Cell culture, osteogenic induction and identification
Osteoblast cell line MC3T3-E1 and endothelial cell line bEnd.3 were purchased from the cell bank of Chinese Academy of Sciences (Shanghai, China), and respectively cultured in Dulbecco's modified Eagle’s medium (DMEM) (Gibco, USA) containing 10% fetal bovine serum (FBS) (Gibco, USA) and 100 U/ml penicillin streptomycin (Amersco, USA) in incubator under the condition of 37 ℃, 5% CO2 and 95% saturated humidity. The whole medium was changed for all cells every three days. OBs were cultured in osteogenic inducing medium composed of DMEM complete medium, 1×10-8M dexamethasone, 50mg/l L-ascorbic acid and 10mM β-Sodium glycerophosphate (Sigma, USA) for 21 days in order to induce OBs to MOBs. Alkaline phosphatase (ALP) staining and Alizarin red S (ARS) staining were respectively performed to identify the osteogenic induction efficiency.
Extraction and identification of exosomes
Before the extraction of exosomes, MOBs were cultured in DMEM with 10% exosome removed FBS for 1 week. Exoquick reagent (Invitrogen, USA) was applied to extract MOB-Exos according to the manufacturer’s protocol with some adjustments. Briefly, the culture supernatant was sequentially centrifuged in a centrifuge tube (Corning, USA) at 2000×g for 30 mins and in a 100 kDa ultrafiltration tube (Millipore, USA) at 4000×g for 30 mins to obtained concentrated cell supernatant. And then, total exosome isolation reagent with 0.5vol of concentrated supernatant was added into the supernatant and well mixed by vortexing. After incubated at 4 ℃ overnight, the mixture was centrifuged at 10000×g for 1 h at 4 ℃. Exosomes were obtained in the deposition at the bottom of the tube after discarding the supernatant, and then resuspended with 1×PBS with 0.01vol of the initial culture supernatant. BCA protein assay kits (Thermo, USA) were applied to determine the concentration of exosomes. And exosomes were identified by Nanosight light scattering technology, transmission electron microscope scanning and Western blot.
Immunofluorescence staining
200 μg MOB-Exos were prepared, subjected to PKH-67 green fluorescent dye (Sigma, USA) and incubated for 10 mins after well mixed by vortexing. An equal volume of 1% BSA were added into the mixture to terminate dyeing. The labeled exosomes were extracted by Exoquick reagent, washed and resuspended with 1×PBS, and then added into the culture medium of bEnd.3. After co-incubated with exosomes for 4 h, the adherent ECs were fixed with 4% neutral paraformaldehyde for 20 mins and subsequently dyed with DAPI for 5 mins. Finally, the labeled ECs were washed and photographed with the inverted phase-contrast fluorescence microscope. As a parallel experiment, micrONTM miRNA mimic and micrOFFTM miRNA inhibitor (RiboBio, China) were applied to detect the transfection efficiency of let-7f-5p mimics and inhibitors. Briefly, 20 μl cy3 labeled miRNA mimics/inhibitors and 20 μl lipo2000 were respectively dissolved in 500 μl DMEM medium and then well mixed and stood for 15 mins. OBs were culture in 6920 μl pure DMEM medium and then subjected to the mixture. After co-incubated for 4 h, the adherent OBs were fixed with 4% neutral paraformaldehyde for 20 mins, subsequently dyed with DAPI for 5 mins and photographed with the inverted phase-contrast fluorescence microscope.
CCK-8 assay
CCK-8 kits (Yeasen, China) were applied to examine the cell proliferation. Briefly, ECs of each group were respectively resuspended with DMEM medium containing 10% FBS without exosomes to density of 5×104 cells/ml. 100 μl of the cell suspension was added into wells of 96-well plates. 3 duplicate were set for each group at each time point, and sterile 1×PBS was added into the blank wells. At 0 h, 24 h, 48 h and 72 h after cell adhesion, 10 μl CCK-8 solution was added into each well respectively, and then incubated at 37 ℃ for 1 h. After the spectrophotometer was set to zero, the light absorption values at 450 nm (A450) of each well were measured, according to which the cell growth curve was drawn.
Wound healing assay
ECs of each group were respectively resuspended with DMEM medium containing 10% FBS without exosomes and inoculated into wells of the 6-well plates in density of 5×105 cells/well. 3 duplicate were set for each group at each time point. ECs were cultured overnight and adherent, and then 200 μl pipette heads were used to draw lines vertically at the well bottom. After wash out the exfoliated cells with 1×PBS, ECs were cultured in exosome-free DMEM medium. The scratches were observed and photographed with inverted phase-contrast microscope at 0h, 24h, 48h and 72h after scribing. The scratch areas and scratch heights were measured in Image J software, and then the average migration widths of cells were calculated by using the formula: average migration width = (0 h scratch area - scratch area at measurement time) / scratch area height.
Transwell migration assay
ECs of each group were respectively resuspended with pure DMEM medium to density of 5×105 cells/ml. 100 μl cell suspension was added into the upper chamber of Transwell chamber (Corning, USA), and 600 μl exosome free medium was added into the lower chamber. After cultured for 24 h, the medium and the cells in the upper chambers were wiped up. ECs migrating to the lower chambers were washed with ddH2O for three times, fixed with methanol for 30 minutes, and stained with 0.1% crystal violet solution for 20 minutes after air drying. Inverted-phase contrast microscope was used to observe and take pictures. Five fields were randomly selected to count the number of migrated cells in Image J software.
Tube formation assay
ECs of each group were respectively resuspended with DMEM medium containing 10% FBS without exosomes to density of 5×105 cells/ml. Matrigel (Corning, USA), 96-well plates and 200 μl pipette tips were placed in 4 ℃ refrigerator for precooling overnight. 50 μl Matrigel was added to each well and incubated at 37 ℃ for 30 mins. Then 50 μl cell suspension was added to each well and cultured for 6 h. The tubule like structures were observed under inverted phase-contrast microscope and photographed. The total segment lengths of tubule-like structures were measured in Image J software.
Real-time Quantitative PCR
Total RNA was isolated by RNA extraction reagents (Servicebio, China) and then subjected to ultramicro spectrophotometer (Thermo, USA) to detect the RNA concentration and purity according to the manufacturer’s instructions. After the RNA concentration was adjusted to 200 ng/μl, RevertAid First Strand cDNA Synthesis Kits (Thermo, USA) were applied in the RNA reverse transcription according to the manufacturer’s instructions. The quantitative PCR was performed with Prism 7300 real-time PCR system (ABI, USA) according to the recommendations. Data were standardized to the threshold cycle values according to the internal control of U6 .The primer sequences were listed on Table 1.
Table 1
List of the primer sequences
RNA name
|
|
primer sequence
|
U6
|
Forward
|
5’- CTCGCTTCGGCAGCACA-3’
|
Reverse
|
5’-AACGCTTCACGAATTTGCGT-3’
|
mmu-let-7f-5p
|
Forward
|
5’-CTCAACTGGTGTCGTGGAGTCGGCAAT
TCAGTTGAGAACTATAC-3’
|
Reverse
|
5’-ACACTCCAGCTGGGTGAGGTAGTAGATTGT-3’
|
mmu-let-7f-5p mimics
|
Forward
|
5’-UGAGGUAGUAGAUUGUAUAGUU-3’
|
Reverse
|
5’-AACTATACAATCTACTACCTCA-3’
|
mmu-let-7f-5p inhibitor
|
Single stranded
|
5’-AACTATACAATCTACTACCTCA-3’
|
DUSP1
|
Forward
|
5’-AGGACAACCACAAGGCAGAC-3’
|
Reverse
|
5’-ATACTCCGCCTCTGCTTCAC-3’
|
DUSP4
|
Forward
|
5’-GTACATCGACGCAGTAAAGGAC-3’
|
Reverse
|
5’-GCTTGACGAACTCAAAAGCCTC-3’
|
β-actin
|
Forward
|
5’-GGCTGTATTCCCCTCCATCG-3’
|
Reverse
|
5’- CCAGTTGGTAACAATGCCATGT-3’
|
Western blotting analysis
Western blotting was performed as previously described[20]. Briefly, cells and exosomes were lysed by RIPA buffer containing protease and phosphatase inhibitors (Sevenbiotech, China). The protein concentration of supernatant was quantified with BCA Protein Assay kit (Thermo, USA), and adjusted to the same with RIPA buffer for each group. Approximately 20 μg total protein of each sample were loaded, subjected to electrophoresis, and then transferred to PVDF membranes (Millipore, USA). Subsequently, the membranes were blocked with with 5% non-fat milk in TBST at room temperature for 2 h, and then respectively incubated with related primary antibodies for CD63 (Invitrogen, USA), CD81 (Invitrogen, USA), Erk1/2 (CST, USA), pErk1/2(Santa Cruz, USA), GAPDH(CST, USA), β-actin(CST, USA) at 4 ℃ overnight. After that, the membranes were washed with TBST for 3 times and incubated with secondary antibodies (CST, USA) at room temperature for 1 h and visualized ECL detection kit (Bio-channel, China). Finally, the photographs of membranes were taken with Fluor Chem E system (Proteinsimple, USA), and analyzed in Image J software.
Dual luciferase reporter assays
The dual luciferase reporter assays were performed with some modifications. Briefly, wild-type and mutant 3′-UTRs of dual specificity phosphatase-1 (DUSP1) and dual specificity phosphatase-4 (DUSP4) were respectively amplified and cloned downstream of the luciferase gene within the pmirGLO vectors (Promega, USA), with the Renilla luciferase plasmid (Promega, USA) co-transfected as a control. After transfected with 1 μg vectors and subjected to 60 μM let-7f-5p mimics, the luciferase activities of the transformed cells were measured with Dual Luciferase Reporter assay kits (Promega, USA) at room temperature according to the manufacturer's instructions.
Statistical analysis
The experimental data were analyzed with graphpad prism 8 software. Unpaired t-test was used for two groups of independent measurement samples, one-way ANOVA test was used for multiple groups of independent measurement samples, and chi square test was used for counting samples. P < 0.05 and P < 0.01 were both regarded as statistical difference.