Overexpression of miR-29b Partially Rescued the Osteogenic Differentiation and Osteogenesis Ability of Ovx-BMSCs

of Rescued the Osteogenic Differentiation and Osteogenesis Ability of Ovx-BMSCs. Abstract Osteoporosis, as a skeletal disorder characterized by gradually reduced bone mineral density, is a severe threat to millions of postmenopausal women. MicroRNAs (miRNAs) are important post-transcriptional factors and play an important role in bone degeneration such as osteoporosis and other bone-related diseases. Here we showed that the expression of miR-29b in bone tissue and bone marrow stromal cells (BMSCs) of Ovx mice is descend and overexpression of miR-29b could partially rescued osteogenic differentiation and bone formation ability of Ovx-BMSCs in vitro and in vivo, respectively. Our studies indicated that miR-29b plays an important role in regulating BMSCs osteogenic differentiation and could contribute to osteoporosis therapy.


Introduction
Osteoporosis, the most common bone remodeling disease, is characterized by a systemic impairment of bone mass and microarchitecture and increases the fracture risk of the patient [1].
Moreover, the osteoporotic fracture often causes further severe disability and premature mortality in the aging population due to impaired bone healing abilty [2,3]. Worldwide, approximately 40% of Caucasian postmenopausal women are expected to suffer from osteoporotic fractures [4]. Among the extensive treatment approaches currently used in clinics for treating osteoporosis, most of them are focusing on antiresorptive agents, which exert their therapeutic effect by slowing down the bone resorption rate.
However, anti-resorptive strategies mainly focus on preventing on-going damage rather than rebuilding damaged bone tissues [5]. Bone-anabolic agents including parathyroid hormone (PTH) and and PTH-related peptide (PTHrP) synthetic analogues such as teriparatide and abaloparatide have been used to increased bone formation and reduces bone fracture risk [6][7][8]. But continuous treatment with PTH may cause bone resorption. Therefore, it is necessary to develop a novel anabolic therapeutic strategy to enhance bone formation.
With the development of regenerative medicine, stem cell-based therapy has attracted much attention for the treatment of bone diseases due to their properties of self-renewal and plasticity [9].
Mesenchymal stem cells (MSCs) such as bone marrow stromal cells (BMSCs) possessing regenerative potential have been considered an ideal cell source for bone formation and repair [10]. However, many studies have demonstrated that osteoporosis affecting the osteogenic differentiation of MSCs [11]. Therefore, it is necessary to establish an approach to rescue the osteogenic differentiation and bone formation ability of MSCs under osteoporotic conditions [12]. microRNAs (miRNAs) are a large class of small, non-coding RNAs that function as repressors of gene expression at the level of post-transcriptional regulation [13]. Previous studies have been demonstrated that miRNA network is involved in regulating the process of bone remodeling and also related to osteoporosis phenomenon [14][15][16]. In this study, a miRNA, namely miR-29b, was identified to be correlated with bone loss in estrogen deficiency-

Cell culture
BMSCs were isolated from the femurs of sham-operated or Ovx mice. The Sham-BMSCs and Ovx-BMSCs were cultured with DMEM medium supplemented with 10% FBS, 100 U/ml penicillin, 100 mg/ml streptomycin, and 2 mM L-glutamine and cultured at 37℃, 5% CO2. The culture medium was changed every 2 or 3 days.
BMSCs at passage 2 or 3 were used in this study.

MiRNA transfection
Ovx-BMSCs were transfected with miR-29b, or their respective negative control (NC, Guangdong Ruibo, China) using siPORT NeoFX transfection reagent (Life Technologies, Carlsbad, CA) according to the instruction of the manufacturer. The transfection reagent (total 5 μl) was diluted in 50 μl Opti-MEM I (Life Technologies) and mixed with 50 nmol/L miR-29b, or equal amounts of its respective negative control. The mixture was added into 6-well plates to transfected BMSCs (1×106 cells/ml). After transfection 6 hours, the medium was replaced with fresh culture medium.

Real-time RT-PCR analysis
Total mRNA was extracted using TRIzol reagent (Invitrogen).
The concentration of total RNA was quantified with a Thermo Scientific NanoDrop™ 1000 ultraviolet-visible spectrophotometer (NanoDrop Technologies, Wilmington, DE). cDNA was synthesized using a Prime-Script TM RT reagent kit (Takara Bio, Shiga, Japan) according to the manufacturer's instructions. The expression of miR-29b and osteogenesis-related marker genes including alkaline phosphatase (ALP) and osteocalcin (OCN) were examined. U6 and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used for normalization of microRNA and mRNA expression levels.
All experiments were performed in triplicate. The PCR primer sequences are displayed in (Table 1).

In vivo bone formation assay
Sham To investigate the neo-bone formation, mice were sacrificed at 8 weeks after operation and the calvarial bone samples were harvested and then subjected to micro-CT observations.

Micro-computed tomography (Micro-CT) analysis
For the calvarial bone analysis, specimens were scanned in scanning mode (voxel size 20 μm, 80 kVp, 500A) to evaluate the bone volume using a micro-CT system (Siemens AG, Germany).
After scanning, three-dimensional images of repaired calvarial bone specimens were reconstructed and the percentage of new bone volume relative to tissue volume was measured using auxiliary software.

Statistics
The experiment data in this study were presented as the mean ± standard derivation (SD). Statistical analysis was performed using one-way ANOVA with Tukey's multiple comparison test to assess the difference among multiple experimental groups. Statistical differences between two groups were determined by the Student's t-test. P<0.05 was used considered statistically significant.  1(A, B)). ALP, as an early differentiation marker, plays an important role in calcium-phosphate mineral formation in bone and OCN is also a marker of mineralization. The expression of ALP and OCN in bone tissue and BMSCs of Ovx mice group was also significantly decreased compared with Sham mice group ( figure 1C). miR-29b, had previously been demonstrated that play a role in osteogenic differentiation, exhibited decreased expression in Ovx mice group than in Sham mice group ( figure 1D).

MiR-29 expression pattern in estrogen deficiency-induced osteoporosis mice
The role of miR-29b in regulating osteogenic differentiation of Ovx-BMSCs was analyzed in the following study. These results indicated that overexpression of miR-29b partially rescued the osteogenic function of Ovx-BMSCs in vitro.

miR-29b treatment enhanced ectopic bone formation ability of Ovx-BMSCs in vivo
To investigate the effects of miR-29b on regulating the BMSCs-

miR-29b-seeded scaffolds repaired calvarial bone defects in mice
To further evaluate whether miR-2b can promote bone formation in suit, a 5-mm critical-sized calvarial bone defects was generated in osteoporotic mice. Ovx-miR-NC-BMSCs or Ovx-miR-29b-BMSCs were mixed with delivery hydrogel and then transplanted into the defect area. Micro-CT was used to detect the neo-bone formation at two time points day 0 and 12 weeks after operation.
The results indicated that the miR-29b transfected group showed more bone tissue regeneration than miR-NC group ( figure 4(A-B)).
Quantitative study revealed that over 50% new bone was formed in Ovx+miR-29b group. Meanwhile, only 30% new bone formation was observed in Ovx+miR-NC group.

Discussion
In this study, we demonstrated that miR-29b is a key regulator of are demonstrated to be involved in osteogenesis related signaling pathways [17]. miRNA mimic or miRNA inhibitors has been considered as candidates to be used as therapies for disease as consequence of osteoblasts and osteoclasts malfunction [18]. The miR-29 family contains three mature miRNAs including miR-29a, miR-29b and miR-29c [19]. Previous study has been demonstrated that miR-29b is a positive regulator of osteogenic differentiation by directly down-regulating osteogenic differentiation inhibitors [20].
Thus, we investigated the potential role of miRNA in regulating bone disease osteoporosis.
In this study, we first demonstrated that the expression of miR- Osteoporosis, especially postmenopausal osteoporosis, has a severe influence in aging population [21]. In current, several drugs such as bisphosphonates, calcitonin and so on have been used to slow the process of bone loss [22][23]. Moreover, administration of stem cells could prevent or reverse the osteoporosis and reduce the susceptibility of bone fractures. In this study, we also investigated the in suit bone formation ability of Ovx-BMSC transfeted with miR-29b using a mouse critical-sized calvarial defect model. Micro-CT analysis showed that miR-29b significantly enhanced the bone regeneration ability of Ovx-BMSCs. Over 50% bone defect area was repaired by the newly formed bone tissue.

Conclusion
This study indicated that overexpression of miR-29b could partially rescued the osteogenic differentiation ability and osteogenesis ability of Ovx-BMSCs both in vitro and in vivo. These findings suggested that miR-29b may be a promising therapeutic target for potent osteoporosis and osteoporotic fracture treatments.

Conflicts of Interest
The authors indicated no potential conflicts of interest.