Microarray-based expression analysis
We isolated peripheral blood exosomes from 7 healthy subjects and 7 DLBCL patients, and then we compared their gene expression profiles with a microRNA microarray. The limma software package was used to identify the differentially expressed genes (DEGs) with |logFC| > 2 and p value < 0.05 as the threshold [16], and the pheatmap package was employed to construct heatmaps of the DEGs. The DLBCL-related microarray profiles of GSE29493 and GSE40239 were retrieved from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/). GSE29493 was used to confirm that micro-124 was differentially expressed in DLBCL samples and seven normal samples. GSE40239 was used to examine the prognostic value of micro-124 in DLBCL samples. The miRbase database (http://www.mirbase.org/), miRDB database (http://mirdb.org), and TargetScan database (http://www.targetscan.org) were employed to predict the miRNAs that might regulate NFATc1. The targeted binding site between NFATc1 and miR-124-3p was predicted through the microRNA database (http://www.microrna.org/). The prognostic genes were obtained from the GEO database (GSE10846) (thresholds: p < 0.05 and HR > 1.5 or < 0.5) [17]. The differentially expressed genes were downloaded from the GEPIA website (http://gepia.cancer-pku.cn/) under the following parameters: log2FC = 1, q-value = 0.01, differential methods = ANOVA, and chromosomal distribution = both [18]. Gene Ontology (GO) analyses were performed using the WebGestalt website (http://www.webgestalt.org/option.php). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were analyzed using Metascape (http://metascape.org/gp/index.html#/main/step1). A protein–protein interaction (PPI) network was constructed using the STRING database (http://www.string-db.org/) with an integrated score of 0.9 [19]. Data were imported into Cytoscape 3.7.2 software to analyze the network. The expression of NFATc1 in The Cancer Genome Atlas (TCGA) database was analyzed using the GEPIA database and Oncomine dataset (www.oncomine.org). Transcription factors were predicted through the hTFtarget website (http://bioinfo.life.hust.edu.cn/hTFtarget#!/).
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 Affiliated 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 confirmed 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 humidified atmosphere at 37°C with 5% CO2. The medium was supplemented with 100 units/mL penicillin and 100 µg/mL streptomycin. BMSCs were extracted and purified from the bone marrow of healthy adults and then cultured in Dulbecco’s modified 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 (1×105 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% confluence, the cells were subcultured again. Then, the hBMSCs were passaged every 2-3 days after purification and amplification. 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 flow 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).
Transfection and lentiviral transduction
DLBCL cells were inoculated into 60-mm dishes (1×106 cells/dish) or 100-mm dishes (2×106 cells/well) 1 day prior to transfection. The cells were transfected with miR-124-3p/NC mimic, miR-124-3p/NC inhibitor, and sh-NFATc1/NC plasmids (GenePharma, Shanghai, China) in accordance with the instructions provided with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) when cell confluence was confirmed to have reached 60–80%. The cells were analyzed and further examined 48 h after transfection. Lentivirus packaging was performed in 60-mm dishes containing pMD2G (1 μg), psPAX2 (3 μg) and pLenti6.3-Luciferase/miR-124-3p (miR-NC/miR-124-3p) (4 μg) plasmids. Twenty-four hours after transfection, the medium was replaced with fresh medium for another 24 h culture period. The changed medium was collected and mixed for target cell infection purposes. Lentivirus-transfected BMSCs were cultured overnight in 24-well plates at a density of 5× 104 cells/well. Before transduction, medium containing 500 µL lentivirus supernatant, 500 µL fresh culture medium, and 8 µg polyacrylamide (Sigma, St. Louis, MO, USA) was used to assist the internalization of virus particles. The plates were then centrifuged at 2100 g at 37 °C for 1 h and replaced with fresh medium.
Dual-luciferase reporter assay
The targeting relationship between miR-124-3p and NFATc1 was verified with a dual luciferase reporter gene assay. The artificially synthesized NFATc1 3’-untranslated region (3’-UTR) gene fragment was integrated into the psiCHECK-2 dual luciferase vector to construct psiCHECK-2-NFATc1-3’-UTR-WT (NFATc1-WT) and psiCHECK-2-NFATc1-3’-UTR-MUT (NFATc1-MUT). The wild-type (WT) and mutant (MUT) plasmids were cotransfected with the miR-124-3p mimic and mimic NC. After 24 h of transfection, the cells were collected and lysed. Luciferase activity was detected using a Dual Luciferase Reporter Kit (Promega, Madison, WI, USA). The experiments were repeated 3 times independently, and all investigations involving at least 6 wells were repeated.
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 confluence 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 filtered through a 0.22 μm filter (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 fixed 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 Purification Kit (Rengen Biosciences Co., Ltd., Liaoning, China). Briefly, 5 µL DiO was incubated with 50 µl reaction buffer solution. Next, the system was cultured with 50 µl exosomes at a concentration of 5×109/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 purified labeled exosomes. The labeled exosomes were cocultured with DLBCL cells in serum-free medium. DLBCL cells were subsequently fixed 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 fluorescence microscope (FV3000, Olympus, Tokyo, Japan).
Coculture and inhibition of exosome secretion
The exosomes extracted from miR-124-3p-transfected hBMSCs were inoculated with DLBCL cells in a 6-well plate for 48 h (Fig. 7a). The cells were then divided into two groups: Exo-miR-NC+ DLBCL and Exo-miR-124-3p + DLBCL. DLBCL cells were washed with PBS three times after incubation, and then subsequent experiments were carried out. miR-124-3p-transfected hBMSCs (5×104) and DLBCL cells (1×105) were cocultured in 6-well plates containing Transwell apical and basolateral chambers (Fig. 8a). The apical chamber was cultured with DMEM containing 10% serum, and the basolateral chamber was cultured in DMEM = containing 15% serum. Cells were cocultured for 72 h. After coculturing, hBMSCs and DLBCL cells were collected and used for subsequent experiments.
The specific 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 quantified using a bicinchoninic acid (BCA) kit (Thermo Fisher Scientific, Rockford, IL, USA). Protein samples (30 μg) were used for sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and subsequently transferred to a polyvinylidene fluoride (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 quantified 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 flow cytometry. According to the manufacturer’s instructions, cells (1 × 106 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 flow 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 × 105 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 × 107) were suspended in 100 μL serum-free culture medium and subcutaneously injected into the upper flank 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 mm3, 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 (5×105 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 paraffin for immunohistochemistry analysis after imaging.
Immunohistochemistry
Immunohistochemical staining was performed using the streptavidin-peroxidase method. Formalin-fixed paraffin-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% H2O2 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 quantified 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 significant difference.