Thrombin-activated platelet rich plasma (PRP) enhanced osteogenic differentiation of bone marrow mesenchymal stem cells by activing autophagy through miR-140-3p/SPRED2 axis

Background Mesenchymal stem cells transplantation gradually become a potential treatment for bone defect in clinic practice. This study aimed to investigate the molecular mechanism of PRP and autophagy for osteogenic differentiation in bone marrow mesenchymal stem cells (BMSCs). Methods Thrombin activated PRP was prepared and the BMSCs were treated with activated PRP with 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. qRT-PCR and Western blotting were used to determine the mRNA expression and protein expression. A luciferase reporter assay was conducted to identied the targeting relationship between iR-140-3p and SPRED2 Subsequently, cell proliferation was detected by MTT and ALP activity was also determined. Alizarin red staining was used for the evaluating the formation of calcium nodules. Results MiR-140-3p expression was found to be inhibited by PRP in a dose-dependent manner, besides, cell proliferation, ALP activity, the expression of COL-I, OPN, Runx2 and OCN, and the formation of calcium nodules related to osteogenic differentiation were enhanced by PRP. Subsequently, we found that PRP activated autophagy and up-regulated SPRED2 expression in BMSCs through suppressing miR-140-3p expression. Moreover, we conrmed that miR-140-3p targeted SPRED2 and negatively regulation its expression. Finally, the ndings showed that inhibition of miR-140-3p enhanced cell proliferation, osteogenic differentiation and autophagy of BMSCs by negatively regulating SPRED2 expression. Conclusion Thrombin activated PRP accelerated osteogenic differentiation of BMSCs by activing autophagy through miR-140-3p/SPRED2 axis.


Introduction
In order to enhance bone regeneration, various approaches, including bone transport and distraction osteogenesis, have been conducted for the repair of bone defect in the last decades [1]. However, these therapies might lead to donor site injury, infection, deformity, surgical risks and even healthy bone loss [2]. Therefore, mesenchymal stem cells transplantation gradually become a potential treatment for bone defect in clinic practice.
Numerous studies revealed that bone marrow mesenchymal stem cells (BMSCs) have the potential to differentiate into osteoblasts, myoblasts, adipocytes and so on under different conditions, representing an alternative reparative cell type [3]. And the osteogenesis of BMSCs is always considered as a potential therapeutic strategy for bone regeneration. However, the molecular mechanisms for differential of BMSCs are still unclear.
In addition to stem cell therapy, platelet rich plasma (PRP) has also been widely applied in various elds, especially bone detects in maxillofacial region and oral cavity [4]. Moreover, PRP at different concentrations showed different potentials to induce chondrogenic differentiation, migration, and proliferation of mesenchymal progenitor cells [5]. Recently, more and more researches show that PRP can be used in the eld of bone regeneration, and it has been involved widely in orthopedic surgery [6]. It was found PRP could be used in treatment of chronic lateral elbow epicondylitis, mandibular degree II furcation defects and tendinopathy [7][8][9]. And the application of PRP is considered as a potential therapeutic strategy in bone repair in the future [10]. However, the underlying molecular mechanisms for PRP in osteogenic differentiation are still unknown. Interesting, PRP was reported to activate autophagy in osteoblast precursor 3T3-L1 [11]. Thus, we wondered the relationship between PRP and autophagy in osteogenic differentiation in BMSCs.
In the present study, we investigated the molecular mechanism of PRP and autophagy in BMSCs. We for the rst time demonstrated that thrombin-activated PRP enhanced osteogenic differentiation of BMSCs by activing autophagy through miR-140-3p/SPRED2 axis. This study might provide more research basis and might give novel study targets for PRP in clinical treatment of bone regeneration.

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 modi ed Eagle Medium (DMEM) including 10% fetal bovine serum (FBS),100 IU/ml penicillin and 100 µg/ml streptomycin. Cells were incubated in a humidi ed atmosphere at 37 °C with 5% CO 2 . 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 × 10 3 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 o C. 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 xed 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 × 10 4 for 7 days, washed with PBS and xed 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).

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 signi cant when P < 0.05.
Subsequently, cells were treated with thrombin-activated PRP at a concentration of 5%, 10% and 20%, respectively. MiR-140-3p was down-regulated in cells treated with PRP in a dose-dependent manner (Fig. 1B). Cell proliferation detected by MTT assay showed that thrombin-activated PRP notably enhanced cell viability in a dose-dependent manner but up-regulated miR-140-3p or inhibited autophagy reversed the effect of activated PRP on cell proliferation (Fig. 1C). ALP activity was also found to be increased by activated PRP in a dose-dependent manner that was reversed by miR-140-3p mimics or 3-MA (Fig. 1D). Besides, osteogenic differentiation related mRNA expression of COL-I, OPN, Runx2 and OCN was signi cantly elevated by activated PRP in a dose-dependent manner but down-regulated by overexpressed miR-140-3p or inhibited autophagy (Fig. 1E). Alizarin red staining illustrated that thrombinactivated PRP obviously accelerated formation of calcium nodules in a dose-dependent manner, which could be reversed by overexpressed miR-140-3p or inhibited autophagy (Fig. 1F). All the results indicated that PRP promotes cell proliferation and osteogenic differentiation of human BMSCs by miR-140-3p inhibition or autophagy activation.
PRP activates autophagy and up-regulated SPRED2 expression of human BMSCs through suppressing miR-140-3p expression We further investigated the regulation of miR-140-3p on autophagy and SPRED2 expression in BMSCs treated with thrombin-activated PRP. As shown in Fig. 2A, the expression of SPRED2 was up-regulated by the treatment of PRP, however, it was reversed by overexpressed miR-140-3p or autophagy inhibition.
Western blotting result also showed that the protein level of Beclin1 and SPRED2, and the conversion rate of LC3 I to LC3 II was signi cantly elevated by PRP in a dose-manner but reversed by miR-140-3p mimics or autophagy inhibitor (Fig. 2B). The above ndings suggested that PRP enhanced autophagy and upregulated SPRED2 expression of human BMSCs through suppressing miR-140-3p expression.

Inhibition of miR-140-3p enhances cell proliferation and osteogenic differentiation of BMSCs by negatively regulating SPRED2 expression
We further studied the molecular mechanism of miR-140-3p for cell proliferation and osteogenic differentiation of BMSCs. Firstly, cells were transfected with si-SPRED2 or miR-140-3p inhibitor, respectively. Both qRT-PCR and western blotting result showed that SPRED2 and miR-140-3p was knockdown successful (Fig. 4A). As shown in Fig. 4B, cell proliferation was suppressed by SPRED2 knockdown. And miR-140-3p inhibitor obviously increased cell proliferation, which was reversed by the transfection of si-SPRED2. ALP activity (Fig. 4C) was also reduced by down-regulated SPRED2. It was also found that miR-140-3p knockdown attenuated ALP activity which could be reversed by si-SPRED2.
Moreover, protein level of COL-I, OPN, Runx2, OCN was decreased by si-SPRED2 compared to NC control in cells treated with 20% PRP (Fig. 4D). Down-regulated miR-140-3p was also proven to enhance the expression of above proteins related to osteogenic differentiation that could be reversed by si-SPRED2. In addition, knockdown of si-SPRED2 suppressed calcium deposition, and miR-140-3p inhibitor accelerated the formation of calcium nodules but it was reversed by si-SPRED2 (Fig. 4E). these nding illustrated that inhibition of miR-140-3p enhanced cell proliferation and osteogenic differentiation of BMSCs by negatively regulating SPRED2 expression.
Knockdown of miR-140-3p enhances autophagy in human BMSCs through negatively regulating SPRED2 expression Finally, the molecular mechanism of miR-140-3p on autophagy in BMSCs was studied. As shown in Fig. 5A, the expression of SPRED2 mRNA was decreased in cells treated with 20% PRP but up-regulated in cells transfected with miR-140-3p inhibitor, which was reversed by the co-transfection of si-SPRED2. Furthermore, western blotting revealed that the expression of SPRED2 and beclin1 as well as the conversion rate of LC3 I to LC3 II was notably decreased in cells treated with PRP, however, the levels of them were elevated in cells transfected with miR-140-3p inhibitor but was decreased by the cotransfection of si-SPRED2. These ndings indicated that miR-140-3p suppression enhanced autophagy in BMSCs through negatively regulating SPRED2 expression.

Discussion
BMSCs are a type of primary stem cells with good differentiation potential, widely using in tissue engineering for translational research and clinical applications [12]. In recent years, several studies reported the roles of various proteins and miRNAs in the treatment of bone defect [13,14], however, the mechanism of miRNA-140-3p, SPRED2 and thrombin in BMSCs as well the target relationship still remains unclear. In the present study, we demonstrated that thrombin-activated platelet rich plasma (PRP) enhanced osteogenic differentiation of bone marrow mesenchymal stem cells through miR-140-3p/SPRED2-mediated autophagy.
MiRNAs are reported to be involved in in a variety of biological processes, including cell viability, apoptosis and differentiation [15]. MiR-140-3p was reported to regulated osteogenic differentiation of different cells in vivo and in vitro studie, besides, overexpressed miR-140-3p suppressed the cell viability and differentiation, and enhanced the apoptosis of preosteoblasts cell MC3T3-E1 [16]. It was also found that miR-140-5p restrained osteogenesis mediated by BMP2 in human mesenchymal stem cells [17]. Yin et al found that miR-140-3p was up-regulated in peripheral blood mononuclear of patients with osteoporosis patients, besides, the in vitro study showed that miR-140-3p regulated the cell proliferation and differentiation of osteoblasts and osteoclasts by targeting PTEN [18]. Another study found that miR-140-3p was up-regulated in patients with osteoporosis in postmenopausal women and it was considered as a potential biomarker [19]. Though PRP was reported to accelerate osteogenic differentiation in mesenchymal stem cells [20], dental stem cells [21], adipose-derived stem cells [22] and so on, the effect of PRP on miR-140-3p was not investigated and no research illustrating the regulation of miR-140-3p for autophagy in BMSCs. In the present study, we found that activated PRP accelerated osteogenic differentiation, cell proliferation and autophagy by inhibiting miR-140-3p expression. However, there is no research illustrating the regulation of miR-140-3p for autophagy in BMSCs.
Circulating evidences showed that Sprouty-related EVH1 domain-2(SPRED2) was potent inhibitors for cytokines and growth factors [23]. SPRED2 was reported to be involved in various diseases [24]. For example, down-regulated SPRED2 induced cardiac arrhythmias and premature death by suppressing autophagy [25]. SPRED2 interacted with LC3 and enhanced autophagy in tumor cells [26]. In addition, SPRED2 was found to induce erythroid differentiation partly through ERK signaling in chronic myeloid leukemia cells [27]. A previous research showed that miR-221-3p could target SPRED2 and SPRED2 regulated LPS-induced lung in ammation via ERK1/2 pathway [28]. However, no studied reported the regulation relationship between SPRED2 and miR-140-3p in BMSCs. Our study for the rst time founder that miR-140-3p negatively regulated SPRED2 expression. Besides, down-regulated miR-140-3p enhanced cell proliferation, osteogenic differentiation and autophagy of BMSCs by negatively regulating SPRED2.

Conclusion
In summary, we for the rst time demonstrated that active PRP accelerate osteogenic differentiation of BMSCs by activing autophagy through miR-140-3p/SPRED2 axis. Our ndings might bring a new novel for the target therapy for PRP in bone defect repair in future clinic practice. The cells were commercially sourced, and the carried work was approved by the Ethics Committee on Henan Provincial People's Hospital.

Consent for publication
Not applicable

Competing interests
The authors declare no con ict of interest.

Data Availability
All data can be obtained from the manuscript or from request to the author.