All animal procedures were performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The Animal Care and Use Committee of Nankai Hospital approved this experimentation. We used forty 294 ± 11 g female 8-week-old Sprague Dawley (SD) rats. As previously reported, PMO model was induced by a rat model of osteoporosis using ovariectomy (OVX). Rats with OVX were randomly and equally divided into four groups: (1) OVX + PBS group; (2) OVX + exosomes group; (3) OVX + exosomes + miR-186 inhibitor; and (4) OVX + miR-186 mimics. 100 µL (about 1013/mL) exosomes were used for the treatment of OVX rats with tail vein injection once a week. And 50 µM miR-186 mimics or inhibitor were used in OVX rats with tail vein injection once a week. All rats were sacrificed at 28 days. Cervical dislocation was used for rat’s euthanasia. And 10% 350 mg/kg chloral hydrate (intraperitoneal injection) was used for anesthesia. Bones of the lower extremities were collected for subsequent experiments.
Cell culture and treatments
Human bone marrow mesenchymal stem cell (hBMSCs) were donated by Professor Xin-long Ma from Tianjin Hospital and cultured for exosome extraction. BMSCs from the bone marrows of OVX or normal rats were also extracted for cellular experiments. The BMSCs were incubated in α-MEM (α-MEM, Gibco, Paisley, UK) medium and supplemented with 10% fetal bovine serum (FBS, HyClone, Logan, UT) and 100 µg/ml penicillin/streptomycin at 37 °C.
The BMSCs from OVX rats were co-cultured with hBMSC exosomes. CCK-8, RT-PCR, Western blotting and RNA-seq assays were performed using the cultured cells.
Exosome isolation, purification, and identification
Exo-free FBS was prepared by ultracentrifugation at 130,000 ⋅ g for 18 h; FBS supernatant was collected. When hBMSC cultures reached 60%-70% confluence under normal culture conditions, exo-free FBS was changed for exosome collection. Next, hBMSC supernatant was collected for another 48 h. Then, exosomes derived from hBMSCs were extracted using exosome extraction kits. Exosome morphology derived from hBMSCs was detected by transmission electron microscopy (TEM). We used DLS to determine exosome size distribution. Specific exosome biomarkers, including Alixs, CD63, and CD81, were analyzed by Western blot.
Exosome labeling with PKH-26
We used PKH-26 (Sigma) to label extracted exosomes. Exosomes extracted from hBMSCs were co-cultured with PKH-26 mixture for 15 min under dark. Next, 10% BSA was used to stop the staining reaction for 1 min. Labeled exosomes were extracted by ultracentrifuge at 100,000 ⋅ g for 1 h at 4 °C.
Cell viability assay
Cell Counting Kit-8 (CCK-8) assays were used to calculate the proliferation rates of BMSCs from OVX or normal rats. Extracted BMSCs were seeded in 96-well plates at 2000 cells per well for three days. Then, 10 µL CCK-8 was added in each well and incubated for 4 hours under dark. An enzyme-linked immunosorbent assay reader was used to detect OD values at 450 nm.
RNA extraction, reverse transcription and q-PCR
Trizol reagent (Invitrogen, Carlsbad, CA, USA) was used for total RNA extraction. A miRNA purification kit was used for miRNA separation. Reverse transcription of cDNA was performed according to manufacturer instructions. Real-time PCR was performed using a PrimeScript RT reagent kit and detected by Applied Biosystems model 7900HT Fast Real-Time PCR System. 2−△△Ct method was used for gene expression calculation.
High-throughput sequencing was performed on a BGUSEQ-500 by BGI, and miRNA expression between the PMO and PMO + exosomes groups were detected. First, the hind limbs of rats in the PMO and PMO + exosomes groups were harvested and frozen using liquid nitrogen. Then, a BioAnalyzer 2100 system was used to quantify the quality of extracted miRNA. Clean-tags of miRNA were mapped to a reference genome library. Differentially expressed miRNA was analyzed and defined by the bioinformatics service of BGI with FDR < 0.05 and p < 0.05.
RNA pull-down assay
We performed RNA pull-down assay with biotinylated miR-186 to detect the relationship between the miR-186 and Mob. BMSCs were transfected with biotinylated miR-186 mimics or biotin-negative control (50 nM) and harvested at 48 h after transfection. The biotin-coupled RNA complex was pulled down by incubating the cell lysates with streptavidin-coated magnetic beads. The abundance of ceRNA in the bound fractions was evaluated by qRT-PCR analysis.
Osteogenic differentiation assay
BMSCs in different groups were cultured in six-well plates containing osteogenic differentiation medium (Cyagen, Suzhou, China). Media were changed every 3 days for 3 weeks. After rinsing, cells were fixed with 4% paraformaldehyde. Mineralized nodules were observed by staining with a 1% alizarin red solution (Cyagen, Suzhou, China) following manufacturer instructions. ALP staining was used to investigate the osteogenesis of BMSCs after induction. Staining images were recorded under an inverted microscope, and ImageJ was used to analyze the results.
Hematoxylin and eosin (HE) staining
Paraformaldehyde was used to fix tibiae from different rats for a week. The fixed samples were washed three times to remove excess paraformaldehyde; then, the tibiae were embedded in paraffin and cut into 5-µm sections. The sections were deparaffinized in xylene and rehydrated through a graded series of ethanol. Hematoxylin and eosin (HE) staining was performed according to manufacturer instructions. Microscopy was used to observe the morphology of cancellous bone in the tibia. We used the left hind leg of rats for HE staining, and all the group tested on the same side.
Transfection of miRNA mimics and inhibitor
According to manufacturer instructions, miR-186 mimics and inhibitors and negative control (50 nM) were purchase from Sangon Biotech and transfected into BMSCs using Lipofectamine™ 2000 Transfection Reagent. Target gene expressions were evaluated using RT-PCR; luciferase intensity was measured using a Dual Luciferase Reporter Assay System 48 hours’ post-transfection. qPCR was used to evaluate the transfection efficiency of miR-186 mimics and inhibitor. The negative control of miR-186 mimics was sense UUCUCCGAACGUGUCACGUTT, antisense ACGUGACACGUU CGG AGA ATT. The negative control of miR-186 inhibitor was CAGUACUUUUGUGUAGUACAA.
Western blotting assay
Protein from cells or bone tissues were extracted using RIPA buffer. Then, 10% SDS-PAGE was used to separate protein lysates. Proteins were electrophoretically transferred to 0.22-µm polyvinylidene difluoride membranes (PVDF, Millipore, USA). The membranes were blocked with 5% milk-TBST for 1 hour at room temperature and incubated overnight at 4 °C with primary antibodies against BMP2 (Abcam, ab214821), RANKL (Abcam, ab239607), YAP (CST, 14074) and β-Actin (CST, 3700) at a 1/1,000 dilution. The membranes were then incubated for 1 hour at room temperature with HRP-linked antibody at a 1/5,000 dilution, and TBST was used to wash the membrane. The band was detected by chemiluminescence and analyzed using ImageJ software.
Dual Luciferase reporter assay
PCR product containing the 5’-flanking sequence of the MOB1 promoter was first inserted into a pGL3-basic vector. Then, 200 ng pGL3-MOB1 Promoter, along with 40 ng pRL-TK Vector (E2241, Promega) were respectively transfected into BMSCs using Lipofectamine 2000. A Dual Luciferase Reporter Assay System was used to detect luciferase intensity. Firefly luciferase activity was normalized to renilla luciferase activity for each transfected well.
SPSS 19.0 statistical software package was used to perform statistical analyses. Analysis of variance was used to detect significant differences between groups. Post hoc multiple comparison test (Fisher’s least significant difference [LSD] test and Tukey's test) was performed for the statistical analysis between every two groups. We used the two independent samples t-test to evaluate results between two groups. Results are reported as means ± SD. Two-tailed P-values 0.05 or less were considered significant.