Patient serum samples
Postmenopausal women aged 65–75 years from two communities in Shanghai, China were enrolled and BMD of the lumbar, vertebra and hip were detected by DXA. This study was approved by the Medical Ethics Committee of Huadong Hospital (2019K055) and informed consent was obtained from all participants. Participants were divided into control group and severe osteoporosis (SOP) group according to BMD results and the history of fragility fractures. In control group, subjects had no fracture history and the T-score of BMD>-1.0, while in SOP group, subjects suffered from fragility fractures in the vertebral spine and/or hip and the T-score of BMD ≤-2.5. Serum levels of calcium, phosphorus, 25-hydroxyvitamin D (25(OH)D), parathyroid hormone (PTH), P1NP and β-CTX were obtained to rule out secondary OP. Participants did not take insulin, sex hormones, glucocorticoids,anti-osteoporosis drugs, such as bisphosphonates, estrogen and progesterone replacement, selective estrogen receptor modulators (SERMs), parathyroid gland hormones or other drugs that will affect bone metabolism. Those who suffered from diabetes, severe cardiopulmonary disease, liver and kidney disease, endocrine and metabolic diseases, autoimmune diseases, malignant tumors and hyperlipemia were excluded. The participants information was listed in Table 1.
Table 1
Characteristics of the participants in this study.
Characteristics | Control, n = 18 | SOP, n = 16 | p-value |
Age (year) | 67.28 ± 5.2 | 67.0 ± 3.2 | 0.86 |
Height (cm) | 157.53 ± 5.8 | 153.0 ± 6.8 | 0.04 |
Weight (kg) | 59.62 ± 6.7 | 53.51 ± 7.4 | 0.02 |
BMI (kg/m2) | 24.02 ± 2.4 | 22.92 ± 3.6 | 0.31 |
Fracture of vertebra (%) | 0 | 68.75 | 0.000 |
Fracture of hip (%) | 0 | 43.75 | 0.000 |
Serum creatinine (umol/L) | 61.8 ± 12.9 | 56.93 ± 8.9 | 0.27 |
AKP (U/L) | 74.6 ± 17.8 | 80.23 ± 25.1 | 0.52 |
Serum calcium (umol/L) | 2.41 ± 0.06 | 2.38 ± 0.11 | 0.53 |
Serum phosphorus (umol/L) | 1.28 ± 0.23 | 1.19 ± 0.13 | 0.25 |
25(OH)D3 (ng/ml) | 30.33 ± 15.11 | 28.79 ± 14.59 | 0.82 |
PTH (pg/ml) | 31.8 ± 9.0 | 45.3 ± 20.2 | 0.14 |
β-CTX (pg/ml) | 482.48 ± 209.58 | 476.79 ± 286.96 | 0.97 |
P1NP (ng/ml) | 61.2 ± 12.4 | 61.1 ± 13.3 | 0.96 |
BMD of LS (g/cm2) | 0.884 ± 0.13 | 0.603 ± 0.06 | 0.000 |
BMD of FN (g/cm2) | 0.705 ± 0.11 | 0.511 ± 0.10 | 0.000 |
BMD of TH (g/cm2) | 0.752 ± 0.21 | 0.612 ± 0.16 | 0.04 |
Serum exosomes isolation
A 3 ml peripheral serum from each participants was collected and exosomes were isolated by traditional differential ultracentrifugation with four steps. At first, serum was diluted with sterile phosphate-buffered saline to 50ml, centrifugation at 3000×g for 30 min was performed, then supernatant was centrifuged at 12,000×g for 45min followed by ultracentrifugation for 2h at 120,000 ×g in 4 ℃. The exosome pellet was re-suspended in lysis buffer or sterile PBS, depending on subsequent experiments.
Transmission electron microscopy (TEM)
The suspension was mixed with an equal volume of 4% paraformaldehyde, and 25ul of the solution was taken up to the loaded copper mesh, dried at room temperature for 20 minutes, and the liquid on the filter screen was blotted from one side with a filter paper, and 30ul of phosphotungstic acid solution was added, stained for 5 min at room temperature, and then was blotted with a filter paper and dried at room temperature. The exosomes were photographed under a transmission electron microscope.
Western blot analysis
Exosomes were lysed in RIPA buffer with 1% phenylmethylsulfonyl fluoride (PMSF) and placed on ice for 10 minutes. Protein was quantified by using BCA protein quantitative kit (Sangon Biotech,Shanghai) according to the instruction. The concentration was adjusted by appropriate amount of radioimmunoprecipitation assay (RIPA) buffer, and sodium dodecyl sulfate (SDS) loading buffer of 1/4 volume was added. Protein samples were loaded, separated on 10% SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE), and transferred to a polyvinylidene difluoride (PVDF) membranes, followed by blocking for 1 hour in 5% non-fat skimmed milk in tris buffered saline with tween (TBST) solution. After blocking, membranes were incubated with primary antibodies against TSG101 (1:1000 dilution, ab125011, Abcam) and CD63 (1:1000 dilution, ab216130, Abcam) respectively overnight at 4℃. Membranes were then washed using TBST for three times and incubated in secondary antibody for 1hour in room temperature. At last, membranes were washed and developed by Tanon3500 gel imaging and photographing system (Tanon Science & Technology Co, Ltd.)
Nanoparticle tracking analysis (NTA)
Isolated pallets were analyzed by the Nanosight NS300 System (Malvern Instruments, UK) to determine the size and quantity of particles. Laser-irradiated nanoparticles are captured for 60 seconds and particle were analyzed by NTA software.
miRNA library construction and sequencing
Serum exosomes were isolated, prepared and sent to BGI-Wuhan (Wuhan, China) for miRNA library construction and next-generation sequencing. For each sample, Clean reads were obtained via removing the low quality reads and aligned with the human genome. Clean reads were further mapped to sRNA in the GenBank and Rfam to analyze their distribution and annotate small RNA sequences. After sequencing by an Illumina sequencer, image analysis, and base identification, the raw reads after quality control were harvested. Clean reads were aligned against known miRNA precursors and mature miRNAs in the miRBase to identify conserved miRNAs.
MiRNA Target Prediction and Relevant Signaling Pathway
Targets of miRNA were predicted by using Targetscan (http://www.targetscan.org) and Gene Ontology (GO) enrichment analysis of target genes was performed using DAVID online tool(https://david.ncifcrf.gov/summary.jsp). The relevant signaling pathways were analyzed using the KEGG database in DAVID.
Cell cultures, transfection and osteogenic differentation
Bone Mesenchymal Stem Cells (BMSCs) were purchased from Cyagen Bioscience Inc and cultured in human bone marrow mesenchymal stem cell basal medium with 10% fetal bovine serum,penicillin-streptomycin and glutamine at 37℃ with 5% CO2. Cells were transfected with 20nM microRNA mimics on day 0 and cultured in human mesenchymal stem cell osteogenic differentiation basal medium with 10% fetal bovine serum, 1%glutamine, 1%ascorbate, 0.2%β-Glycerophosphate and 0.01% dexamethasone from day1 to day7 to induce osteogenic differentiation. Mediums were changed every 2 days.
Alkaline phosphatase (ALP) activity assay
ALP activity was examined by using Alkaline Phosphatase Assay Kit (ab83369, Abcam) in bone mesenchymal stem cells on day 7 after transfecting with related miRNA mimics or vehicles. 5mg pNPP was dissolved in solution with 0.1 M glycine, pH 10.4, 1 mM MgCl2 and 1 mM ZnCl2. Cell culture medium was discarded and 100ul pNPP solution was added 15 minutes. The absorbance was examined at 405 nm.
Statistical analysis
Numerical data was presented as the mean ± standard deviation. Statistical analysis was performed using SPSS and correlations were analyzed using Spearman data. P<0.05 was considered statistically significant.