The IDD-related gene expression dataset GSE63492 was retrieved from Gene Expression Omnibus (GEO) database (GSE63492) , which included microRNA expression profiling of human NP derived from 5 patients with IDD in comparison with those derived from 5 healthy cadaveric discs as normal control. The limma 3.26.8 package was employed for differential analyses . Differentially expressed microRNAs (DEMs) were screened out based on the set P Value < 0.05 and Log FoldChange > 2. Analyses were performed using R (v 3.6.1) with Rstudio (1.2.5019). Afterwards, miRNA-target gene prediction databases including miRTarBase (http://mirtarbase.mbc.nctu.edu.tw/php/download.php), DIANA (http://diana.imis.athena-innovation.gr/DianaTools/index.php?r=microT_CDS/index), miRDB (http://www.mirdb.org/), miWalk (http://mirwalk.umm.uni-heidelberg.de/) and TargetScan (http://www.targetscan.org/vert_72/) were employed to predict Genes that might be regulated by the interested miRNA. The predicted results were analyzed using Jvenn (http://jvenn.toulouse.inra.fr/app/example.html).
Isolation and culture of nucleus pulposus cells
Degenerative NP tissues from 4 males and 6 females, aged 32–69 years (average 53.9 years), were collected from patients undergoing surgery for IDD (Additional file 1). Healthy NP tissues were collected from four non-IDD patients aged 16 to 22 years (average: 19 years), who underwent idiopathic scoliosis (IS) surgery or anterior decompression surgery due to fresh traumatic lumbar fractures with neurological deficits. These patients underwent conventional lumbar magnetic resonance imaging (MRI) scans before surgery. According to the Pfirrmann classification , the degree of disc degeneration was graded based on T2-weighted images. Specimens were cut into small parts (approximately 1–2 mm3) immediately for various experiments. A portion was immediately immersed in RNA later (Invitrogen, Carlsbad, CA, USA) and frozen in liquid nitrogen for RNA analysis. The other part was immediately immersed in 1x phosphate buffered saline (PBS) for cell separation. The NP tissues were cut into small pieces of 1–2 mm3, washed 3 times with PBS supplemented with penicillin and streptomycin, and then digested with 0.5% type II collagenase (Sigma, St Louis, MO, USA) for 6 hours. The tissue debris was then removed through a 75-µm filter. The resulting cells were centrifuged at 250 × g for 10 minutes, and then resuspended in Dulbecco’s modified Eagle medium/F12 (DMEM/F-12) medium (Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS) (Gibco) and 100 U/ml penicillin-streptomycin. Finally, the NPCs were incubated at 37 °C in a humidified atmosphere of 5% CO2. After one week, the cells adhering to the well were considered as primary NPCs. The cells from the second passage were used for further experiments.
Dual luciferase reporter gene assay
The target relationship between miR-4450 and ZNF121 was initially predicted by the biological prediction tool miRDB (http://www.mirdb.org/) and further verified using the dual luciferase reporter gene detection method. The target gene ZNF121 dual luciferase reporter gene vector and the mutated miR-4450 binding site were constructed and named pGL3-ZNF121-Luc wild type (WT) and pGL3-ZNF121-Luc mutant (MUT), respectively. Then the two reporter plasmid pGL3-Luc vectors (Promega, Madison, Wisconsin, USA) were co-transfected with miR-4450 mimic and NC mimic into HEK293T cells (Cell Resource Center, Shanghai Institute of Biological Sciences, Chinese Academy of Sciences, Shanghai, China). Twenty-four hours after transfection, the luciferase activity was detected using a dual luciferase reporter gene detection system (Dual-Luciferase® Reporter Assay System, E1910, Promega, Madison, WI, United States). Briefly, cells were washed with PBS and lysed with the recommended volume of 1X Passive Lysis Buffer (PLB). The culture vessel was gently shaken / rock at room temperature for 15 minutes. The lysate was then transferred to a test tube or vial, centrifuged at 12,000 rpm for 1 minute, and the supernatant was collected. Next, 100 µL of LAR II (Luciferase Assay Substrate in Luciferase Assay Buffer II) equilibrated to room temperature was added to the photometer tube, and carefully draw 20 µL of cell lysate into the photometer tube, The firefly luciferase activity was then measured by shaking gently. Thereafter, 100 µL of Stop & Glo® reagent equilibrated to room temperature was added to measure Renilla luciferase activity. The relative luciferase activity was expressed as the ratio of firefly luciferase activity to Renilla luciferase activity. Luciferase activity was detected using a GloMax® 20/20 luminometer with a programmed photometer to provide a pre-read delay of 2 seconds, followed by a measurement time of 10 seconds. The experiment was repeated three times independently.
NPCs in vitro inflammation induction and transfection
NF-α is a key pro-inflammatory cytokine that could induce NPC apoptosis . Therefore, in ivtro, TNF-α was usually used as an inducer of a model of IDD. TNF-α (10 ng/ml, 24 h) induced apoptosis of degenerative NPCs (DNPCs), and successfully established an apoptosis model suitable for subsequent experiments. TNF-α was used in subsequent experiments. The DNPCs were treated with negative control (NC) of miR-4450 mimic (NC-mimic), miR-4450 mimic, NC of miR-4450 inhibitor (NC-inhibitor), miR-4450 inhibitor, NC of Vector-ZNF121 (Vector-ZNF121 NC) and Vector-ZNF121. miR-4450 mimic and miR-4450 inhibitor were purchased from GenePharma (Shanghai, China). The respective sequence of miR-4450 mimic and miR-4450 inhibitor was 5’- UGGGGAUUUGGAGAAGUGGUGA-3’ and 5’-UCACCACUUCUCCAAAUCCCCA-3’. The pCMV6-AC-GFP and pCMV6-AC-GFP-ZNF121 plasmids were purchased from Origene Company and were used as Vector-NC and Vector-ZNF121 respectively. The open reading frame (ORF) of ZNF121 cloned into this vector will be expressed in mammalian cells as a C-terminal green fluorescent protein (GFP) tagged protein. GFP (maximum excitation/emission = 482/502 nm) was mainly used for applications where fast appearance of bright fluorescence is crucial. The transfection procedure of DNPCs was performed according to the manual of Lipofectamine 2000 (11668-019, Invitrogen Inc., Carlsbad, CA, USA). DNPCs were seeded in a 12-well culture plate at a density of 1.5 × 105 cells. After incubating overnight, cells were transiently transfected with Vector-ZNF121 NC (1.5 µg/well) or Vector-ZNF121 (1.5 µg/well). Cells were incubated in a CO2 incubator at 37 °C for 6–8 hours, and then cultured for 24 hours in fresh complete medium (DMEM/F-12 with 10% FBS and 100 U/ml penicillin-streptomycin) for further experiments.
Isolation and culture of PLMSCs
After signing the written consent, the placentas were collected from 11 female donors immediately after selective caesarean section without artificial labor, premature rupture of membranes, chromosomal abnormalities or chorioamnionitis. The average maternal age was 29 years (between 26–33 years), and the average gestational age is 37.5 weeks. The average placenta weight is 501.23 grams. Placental tissues (3 × 3 × 1 cm size) were dissected under sterile conditions. The tissues were washed thoroughly with 1x PBS pH 7.4 and cut into small pieces (about 1–2 mm3). Subsequently, the tissue was digested with 1.6 mg / ml collagenase (Sigma-Aldrich, USA) and 200 mg / ml deoxyribonuclease I (Sigma-Aldrich, USA) with shaking at 37 °C for 4 hours. After washing twice with PBS, the cells and all pellets were resuspended in MSC growth medium [Dulbecco's Modified Eagle Medium (DMEM; Gibco) supplemented with 10% human serum or 10% fetal bovine serum (FBS; Gibco®, USA)] and plated in a 25 cm2 tissue culture flask (Costa, Corning, USA). The flask was placed in a humidified tissue incubator with 5% carbon dioxide and kept at 37 °C. The medium was replaced every 3–4 days. The cells were continuously observed until the developmental colonies of fibroblast-like cells were formed. Adherent cells (approximately 80–90% confluence) were subcultured with 0.25% trypsin-EDTA (Gibco®, USA) and re-plated at a density of 1 × 104 cells/ cm2 for further expansion. Some batches of continuous subcultured cells were cryopreserved in freezing medium (90% FBS and 10% DMSO) and stored in liquid nitrogen for future use.
Characterization of hPLMSCs
PLMSCs at passage 3–5 cultured in DMEM supplemented with FBS or HS were detached with 0.25% trypsin-EDTA and washed twice with PBS. In each sample, 4 × 105 cells were resuspended in 50 µl PBS and incubated with 10 µl fluorescein isothiocyanate (FITC) or phycoerythrin (PE)-conjugated antibodies against CD34 (Bio Legend, USA), CD44 (Bio Legend, USA), CD45 (USA, Bio Legend), CD73 (USA, Bio Legend), CD90 (USA, Bio Legend) or CD105 (USA, BD Bioscience), placed in a dark environment at 4 ℃ for 30 minutes. After washing with PBS, the cells were fixed with PBS containing 1% paraformaldehyde. Positive cells were identified by comparison with isotype matching controls [PE-conjugated mouse immunoglobulin G1 (IgG1) and FITC-conjugated mouse immunoglobulin G2a (IgG2a)]. At least 20,000 labeled cells were collected and analyzed using a flow cytometer (FACS caliburTM, Becton Dickinson, USA), and then the results were analyzed using FlowJo V10 software.
Lentivirus infection in PLMSCs
By the miRNA retroviral lentiviral vector system (Invitrogen Inc., Carlsbad, CA, USA), recombinant lentivirus containing the antagomiR-4450 sequence and negative recombinant lentivirus without the antagomiR-4450 (antagomiR-NC) were prepared. The synthesized antagomiR-4450 sequence was cloned into the pLenti-C-mGFP-P2A-Puro miR plasmid of the retroviral expression vector system (OriGene Inc., USA) and transformed into DH5α competent cells. Then the pLenti-C-mGFP-P2A-Puro antagomiR-4450 plasmid DNA was prepared and purified. Using liposomes, the recombinant plasmid and the packaging plasmid containing the target gene were co-transfected into HEK293T cells to construct an experimental lentivirus. Similarly, the plasmid without the target gene and the packaging plasmid were co-transfected into HEK293T cells to construct NC lentivirus. After incubating in 5% CO2 at 37 °C for 4–6 hours, the cells were further cultured in DMEM supplemented with 10% FBS for 72 hours. The virus-containing supernatant was collected and then filtered with a 0.45 µm cellulose acetate filter (Merck Millipore, Burlington, MA, USA) and stored at -80 °C. Subsequently, PLMSCs in the logarithmic growth phase were inoculated into a 6-well plate at a seeding density of 1 × 106 cells/mL and incubated for 24 hours at 37 °C with 5% CO2. A lentivirus solution was prepared by dilution with complete medium supplemented with 10% FBS, and Coagel was added at 5 µg/mL to enhance infection ability. After setting the NC group, 24 hours after infection, the medium was replaced with fresh complete medium. After 72 hours of further cultivation, exosomes were extracted for subsequent experiments.
Exosome purification and tracking analysis
The cells were cultured in medium supplemented with 10% exosome-depleted FBS (ultracentrifugation at 100,000 × g overnight) for 48–72 h. The supernatant was collected by differential centrifugation methods. The supernatant was collected and centrifuged at 300 × g for 10 minutes to remove the cell pellet. The supernatant was then centrifuged at 2,000 × g for 10 minutes to remove dead cells, and at 10,000 × g for 30 minutes to remove cell debris. The exosomes were then harvested by centrifugation at 100,000 × g for 70 minutes. The exosome pellet was washed twice by resuspending in 20 ml PBS and ultracentrifuging at 100,000 × g for 70 minutes (Sorvall SureSpin 630 rotor). A nanoparticle characterization system (NanoSight NS-300 instrument, Malvern, UK) equipped with a blue laser (405 nm) was used to characterize vesicles in real time.
The exosomes purified as described above were fixed in 200 mM phosphate buffer (pH 7.4) containing 2% PFA (w/v). The fixed exosomes were dropped onto the grid of the carbon coating of Forval and dried at room temperature for 20 minutes. After washing with PBS, the exosomes were fixed in 1% glutaraldehyde for 5 minutes, washed in water and stained with saturated aqueous uranyl oxalate solution for 5 minutes. The samples were then embedded in 0.4% (w/v) uranyl acetate and 1.8% (w/v) methyl cellulose, and incubated on ice for 10 minutes. Then the excess liquid was removed. The grid was dried at room temperature for 10 minutes and observed using an electron microscope (Model 910, Carl Zeiss) at 20,000 and 50,000 magnifications.
Uptake assay of exosomes by NPCs
The purified exosomes were directly labeled with 1 µM Vybrant Cell Tracers DiL (Life Technologies) by incubating at 37 °C for 30 minutes. The labeled exosomes were washed twice in 20 ml PBS, collected by ultracentrifugation as described above, and resuspended in PBS. We verified that no dye contamination occurred in the DiL-labeled exosome preparation by ultracentrifugation as described above. NPCs were stained with 1 µM DiO (Life Technologies) and seeded into the 8-well chamber and incubated with DiL-labeled exosomes (1 × 1010 particles/ ml) at 37 °C for 24 h under 5% CO2.
Exosome treatment in vitro
The DNPCs were seeded into 6-well or 12-well plates the day before treatment. When the cell concentration reached 70%, 1 × 1010 particles/ml exosomes (Exo-AntagomiR-NC or Exo-AntagomiR-4450) were introduced into the DNPC medium. DNPCs treated with PBS were regarded as blank controls. After 48 hours of treatment, cells were collected for later use.
BrdU cell proliferation assay
Cell proliferation rate was evaluated using a commercially available 5-bromo-2-deoxyuridine (BrdU) immunohistochemistry kit (Abcam). This method was based on the incorporation of BrdU into the newly synthesized DNA chain of proliferating cells. All steps were performed according to the manufacturer's manual. BrdU analysis was used to study the role of miR-4450, ZNF121 and related exosomes in DNPC proliferation. In short, the cultured cells (1 × 105 /well NPCs) were seeded into a 4-well chamber (Thomas Scientific) and incubated with 10 ng / mL TNF-α for 24 hours, and then treated with relevant reagents. The vector or lentivirus was transfected and incubated for 24 hours or exosomes treated and incubated for 48 hours, then the medium was removed, the cells were washed twice with 1xPBS, and labeled with 10 µM BrdU at 37 °C for 6 hours. Cells were fixed with 4% paraformaldehyde (Sigma-Aldrich; Merck-Millipore) for 30 minutes and incubated with biotinylated anti-BrdU antibody for 90 minutes at room temperature. Subsequently, streptavidin-HRP conjugate and diaminobenzidine (DAB) substrate were added, and the slides with cover slips were observed under a microscope. The BrdU incorporation was quantified by ImageJ 1.52a software (National Institutes of Health, USA). The cell proliferation rate was expressed as the number of positively stained cells divided by the total number of cells (set at 100%).
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazole bromide (MTT cell proliferation assay kit; Abcam) was used to measure cell viability. Cells (1 × 105 / well) were seeded into 96-well microplates and exposed to the indicated reagents at 37 °C for 48 hours before adding MTT. After treatment, the medium was carefully aspirated, and then 50 µL of serum-free medium and 50 µL of MTT reagents were added into each well, and incubated at 37 °C for 3 hours until a purple precipitate was visible. After incubation, 150 µL of MTT solvent was added to each well. The absorbance at 590 nm was measured by a microplate reader (EL × 800 Absorbance Microplate Reader, USA). All measurements were repeated three times.
Cell migration assay
The cell migration rate was measured using a transwell chamber (BD Biosciences, Franklin Lakes, NJ, USA). Trypsin-digested DNPCs were diluted to a final concentration of 2 × 106 cells/ml in serum-free medium, and 100 µl of cell suspension was added to the upper chamber, while 0.6 ml of DMEM containing 10% FBS was added to the lower chamber. DNPCs were transfected with vector or lentivirus and incubated for 24 hours, or treated with exosomes and incubated for 48 hours, then the medium was removed and the cells were washed twice with 1xPBS. After removal of the medium, chambers were incubated with 4% paraformaldehyde (Sigma-Aldrich; Merck-Millipore) for 15 minutes to fix the migrated cells on the lower side of the insert filter membrane, and the cells on the upper side of the filter membrane were removed with cotton swabs. The cells on the underside of the filter membrane were stained with 0.1% crystal violet (Sigma-Aldrich; Merck-Millipore) for 10 minutes. Then, after washing with 1 × PBS (Gibco; Thermo Fisher Scientific, Inc.), the cells on the lower side of the filter membrane were clearly visible and counted under a microscope (IX-70; Olympus Corporation, Tokyo, Japan). The cells that have migrated through the membrane were stained and counted, and NPC migration was expressed as the total number of cells that have migrated.
Annexin-V propidium iodide (PI) staining
Apoptosis detection kit (abcam) of Annexin V-FITC was used for the detection of cell apoptosis. After 48 hours of treatment, the cells were detached with trypsin (YB15050057, Yubo Biotechnology Co., Ltd., Shanghai) free of ethylenediaminetetraacetic acid (EDTA). DNPCs (1–5 × 105) were collected by centrifugation, washed once with cold serum-containing medium, and then resuspended in 500 µl 1X Binding Buffer. 5 µl of Annexin V-FITC and 5 µl of propidium iodide (PI 50 µg / ml) were added to the suspended cells, and incubated for 5 minutes at room temperature in the dark. Then, FITC signal detector (usually FL1) was used to perform annexin V-FITC binding analysis on the prepared sample by flow cytometry (Ex = 488 nm; Em = 530 nm) within 1 h. The phycoerythrin emission signal detector (usually FL2) performed PI staining analysis. To compensate and do gate settings, a negative control (binding buffer alone, unstained), a positive control and single staining of annexin V-FITC (no PI) and PI (no annexin V-FITC) were used. Usually, FITC Annexin V and PI double negative (feasible or no measurable apoptosis) were used to track cells, FITC Annexin V positive and PI negative (early apoptosis, membrane integrity) were employed to trace early apoptotic cells, and finally FITC Annexin V And PI positive (final stage apoptosis and death) were applied to trace late apoptosis or dead cells.
Establishment of a mouse model of IDD
Forty 15-week-old C57BL/6 male mice were obtained from the Experimental Animal Center of the Health Science Center of Xi'an Jiaotong University, and the study protocol passed the review and approval of the Ethics Committee of the Health Science Center of Xi'an Jiaotong University. The mice were randomly divided into sham operation, IDD + PBS, IDD + Exo-AntagomiR-NC and IDD + Exo-AntagomiR-4450 groups, 10 mice each group. The mice were placed in a room under standard laboratory conditions (temperature controlled at 21 ± 1 °C) and reared with a normal 12-hour light/12-hour dark cycle. The research protocol involving animal procedures was consistent with the guidelines of the Rush Institutional Animal Care and Use Committee (IACUC) and the IACUC Health Science Center of Xi’an Jiaotong University. All surgical operations were performed according to the protocol by Shi et al . The mice (15 weeks old) were placed in a supine position and anesthetized with oxygen containing 1.5% isoflurane (Abbott Laboratories, North Chicago, USA) through the mask at a rate of 2 L/min. After shaving and disinfecting the skin, use a #15 blade scalpel to make a longitudinal incision on the outside of the left abdomen with a length of about 1.5 cm. The peritoneum was separated from the subcutaneous fat and pulled to the right to expose the space between the back of the peritoneum and the left psoas muscle. Through this space, the posterior peritoneum was gently pulled to the right to make the spine visible and easily accessible. Use the pelvic edge as an anatomical landmark to identify L5/6 and L6/S1 IVDs. To induce IDD, first, under the guidance of a polyethylene stopper sleeve (BD Intramedic™ polyethylene tube; BD, Franklin Lakes, NJ), a sharp micro-scalpel was used to puncture the IVD at a depth of 0.7 mm. Through the same incision, another micro-scalpel with a bending point was used for a second puncture to destroy and remove the nucleus pulposus tissue. Except for the absence of IVD puncture, the mice in the sham operation group received all the operations before IDD surgical puncture. Then the IDD + PBS, IDD + Exo-AntagomiR-NC and IDD + Exo-AntagomiR-4450 were injected with 2 ul PBS, 2 ul 1 × 1010 particles/ml Exo-AntagomiR-NC and Exo-AntagomiR-4450, respectively. Then, the skin incision was sutured with 4 − 0 vein suture. After the operation, penicillin (Beijing bayerdi biotechnology Co. LTD) was injected into the thigh muscle of the mice, 40,000 units/kg per day according to the instructions for 3 consecutive days. On the 42nd day after surgery and injection, the mice were sacrificed with carbon dioxide. After removing the fur and tendon tissue, under a dissecting microscope (VistaVision, VWR International, Radnor, Pennsylvania), the IVD tissue was separated from the adjacent cartilage endplate and bone with a scalpel. The specimen was immediately immersed in RNA later and frozen in liquid nitrogen for RNA analysis.
The gait was recorded using the DigiGait 9.0 analysis system (Mouse Specifics, Inc.; Quincy, MA) . In short, the mouse ran at a specific speed on a transparent flat treadmill, and the camera captured ventral images. For each measurement, the animal ran for a maximum of 30 seconds, of which 5 seconds (equivalent to 10 consecutive steps) were used for analysis. Each mouse underwent a habituation test before the experimental test, and the animal was required to walk at a slightly faster speed (10 cm/sec) for 10 seconds. DigiGait image analysis software automatically defined each paw area, generated a waveform describing the forward/backward movement of each limb in a continuous stride, and determined the time period during which each paw contacted on the treadmill was the standing phase, and the middle period was the swing phase. We could also calculate posture and movement gait measurements, including stride time, stride length, and paw area. Brake and propel times were defined as the time before and after the maximum paw area during the stance phase, and the paw angle was expressed as the angle of the paw relative to the long axis . The symmetry index is defined as the absolute value of the difference between the contralateral hindlimbs divided by its average value. Before conducting the experimental tests, all settings (ie camera, lighting, belt speed) were optimized. After studying a series of speeds (Study 1) and reproducibility (Study 2), the treadmill speed used for the subsequent research (Study 3) was set to 15 cm/sec. It also recorded that the mouse treadmill task was not in compliance (ie, refuses to perform or complete the treadmill running task, which was considered to be the mouse's inability or reluctance to perform more than 2 consecutive steps). Data were recorded at the time points of day 07 and day 42 after surgery and injection. Considering that IDD mainly affected two hind legs, and the interference of any one of the four legs would affect the remaining legs, so the right hind leg which was researched in multiple studies was selected for analysis.
Fluorescence molecular tomopraphy (FMT)
Inflammation-related factors and MMPs were monitored by FMT in vivo at day 21 and 42 after surgery and injection, respectively. FMT could perform real-time three-dimensional quantitative analysis of the distribution of fluorescent dyes in living animal tissues. 24 hours before imaging, mice were injected with single dose of ProSense 680 or MMPSense 680 fluorescent imaging agents (PerkinElmer, Waltham, Massachusetts, USA). ProSense detected changes in the activity of lysosomal cathepsin (a protease), which could indicate many processes related to disease, such as cancer, inflammation, arthritis, etc. Matrix metalloproteinases (MMPs) were calcium-dependent zinc-containing endopeptidases, responsible for the degradation of most extracellular matrix proteins, and were associated with a variety of diseases, including inflammation and arthritis. MMPSense fluorescent agent was designed to detect the activity of MMPs (MMP-2, MMP-3, MMP-9 and MMP-13) and to evaluate many disease-related processes, including cancer progression, rheumatoid arthritis, lung disease, etc. Moreover, MMPSense also was used to evaluate the therapeutic efficacy of MMPs and potential drug candidates. The mice were anesthetized by intraperitoneal injection of ketamine (75 mg/kg) and medetomidine (1 mg/kg), placed in the imaging cassette, and then imaged using the VisEn FMT optical imaging system (PerkinElmer, Waltham, MA). A near-infrared (NIR) laser diode emitting continuous wave radiation at wavelengths of 670 nm transilluminated the lower body of each animal from posterior to anterior, and both excitation and emission signals were detected by a charge-coupled device camera and appropriate band-pass filters. The fluorescence intensity was quantified by evaluating the total radiation efficiency of the signal in the region of interest (ROI) ([photon/sec]/[mW/cm2]). The ROI was a 12.3 mm2 rectangle. It was selected from the anatomy of the disc segment on the grayscale photo of the mouse. Therefore, the ROI selection included the entire disc and was not affected by the fluorescent signal. A threshold of 10% of the maximum fluorescence in each reconstructed volume was used. The peak concentration (nmol/L) and total amount (pmol) of the fluorescent dye was automatically calculated relative to the internal standard generated by the appropriate dye of known concentration.
Enzyme linked immunosorbent assay (ELISA)
The expression of TNF-α and MMP13 was determined according to the operational manual provided by ELISA kit (Neobioscience Biotechnology Co., Ltd., Shenzhen, Guangdong, China). The optical density (OD) value of each well was determined at a wavelength of 450 nm within 20 minutes.
Reverse transcription quantitative polymerase chain reaction (RT-qPCR)
According to the manufacturer's instructions, total RNA was extracted from cells or tissues using Trizol reagent (Invitrogen) and quantified spectrophotometrically at 260 nm. The acceptable optical density 260/280 ratio was between 1.8 and 2.0. RNA quality was also determined by 1% agarose gel electrophoresis and stained with 1 mg/ml ethidium bromide. RNA was incubated with RNase-free DNase (Promega) to remove residual genomic DNA. The expression level of miR-4450 was quantified by Taq-Man microRNA analysis (Life Technologies) specific for mature miR-4450. Briefly, 10 ng RNA was transcribed by TaqMan microRNA RT kit (Life Technologies), and real-time PCR was performed using TaqMan MicroRNA Assays. The ubiquitously expressed miRNA snRNA U6 was used as an endogenous control. Levels of mRNA were quantified by real-time PCR using SYBR Green PCR Master Mix (Qiagen). Real-time PCR was performed on the ABI Prism 7900HT fast system (Applied Biosystems). Primers of ZNF121 were designed by Primer 5 software according to the related sequences provided by GeneBank, and the primers of miR-4450 were designed and synthesized by Shanghai Sanyuan Bioengineering Technology and Service Co., Ltd. (Shanghai, China). BLAST software was used to design primers for downstream target genes by homology analysis. The primer sequences were shown in Additional file 2. Amplifications were performed in a total volume of 20 µl with 1 µl cDNA. RT-qPCR was conducted with the following condition: 1 cycle of pre-denaturation at 95 °C for 15 min, and 45 cycles of denaturation at 94 °C for 15 s, annealing at 55 °C for 30 s and extension at 70 °C for 30 s. The relative mRNA expression was quantified by a comparison of the cycle threshold (Ct) values. The relative transcript level was calculated using the 2−ΔΔCt method and normalized to the internal reference gene. Each experiment was repeated three times.
Western blot analysis
Exosomes and cells were lysed using RIPA lysis buffer (Thermo Scientific), then total protein was collected and protein concentration was determined using BCA kit (20201ES76, Yasen Company, Shanghai, China). Next, 20 µg/well protein was loaded and separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for 1.5 h. The protein was then transferred to a polyvinylidene fluoride (PVDF) membrane and then sealed with 5% skim milk. Thereafter, rabbit polyclonal antibodies against β-actin (ab8227, 1: 1000), CD9 (ab223052, 1: 500), CD63 (ab134045, 1: 400), ZNF121 (PA5-63043, 1: 200), MMP13 (ab39012, 1: 500), cleaved-Caspase3 (c-Caspase3, ab2302, 1: 400) and collagenⅡ (COL2, ab34712, 1: 500) were incubated with the membrane at 4 °C overnight. The membrane was then incubated with the secondary antibody goat anti-rabbit IgG H & L (488) (ab150077, 1: 1000) at room temperature for 1 hour. All the above antibodies were purchased from Abcam Inc. (Cambridge, UK) except for ZNF121 which was from Invitrogen Company. Next, the membrane was imaged using an Odyssey fluorescence scanner (LI-COR Biosciences, Lincoln, Nebraska, USA). Finally, ImageJ was used to analyze the relative protein expression by scanning the protein strips in gray. Each reaction was repeated three times.
All data analyses were performed using SPSS 21.0 software (IBM Corp., Armonk, NY, USA). The measurement data were expressed as mean ± standard deviation. T-test was applied to the comparison of two groups. For data comparison between multiple groups, one-way analysis of variance (ANOVA) was used, and Tukey test was used for post-hoc test. P < 0.05 was considered a statistically significant indicator.