Isolation and culture of ADSCs
The Institutional Animal Care and Use Committee of Southeast University approved the protocol for the use of animals in this study. Adipose tissues were collected from the inguinal fat pad from 2-3-week-old C57BL/6 mice (SLAC, Shanghai, China), and then rinsed in phosphate-buffered saline (PBS) and cut into 1×1mm pieces. The collected tissues were digested by collagenase type II (Sigma-Aldrich, USA) at 37 °C for 1 h. After digestion, tissues were centrifuged at room temperature (1000rpm, 5min) and the resultant cell pellet was re-suspended in Dulbecco’s modified Eagle’s medium (DMEM) at the cell density of 5×106/ml. Cells were then cultured at 37℃ under a 5% CO2 atmosphere. The culture medium was replaced every 2-3 days and cells after 3 passages were used in the present study.
Induced differentiation potential of isolated ADSCs
ADSCs were cultured in a 24-well plate at a density of 4×104 cells/well with basic culture medium (DMEM-LG) containing 10% FBS, 100 U/ml penicillin and 100mg/ml streptomycin. They were subjected to induced differentiation by culturing them in osteogenic (Cyagen Biosciences, Rasmx-90021), adipogenic (Cyagen Biosciences, Rasmx-90031) and chondrogenic (Cyagen Biosciences, Rasmx-90041) medium, respectively. The outcomes were evaluated by Alizarin Red, Oil Red O and Alcian Blue staining, respectively.
Isolation and characterization of exosomes
A Total Exosome Isolation kit (Invitrogen, 4478359, USA) was applied to isolate the total exosomes from the supernatant of ADSCs culture medium according to the manufacturer’s protocol. The experimental procedures were conducted as described before [24]. Bicinchoninic acid assay (BCA) protein assay kit was used to measure the concentration of isolated exosomes. The protein levels of CD9, CD63, CD81 and Alix (representative markers of exosomes) were then detected.
Exosomes uptake assay
ADSCs-Exos were labeled with PKH26 (Sigma Aldrich, USA) according to the manufacturer’s protocol. Isolated exosomes were resuspended in diluent C (1ml). Then 6 μl PKH26 was added into diluent C (1ml). The ADSCs-Exos and PKH26 solutions were mixed for 30 s and then centrifuged (120000×g, 2h, 4℃). Exosomes were resuspended in the complete culture medium and the PKH26-labeled ADSCs-Exos solution was added into MC3T3-E1 cells for incubation. After 24-48h of culture, MC3T3-E1 cells were fixed with 4% formaldehyde for 10 min. DAPI was used to stain the nuclei. Cells were finally observed under a confocal microscope.
MC3T3-E1 cells culture
MC3T3-E1 cells were provided by the American Type Culture Collection (ATCC, USA) and cultured with α-modified essential medium (Gibco, USA) at 37℃ under a 5% CO2 atmosphere. MC3T3-E1 cells were cultured in 6-well plates at the concentration of 1×106 cells/well. After 24-48 h of culture, MC3T3-E1 cells were transfected with anti-miR-NC, anti-miR-141-5p, miR-141-5pormiR-NC, which were obtained from GenePharma (Shanghai, China). After transfection, MC3T3-E1 cells were treated with different doses of TNF-α (0, 1, 2.5, 5, 10ng/ml). After 24-48 h of culture, MC3T3-E1 cells were then co-cultured with ADSCs-Exos (25 μg/ml, 50 μg/ml, 100 μg/ml) or KCNQ1OT1-Exos (25 μg/ml) for 24-48 h.
Cell Viability Assay
The MC3T3-E1 viability was determined by the Cell Counting Kit-8 (CCK-8, Dojindo, Japan) assay. Briefly, cells were seeded into 96-well plates (2×104cell/ml) and incubated for 24h. Then, 10 μl CCK-8 solution was added into each well for incubating 1-2 h (37 °C, 5% CO2). Subsequently, the optical density (OD) value was measured at 450 nm using spectrophotometer (Bio-Rad, USA).
Cell apoptosis assay
MC3T3-E1 cell apoptosis was evaluated by Annexin V-FITC/PI Apoptosis Assay Kit (Keygen Biotech, China). Briefly, MC3T3-E1 cells were collected and washed with PBS. A total of 500 μl binding buffer was added to suspend cells. Firstly, 5 μl annexin V-FITC was added, and then 5 μl propidium iodide was added for incubation for 5-15min in the dark at room temperature. Cell apoptosis was analyzed by flow cytometry (Becton-Dickinson, FACS Calibur, USA).
Western blot analysis
The total protein of MC3T3-E1 cell was extracted using RIPA lysis buffer (Beyotime, China) and quantified by BCA assay (Beyotime, China). Equal amounts of proteins (100μg) were separated via BeyoGel™ Plus PAGE (Beyotime, China) and then transferred to a PVDF membrane (Millipore, USA). After transferring, the membranes were blocked with 5% fat-free milk for 1h. The membranes were incubated with primary antibodies (Bax, ab32503, Caspase-3, ab32351, cleaved- Caspase-3, ab32042), which were purchased from Abcam (USA) and GAPDH, 10494-I-AP which was obtained from Proteintech (China) at 4°C overnight. The membranes were incubated with the second antibodies (Goat anti rabbit IgG HRP SE134, Goat anti mouse IgG HRP SE131, Solarbio, China) at 37°C for 1h. The ECL system (CLINX, China) was used for exposing protein bands. The intensity of the bands was analyzed using Image lab (version 3.0, Bio-Rad, USA).
Gene expression analysis using quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was extracted from cells using TRIzol reagent (Invitrogen, USA) according to the manufacturer’s protocol. For detecting miR-141-5p expression, MicroRNA cDNA Synthesis Kit (Vazyme, China) was applied for reverse transcription of cDNA, followed by qRT-PCR analysis. U6 was employed as the loading control. The primers of miR-141-5p and U6 were purchased from GeneCopoeia (Guangzhou, China). Meanwhile, the expression of lncRNA-KCNQ1OT1 was evaluated by Prime Script TM Master Mix (Takara, Japan) and GAPDH was employed as the loading control. The data was calculated using the 2−DDCt method. Primers used in this study were shown below:
lncRNA-KCNQ1OT1, F: 5’-TTGGTAGGATTTTGTTGAGG-3’ and
R: 5’-CAACCTTCCCCTACTACC-3’;
GAPDH, F: 5-TGGATTTGGACGCATTGGTC-3 and
R: 5-TTTGCACTGGTACGTGTTGAT-3.
Dual-luciferase reporter assay
The wild-type (WT) sequence of lncRNA-KCNQ1OT1 containing the miR-141-5p binding sites (KCNQ1OT1-WT) and the mutant sequence (KCNQ1OT1-MUT) were cloned into pMIR vectors (Promega, USA) respectively. MC3T3-E1 and HEK293 cells were co-transfected with miR-141-5p or miR-NC and KCNQ1OT1-WT or KCNQ1OT1-MUT and kept for 24 h. The luciferase activity was detected using Dual-Luciferase Reporter Assay System (Promega, USA) in the dark.
Statistical Analysis
Statistical analysis was performed with SPSS 20.0 software (IBM, Armonk, NY, U.S.A.). All quantitative data were described as mean ± SD. One-way ANOVA was used to analyze the statistical differences among three or more groups while unpaired Student’s t test was applied to analyze the statistical differences between two groups. P< 0.05 was considered statistically significant.