Mechanical stimulation regulates differentiation of bone marrow stem cells by modulating miR-140-5p via TGFβ1/Smad2 signaling pathway CURRENT STATUS:

Background: Osteoporosis is a common progressive bone disease that drastically impairs patient health, independent mobility, and quality of life, and there is an urgent need for improved preventive and therapeutic strategies. A shift in bone marrow mesenchymal stem cell (BMSC) differentiation from osteogenic to adipogenic may contribute to disease pathogenesis. Mechanical stress on BMSCs is reported to promote osteogenesis, so we examined the effects of mechanical stimulation on BMSC differentiation and associated signaling pathways. Methods: A sinusoidal tensile stress loading device was developed and examined effects of mechanical (stretch) stimulation on cultured BMSC (isolated from Sprague-Dawley rats) phenotype under osteogenic and adipogenic culture conditions. Osteogenic differentiation of BMSCs was assessed by alkaline phosphatase (ALP) staining and expression of Runx2 and BMP2, while adipogenic differentiation was evaluated by oil red O staining and expression of PPARγ and C/EBPα. Results: It demonstrated that appropriate mechanical stimulation could promote osteogenic differentiation of BMSCs and inhibit differentiation into adipocytes. The mechanic stimuli could inhibit the expression of miR-140-5P in BMSCs, and the overexpression of miR-140-5P inhibited the osteogenic differentiation, whereas the inhibition of miR-140-5P promoted the osteogenic differentiation. Further, both mechanical stimulation and miR-140-5p knockdown promoted TGFβ1/Smad2 signaling, while miR-140-5p overexpression downregulated TGFβ1/ Smad2 signaling. Conclusions: Appropriate mechanical stimulation promoted osteogenic differentiation and inhibited the adipogenic differentiation of BMSCs by lowering miR-140-5p expression, which in turn upregulates the TGFβ1/Smad2 signaling pathway. Our results provide a foundation for the development of effective strategies to promote bone remodeling, thereby lowering the burden of osteoporosis. protein These results suggest osteogenic differentiation inhibit adipogenic differentiation of BMSCs.


Background
Osteoporosis is a common progressive disease characterized by bone fragility due to the degradation of bone microstructure and decreased bone mass [1], which severely impacts health, mobility, and general quality of life [2]. Although the precise pathogenesis remains unclear, the fundamental mechanism underlying osteoporosis is an imbalance between bone formation and resorption [3]. 3 Osteoblasts derived from bone marrow mesenchymal stem cells (BMSCs) serve to replenish bone tissue. However, BMSCs can differentiate into a variety of cell types aside from osteoblasts, including chondrocytes, myocytes, and adipocytes [4,5]. In general, the shift in the differentiation from the osteogenic to adipogenic lineage increases with age, which may impair bone remodeling and reduce osteoblast formation, leading to the development of osteoporosis [6,7].
Accordingly, most current drug treatments for osteoporosis inhibit bone resorption, accelerate bone formation, or promote bone mineralization. However, long-term use of these drugs has certain limitations. For instance, hormone replacement therapy may increase the risk of cardiovascular diseases [8], while bisphosphonate therapy may cause severe bone turnover suppression and mandible osteonecrosis [9]. Thus, there is a critical need for improved preventive and therapeutic strategies for osteoporosis.
Most tissues are subjected to mechanical stimuli that regulate biological functions such as proliferation and differentiation [10]. Previous studies have shown that lack of exercise and the associated reduction in stem cell mechanical stimulation decreases osteogenic differentiation and increases the risk of osteoporosis [11,12]. Indeed, our previous studies have demonstrated that mechanical stress can promote osteogenic differentiation and inhibit adipogenic differentiation of BMSCs [13,14]. Further understanding of how mechanical stimuli, especially mechanical stress, affect skeletal tissue differentiation will provide insight into bone repair processes that may be exploited for novel therapeutic strategies. Several studies examining the relationships between mechanical stimulation and molecular expression profiles during bone healing have reported that non-coding microRNAs (miRNAs) participate in the regulation of skeletal growth and development. For instance, miR-140-5p has been reported to regulate cartilage development and bone homeostasis as well as contribute to age-related joint disease [15]. MicroRNA-140-5p was also found to be upregulated in murine primary osteoblasts and a vitamin D-treated osteoblast cell line [16]. A very recent study provided evidence that miR-140-5p regulates temporomandibular joint osteoarthritis (TMJ-OA) pathogenesis through the TGF-β/Smad2 signaling pathway [17]. Furthermore, miR-140-5p was reported to promote osteogenesis of ACSs by directly regulating toll-like receptor 4 (TLR4) and bone 4 morphogenic protein 2 (BMP2), resulting in enhanced fracture healing and bone formation in the atrophic nonunion rat model [18]. However, is still unknown whether miR-140-5p influences osteogenic or adipogenic differentiation of BMSCs in response to mechanical stimulation. The purpose of this study is to investigate the effects of mechanical stimulation on osteogenic and adipogenic differentiation of BMSCs and to explore the underlying molecular mechanisms, including the contributions of miR-140-5p and TGF-β/Smad2 signaling.

Isolation and culture of rat BMSCs
Bone marrow stem cells were isolated from Sprague-Dawley rats (Experimental animal center of Nanfang Hospital male or female, 80-100g. All rats were killed by cervical dissection, and the bodies were collected and burned by environmental health management department of Guangdong province.) by flushing the femurs and tibias with DMEM-LG medium (Gibco, Langley, OK, USA) supplemented with 10% defined fetal calf serum (Gibco), 100 U/ml penicillin, and 100 μg/ml streptomycin (North China Pharmaceutical Factory, China) (termed general medium, GM). Isolated cells were plated in the some medium on 25 cm 2 flasks and incubated at 37 °C under a humidified atmosphere containing 5% CO 2 . After 24 h, non-adherent cells were removed by washing with PBS, and fresh GM was added to allow for further growth. The culture medium was changed every 2-3 days thereafter. When the cells reached 80%-90% confluence, they were washed with PBS, detached by 0.25% trypsin, and subcultured in new 25 cm 2 flasks at 1 × 10 4 cells/cm 2 . Cells were collected at the second or third generation and sent for flow cytometry analysis (BD, Franklin Lakes, NJ, USA) of CD29, CD34, CD44, and CD45 expression for confirmation of BMSC phenotype [13,14].

Induction of osteogenesis or adipogenic differentiation and delivery of mechanical stimuli
Bone marrow stem cells at the logarithmic growth phase were collected, washed, and resuspended in DMEM at 1×10 5 cells/ml. Cells were seeded onto Bioflex 6-well plates at 1.5-2 ml per well and incubated under 5% CO 2 at 37 °C. Control cultures were refreshed with basal medium every 48 h.

Alkaline phosphatase and oil red O staining
An ALP staining kit (Tiangen Biotech Co. Ltd., Beijing, China) was used for determination of osteogenic differentiation according to the manufacturer´s instructions. Briefly, cells were fixed with 4% paraformaldehyde in PBS for 12 min at room temperature then stained with 1-2 ml/well ALP solution for 30 min at room temperature. The solution was aspirated and the cells were washed with distilled water and observed under light microscopy for ALP-positive cells. For determination of adipogenic differentiation, cells were fixed with 10% neutral buffered formalin for 1 h at room temperature, incubated with 60% isopropanol for 1 min, and then stained with oil red O for 15 min. Positively stained lipid droplets (red) were visualized under light microscopy.

RNA Extraction and RT-PCR Analysis
The expression levels of osteoblastic and adipocyte genes were analyzed by quantitative real-time PCR. Briefly, BMSCs were harvested and RNA was extracted using Trizol Reagent. Total RNA was reverse transcribed to cDNA using kit K1622 (Thermo Scientific). Real-time PCR assays were performed using All-in-One™ qPCR reagents (Genecopoeia, Guangzhou, China) with specific primers.
Total RNA was then extracted for RT-PCR analysis and proteins were extracted for western blot analysis as described.

Luciferase assays of miR-140-5p expression
The putative target sites for miR-140-5p on the 3'-UTR of TGFβR1were predicted using bioinformatics tools. HEK293 cells were seeded on 24-well plates and cultured until 60% confluent. The cells were then transfected with a vector encoding wild type or mutated TGFβR1 3'-UTR using the X-treme GENE™ HP DNA Transfection Reagent. After incubation for 48 h, the cells were lysed in 1× Passive Lysis Buffer and luciferase activities measured using the Dual-Luciferase® Reporter Assay System (Promega, Madison, WI, USA) according to the manufacturer´s instructions.

Statistical analysis
All data shown are expressed as mean ± standard deviation (SD) of at least three independent experiments. Group means were compared by independent sample t-tests using Prism version 6 (GraphPad software). A p ≤ 0.05 (two-tailed) was considered statistically significant for all tests.

Effects of mechanical stimulation on BMSC differentiation
Bone marrow stem cells were induced towards osteogenic or adipogenic differentiation using specific culture conditions with or without additional mechanical stimulation (stretch stress). After 5 days in culture, RT-qPCR and western blotting (WB) were performed to measure expression levels of the BMSC osteogenic markers Runx2 and BMP2 and the adipogenic markers PPARγ and CEBPα. As demonstrated in Figure 1A

Expression of miR-140-5p during osteogenic and adipogenic differentiation of BMSCs
To explore the possible molecular mechanisms underlying the effects of mechanical stimulation on BMSC differentiation, we first assessed the expression levels of miR-140-5p. As shown in Figure 2a and b, miR-140-5p expression gradually decreased and reached a nadir on the third day of osteogenic differentiation. Alternatively, miR-140-5p expression increased significantly during adipogenic differentiation, reaching a peak on the third day, followed by a slight decrease. On day 3, the expression level of miR-140-5p was 20% of control in the osteogenic condition and 5.6-fold higher than control in the adipogenic condition. Thus, miR-140-5p expression app-ears to suppress osteogenic and (or) promote adipogenic differentiation.
Collectively, these results indicate that the expression level of miR-140-5P is downregulated during osteogenic differentiation of BMSCs and further inhibited by appropriate stretch stress.

BMSCs
To provide further evidence for miR-140-5p function in BMSC osteogenic and adipogenic differentiation, we examined the effects of culture condition and stretch stress in BMSCs transfected with a miR-140-5p mimic. RT-PCR on day 3 of induction confirmed miR-140-5p overexpression ( Figure   3Aa). Moreover, expression levels of the osteogenic marker genes Runx2 and BMP2 were significantly lower in BMSCs overexpressing miR-140-5p than osteogenic BMSCs transfected with control vector (Figure 3Ac-d), suggesting that miR-140-5p negatively regulates osteogenic differentiation of BMSCs.
Further, under adipogenic differentiation conditions, expression levels of the adipogenic marker genes PPAR and C/EBPα were significantly upregulated in BMSCs overexpressing miR-140-5p (Figure 3Bab). After 7 days of differentiation under osteogenic conditions, BMSCs overexpressing miR-140-5p (A1 group) also exhibited lower ALP staining intensity than osteogenic BMSCs expressing control vector (A2 group) (Fig. 3C). Conversely, under adipogenic culture conditions, BMSCs overexpressing miR-140-5p (C1 group) demonstrated more intense oil red O staining than adipogenic BMSCs expressing control vector (C2 group). These results suggest that miR-140-5p promotes adipogenic differentiation of BMSCs rather than osteogenic differentiation. The participation of TGFβ1/Smad2 signaling pathway Furthermore, we try to find out the pathway signal targeted to the mechanism that miR-140-5p regulate to BMSCs differentiation. With the help of biomedical date source website, we decided to choose TGF-1/Smad2 as our target signal pathway to perform further research. Firstly, luciferase reporter assay was performed to detect the direct binding between miR-140-5P and TGFβr1. We found that ectopic expression of miR-140-5P significantly decreased the luciferase signal of 3' UTR of WT TGFβr1 compared to the miR-NC. This suppressive effect was abolished by mutated miR-194 binding site of 3' UTR TGFβr1. Secondly, The WB expression of TGF-1/Smad2 signal transduction pathway markers significantly downregulate in the BMSCs with adipogenic as significantly upregulated with osteogenic ( Figure 5). These results indicated the participating role of TGFβ1/Smad2 signaling pathway in the regulatory role of miR-140-5P on the BMSCs differentiation.

The expression level of TGF-1/Smad2 signal transduction pathway markers in BMSCs change as miR-140-5p overexpression and inhibition under mechanical stimulation
To explore the connection between miR-140-5P and TGF-1/Smad2 signal pathway, we transfected the miR-140-5P mimic and inhibitor into BMSCs separately and found TGF-1/Smad2 pathway were influenced by the change activity of miR-140-5P. As mentioned above, during the osteogenic differentiation of BMSCs, the activity of TGFβ1/Smad2 marks was significantly promoted under stress.
As shown in Figure 6A, this effect was further promoted when agonizing miR-140-5p, which was antagonized in inhibiting miR-140-5p. Conversely, it was observed in adipogenic differentiation that stress inhibited the activity of TGFβ1/Smad2 channels, while the effect is antagonized by a 140-5p inhibitor ( Figure 6B). This suggests that the regulation of the TGF-1/Smad2 pathway by miR-140-5p is involved in the regulation of stress on the differentiation of BMSCs.

Discussion
Osteoporosis is a systemic bone disease characterized by reduced bone mineral density and bone mass [21]. During the development of osteoporosis, the proportion of BMSCs differentiated into solitary cells is reduced and bone marrow is gradually replaced by adipose tissue [22]. Previous studies have shown that adipocytes and osteoblasts share a common progenitor cell [23]. Under the appropriate mechanical (stretch) stimulation, a variety of extracellular matrix components are secreted in the stretch zone and BMSCs differentiate into osteoblasts [24]. Consistent with previous research results, we demonstrate that appropriate mechanical stimulation can promote osteogenic differentiation of BMSCs and inhibit the adipogenic differentiation of BMSCs [13,14]. Adipogenic differentiation was associated with enhanced expression of miR-140-5p and concomitant downregulation of TGFβ1/Smad2 signaling components (TGFβ1, TGFβR1, and Smad2), while both mechanical stimulation and osteogenic culture suppressed miR-140-5p expression and upregulated expression of TGFβ1/Smad2 signaling components.
Stress exists in all intracellular environments, regulating cell biological functions, such as cell proliferation and differentiation, and appropriate stress stimulation is particularly important in bone regeneration after fracture [25]. To further study the effects of mechanical stimulation on the differentiation of bone marrow stem cells in vitro, we developed a sinusoidal tensile stress loading machine suitable for stretch stimulation of cultured BMSCs [20]. Our results clearly show that appropriate mechanical stimulation can promote osteogenic differentiation of BMSCs as indicated by deeper ALP staining and upregulation of osteogenic markers Runx2 and BMP2. Runx2 is a member of the Runxx family of transcription factors and acts as a specific transcription factor for bone cells during development and remodeling [26], while BMP-2 is a member of the TGFβ structure-related protein superfamily that can induce bone and cartilage formation in combination with bone conduction carriers such as collagen and synthetic hydroxyapatite [27]. Our results also demonstrated that the same extent of mechanic stimuli inhibited the adipogenic differentiation of BMSCs, indicated by the lower red oil O staining, as well as upregulated expressions of osteogenic gene markers including PPAR-γ and C/EBPα, which are the most important transcription factors in MSCs during adipogenic differentiation. Our results were consistent with previous study by Turner et al., which showed that the appearance of intracellular lipid droplets was delayed in the stress group during the differentiation and maturation of MSCs into adipocytes [28], and supported the statement that shear stresses at physiological levels can differentiate MSCs into endothelial cells by inducing the expression of endothelial-cell-specific markers [29].

Acknowledgement
We sincerely appreciated all workers in this study

Conflicts of interest:
None Consent for publication:

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