomas [29].MiR-124-3p is frequently hypermethylated in non-Hodgkin's lymphoma (NHL), with a heterochromatic histone con guration.Hypermethylation results in the inactivation of miR-124-3p and the activation of downstream oncogenes, leading to tumorigenesis [30].It has been reported that miR-124-3p inhibits cell proliferation and promotes apoptosis by suppressing the expression of MYC and BCL2 by directly targeting NF-κB p65 in DLBCL cells [14].In ovarian cancer, miR-124-3p secreted by ovarian surface epithelial cells can be transferred via exosomes to cancer-associated broblasts and inhibit the transition from normal broblasts to cancer-associated broblasts by targeting sphingosine kinase 1 (SPHK1) [31].Our results also suggest that miR-124-3p can target and negatively regulate the expression of the transcription factor NFATc1.NFATc1 can directly upregulate the cMYC gene by binding to the cMYC promoter, and the upregulation of cMYC expression is closely related to the growth and survival of tumor cells [32,33].In human acute myelogenous leukemia, high NFATc1 expression is associated with a poor prognosis [34].However, NFATc1 overexpression is associated with good overall survival, and the ectopic expression of NFATc1 inhibits hepatic cancer proliferation [35].Our results suggest that NFATc1 can promote the expression of c-MYC.Moreover, increased NFATc1 expression has been correlated with an advanced tumor stage, a high IPI score and a poor prognosis in DLBCL patients.
Pham et al. found a novel epigenetic chromatin remodeling mechanism for NFATc1 in the pathophysiology of aggressive lymphoma B cells.NFATc1 regulates the c-myc oncogene in DLBCL cells through a chromatin remodeling mechanism that involves recruitment of the SWI/SNF ATPase Brg-1 chromatin remodeling complex [15].Gong et al. concluded that miR-124-3p could target NFATc1 in the chondrogenesis process [36].Until now, there has been no research focusing on miR-124-3p regulating NFATc1 in DLBCL.The bioinformatics analysis in the current study showed that NFATc1 was a target of miR-124-3p in DLBCL, which was further veri ed with a dual luciferase reporter gene assay.hBMSCs have self-renewal ability and multilineage differentiation potential and are able to differentiate into multiple cell types, including osteoblasts, adipocytes and chondroblasts [37].
They mainly act as immunosuppressive mediators, as evidenced by the upregulation of PD-1 in T cells [40].In addition, DLBCL exosomes promote cell proliferation, the migration of stromal cells and angiogenesis.It has also been demonstrated that exosomal miRNA-99a-5p and miRNA-125b-5p contribute to DLBCL chemoresistance [41].Koch R et al [42] reported that DLBCL possesses a selforganized infrastructure comprising side population (SP) and non-SP cells.SP cells exhibit autonomous clonogenicity, and their clonogenic ability can be transported to non-SP cells by EXO-mediated Wnt signaling.Recent evidence has shown that miRNAs loaded into exosomes can be delivered to cancer cells and function as suppressors in vitro and in vivo.For instance, hBMSC-derived exosomal miR-205 retards prostate cancer progression by inhibiting RHPN2 [43].MiR-155-5p secreted by melanoma cell exosomes can induce the proangiogenic switch of cancer-associated broblasts via the SOCS1/JAK2/STAT3 signaling pathway [44].MiR-34a in hBMSC-derived exosomes inhibits glioblastoma cell proliferation, invasion, migration and tumorigenesis in vitro and in vivo and promotes chemosensitivity to temozolomide by silencing MYCN [20].In the present study, we found that miR-124-3p transferred by BMSC-derived exosomes could be delivered to DLBCL cells and inhibit tumor growth both in vivo and in vitro via the downregulation of NFATc1.

Conclusion
In summary, the results of the study present show that miR-124-3p, when delivered by hBMSCs via exosomes, is able to inhibit proliferation and induce apoptosis in DLBCL cells by downregulating NFATc1.These ndings likely highlight the potential of miR-124-3p as a novel molecular target for DLBCL treatment.However, the e ciency of miR-124-3p delivered from hBMSCs to DLBCL cells via exosomes needs to be validated and improved.Further research with more samples and more cell lines is required to substantiate our ndings.
Bioinformatics predicted NFATc1 as the target of miR-124-3p.a A total of 1027 genes were identi ed as miR-124-3p-related genes analyzed by three miRNA-mRNA relation prediction databases (TargetScan, miRTarBase, and mirRDB).b The 1027 miR-124-3p-related genes were intersected with the differentially expressed genes (GEPIA website) and prognostic genes (GSE10846) in DLBCL.We obtained 237 miR-124-3p-targeted genes in DLBCL.c GO functional enrichment analysis of the 237 miR-124-3p-targeted genes.The results revealed that biological regulation, metabolic process, and response to stimulus were the main biological processes.These genes are related to various cellular components, including membrane, nucleus, and membrane-enclosed lumen.Concerning molecular function, the major activities of these genes include protein binding, ion binding, and nucleic acid binding.d The 237 miR-124-3prelated genes are displayed.e Protein-protein interaction (PPI) network analysis showed that NFATc1 was one of the hub genes that exhibited the greatest number of interactions analyzed using the STRING database and cytoHubba software.f Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis suggested that the 237 miR-124-3p-related genes were mainly concentrated in the signaling pathways "blood vessel development", "regulation of cell adhesion" and "positive regulation of cell cycle".The expression of the NFATc1 gene was decreased when miR-124-3p was overexpressed, whereas this trend was reversed when miR-124-3p was inhibited.The data are expressed as the mean ± SD; comparisons between two groups were analyzed using unpaired t-tests, and the experiment was repeated three times independently (*P < 0.05, **P < 0.01).