Preparation of Thrombin Activated Platelet-Rich Plasma(PRP)
For the preparation of PRP, human blood was collected from 8 donors (150 mL of venous blood each) and mixed with 10% acid-citrate-dextrose as an anticoagulant. The whole blood was centrifuged for 25 min and PRP was extracted from the buffycoat into an empty sterile syringe. For the activation, bovine thrombin was added to PRP at 1:5(vol/vol) and incubated for 30 minutes at 37 ˚C. Then the sample was centrifuged at 4000 rpm for 5 min, and the supernatant was collected and mixed with serum-free DMEM solution to prepare PRP at a concentration of 5%, 10% and 20%.
Cell Culture and Transfection
The human bone marrow stem cells (BMSCs) were obtained from ATCC (Rockville, MD, USA) and cultured in Dulbecco's modified Eagle Medium (DMEM) including 10% fetal bovine serum (FBS),100 IU/ml penicillin and 100 µg/ml streptomycin. Cells were incubated in a humidified atmosphere at 37 °C with 5% CO2. BMSCs were treated with activated PRP at different concentration and transfected with miR-140-3p vector (mimics or inhibitor), si-SPRED2 or co-transfected with miR-140-3p inhibitor and si-SPRED2, respectively.
MTT Assay
The cell viability was detected by MTT assay at 0 h, 24 h, 48 h, 72 h and 96 h after the treatment. BMSCs were planted into 96-well microplates with the density of 5 × 103 and incubated for 48 h. subsequently, 10 µL of 5 mg/mL MTT solution was added to each well and incubated for another 4 h at 37 oC. The supernatant was discarded and replaced with 150 µl DMSO. Finally, the absorbance was measured was measured at 490 nm by using a microplate reader (DNM-9602; Pe rlong, Beijing, China). Each experiment was performed in triplicate and repeated at least three times.
Alizarin Red Staining and Alkaline Phosphatase (ALP) Activity
BMSCs were fixed with 4% paraformaldehyde and incubated with 1% alizarin red S solution (Beyotime, Shanghai, China) for 10 min at room temperature. Excess stain was eliminated using PBS washing. Calcium-bound stain was collected with 100 mM CPC and absorbance value was measured at 405 nm by a microplate reader.
For the detection of ALP activity, BMSCs were cultured in 24-well plates at a cell density of 1 × 104 for 7 days, washed with PBS and fixed with 4% paraformaldehyde. Protein levels were determined by a BCA protein assay kit and the ALP activity was measured by using a ALP detection kit (Wako Chemical, Richmond, VA).
Reverse Transcription-Quantitative Polymerase Chain Reaction (qRT-PCR)
In brief, Total RNA was extracted from BMSCs using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). For the detection of miRNA expression, RNA was firstly converted into cDNA by using a iScriptTM cDNA Synthesis Kit (Bio-Rad, Reinach, Switzerland). A SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) on a ABI Prism 7300 (Applied Biosystems) was used for quantification. For the detection of mRNA expression, DyNamoTM cDNA synthesis kit (Fermentas) was used for the reverse transcription. PCR reaction and analysis was performed on an ABI Prism 7500 sequence detection system (PE Applied Biosystems) with a DyNamo SYBR1 Green qPCR kit (Finnzymes, Espoo, Finland). U6 and GAPDH were used as internal controls for miRNA and mRNA, respectively. The following primers were used in PCRs: F 5'-GCTTGGTGGGCTTCTGGT-3' and R 5'-CCCGGTATCCTGTCCTGTCCGTGGT-3' for miR-140-3p; F 5'-CTCATCCATGGTGAACGACAGAA-3’ and R 5’-TGTCAAAGGCTCGGGCATC-3’ for SPRED2; F 5'-GAGAGCATGACCGATGGAT-3' and R 5'-ATGTTTTGGTGGTTCAGGAGG-3' for COL-1; F 5'-GTGCAGCCTHGTGTCCAAG-3' and R 5'-GTCAGCCAACTCGTCACAGT-3' for OCN; F 5′-CTGAACGCGCCTTCTGATTG-3′ and R 5′-ACATCGGAATGCTCATTGCTCT-3′ for OPN; F 5′-GGAGTGGACGAGGCAAGAGTTT-3′ and 5′-AGCTTCTGTCTGTGCCTTCTGG-3′ for Runx2; F 5′-TGCGGGTGCTCGCTTCGGCAGC-3′ and R 5′-CCAGTGCAGGGTCCGAGGT-3′ for U6; F 5'-ACAGAGCCTCGCCTTTGC-3' and R 5'-GAGGCGTACAGGGATAGCAC-3' for β-actin. The relative expression levels of miRNA or mRNA was calculated by the 2−ΔΔCq method.
Western Blotting
Protein was extracted by RIPA lysis buffer (Thermo Scientific, U.S.A.) from BMSCs and determined using a BCA Protein Assay Kit (ComWin Biotech, Beijing, China). Equal amount of proteins was loaded on 10% SDS-PAGE gels, transferred to PVDF membranes and blocked with 5% non-fat milk at room temperature for 1 h. Afterwards, membranes were subjected to immunoblotting with primary antibodies at 4˚C overnight, followed by horseradish peroxidase-conjugated secondary antibodies at 37˚C for 1 h. The primary antibodies were listed as follows: SPRED2(ab153700, 1/500)), Beclin1 (ab207612, 1/2000) and LC3 I/II (ab62721, 1/1000) (all from Abcam, Cambridge, MA, U.S.A). β-actin was used as a control. Immunoreactive bands were visualized using an enhanced chemiluminescence detection system (Amersham Biosciences, Piscataway, NJ, USA) and quantified using AlphaEase FC software.
Luciferase Reporter Assay
Two types of 3′-UTRs (WT and MUT) of SPRED2 sequences containing the binding sequence of miR-140-3p were amplified and inserted into the p-MIR-report plasmid (Ambion, Austin, TX, USA). Subsequently, BMSCs were transfected with miR-140-3p vector (mimics or inhibitor) and SPRED2 luciferase reporter plasmid (WT- SPRED2 or MUT- SPRED2) or NC (mimics or inhibitor) using Lipofectamine 2000 (GeneChem, Shanghai, China). The luciferase activity was detected using luciferase assay kits (Promega, Madison, Wisconsin, U.S.A.) after the transfection of 48 h. Luciferase activity of cells was normalized to Renilla luciferase activity.
Statistical Analysis
All calculations were performed using SPSS 18.0. The continuous data was expressed as mean ± SD. Comparison between two groups was made using the Student t-test, while, comparison among three or more groups was perfomed using one-way analysis of variance (ANOVA) following by Turkey analysis. The comparison for rates was made by Chi square test. The differences were considered statistically significant when P < 0.05.