miR‐542‐3p prevents ovariectomy‐induced osteoporosis in rats via targeting SFRP1

Secreted frizzled‐related protein‐1 (SFRP1) is a negative regulatory molecule of the WNT signaling pathway and serves as a therapeutic target for bone formation in osteoporosis. In this study, we first established an ovariectomized (OVX) rat model to simulate postmenopausal osteoporosis and found significant changes in miR‐542‐3p and sFRP1 expression by RNA sequencing and qRT‐PCR. In addition, there was a significant negative correlation between miR‐542‐3p and sFRP1 mRNA levels in postmenopausal women with osteoporosis. We found that miR‐542‐3p inhibited the expression of sFRP1 mRNA by luciferase reporter assay. When the miR‐542‐3p binding site in sFRP1 3'UTR was deleted, it did not affect its expression. Western blot results showed that miR‐542‐3p inhibited the expression of SFRP1 protein. The expression of SFRP1 was significantly increased in osteoblast‐induced mesenchymal stem cells (MSC), whereas the expression of miR‐542‐3p was significantly decreased. And miR‐542‐3p transfected MSCs showed a significant increase in osteoblast‐specific marker expression, indicating that miR‐542‐3p is necessary for MSC differentiation. Inhibition of miR‐542‐3p reduced bone formation, confirmed miR‐542‐3p play a role in bone formation in vivo. In general, these data suggest that miR‐542‐3p play an important role in bone formation via inhibiting SFRP1 expression and inducing osteoblast differentiation.


| INTRODUCTION
Osteoporosis is a bone metabolic disease characterized by a decrease in bone mass and bone mineral density (BMD), and a degradation of bone tissue microstructures, which lead to an increase in bone fragility and fracture incidence (Kanis et al., 2013;Roux & Briot, 2017). The main pathogenesis of osteoporosis is caused by bone remodeling disorders, which are due to osteoclast-mediated bone resorption rates higher than osteoblast-mediated bone formation (Bidwell, Alvarez, & Childress, 2013). The incidence of osteoporosis in the elder population is higher (senile osteoporosis) (Li et al., 2017). In particular, postmenopausal women (postmenopausal osteoporosis) are prone to osteoporosis because of estrogen deficiency is associated with excessive bone resorption and poor bone formation (Epstein, 2006;Heiss et al., 2012). However, the detailed mechanisms of estrogen deficiency in postmenopausal osteoporosis have not been fully understood.
The canonical Wnt/β-catenin signaling pathway plays a vital role in activating bone formation and resorption genes transcription, and then regulate osteoblast differentiation, proliferation, survival, and bone formation (Zhang & Drake, 2012). SFRP1 is homologous to the extracellular cysteine-rich domain of the WNT receptor Frizzled, but lacks the intracellular and transmembrane domains (Yang et al., 2009).
It has been reported that competition with extracellular Wnt binds to the Frizzled receptors, and further antagonizes Wnt signaling by direct binding to Wnt proteins (Gaur et al., 2006;Hausler et al., 2004).
Previous studies have shown that SFRP1 plays an important role in osteoblast differentiation, trabecular bone formation and bone fracture healing (Bodine et al., 2004;Bodine, Seestaller-Wehr, Kharode, Bex, & Komm, 2007;Gaur et al., 2006Gaur et al., , 2009. Thus, the development of inhibitors against SFRP1 is a viable method of stimulating bone formation in metabolic bone diseases, osteoporosis and aging (Baron & Rawadi, 2007;Gaur et al., 2009). MicroRNAs (miRNAs) is a class of small non-coding (18-25 nucleotides) single-stranded RNAs that binds to an incomplete or complete base pairing of a specific sequence in the 3′ untranslated regions (3′UTRs) or CDS of mRNAs, then induces either translational repression or cleavage of the target mRNAs (Noma et al., 2004;Schramke & Allshire, 2003). MiRNAs play important roles in a variety of biological processes, such as cell proliferation, differentiation, apoptosis, and tumorigenesis (Yang et al., 2013;Yin et al., 2013;Zhang, Wei, & Xu, 2013). Recently, numerous studies have revealed that miRNAs play critical roles in bone homeostasis. For example, miR-433-3p plays an critical role in DKK1/WNT/beta-catenin pathway by reducing DKK1 expression and inducing osteoblast differentiation (Tang, Lin, Wang, & Lu, 2017). MiR-23a cluster regulates osteoblast differentiation by attenuating Prdm16 expression level to modulate the TGF-β signaling pathway (Zeng et al., 2017). However, the role of miRNAs in the pathogenesis of osteoporosis remains to be further studied.
In this study, we found that sFRP1 and miR-542-3p were negatively correlated in postmenopausal osteoporosis patients. Then, miRNA target analysis and experiments showed that sFRP1 was a target of miR-542-3p. Further studies have shown that miR-542-3p plays a vital role during the process of MSC differentiation. In general, our study show that miR-542-3p play an important role in bone formation by targeting SFRP1 and inducing osteoblasts differentiation.
2 | RESULTS 2.1 | MiR-542-3p is differentially expressed and negatively correlated with SFRP1 in postmenopausal osteoporotic patients We chose estrogen deficient ovariectomized (OVX) rats model to mimic postmenopausal osteoporosis, because it is close to postmenopausal bone loss (Kalu, 1991). A total of 45 or 90 days after surgery, the bone mineral density (BMD) of right femoral in OVX rats was lower than that in sham operation group (Sham) (p < 0.05) ( Table 1).
Compared with sham rats, OVX rats also showed increased trabecular separation (Figure 1). It indicated that ovariectomy-induced osteoporosis in rat model has been established.
To compare the mRNAs expression profiles in the MSCs from OVX rats and Sham rats, three subjects of each subgroup were selected for RNA-seq assays. mRNAs with log 2 (fold change) >3-fold were determined as putative candidates. As shown in Table 2, six mRNAs were up-regulated and 22 mRNAs were down-regulated. In the dramatically up-regulated mRNAs, SFRP1 is an antagonist of Wnt signaling and play an important role in maintaining bone homeostasis (Baron & Rawadi, 2007;Gaur et al., 2009). So we selected sFRP1 as a candidate gene. The expression of SFRP1 was detected in three subjects using western blot and qRT-PCR assay, and the results were consistent with RNA-seq expression (Figures 2a and b) Figure 2c). Finally, the relationship between miR-542-3p and sFRP1 expression levels in postmenopausal osteoporotic patients was analyzed and plotted on the graph shown in Figure 2d. Correlation analysis showed a significant negative correlation between sFRP1 and miR-542-3p expression (Figure 2d).

| MiR-542-3p directly targets sFRP1
It has been demonstrated that miRNAs regulate the mRNA expression of by binding to the 3′-UTR or amino acid coding sequence (CDS) of target gene (Tay, Zhang, Thomson, Lim, & Rigoutsos, 2008). Based on the bioinformatics online analysis software (TargetScan), miR-542-3p was predicted to target in the 3′UTR of sFRP1.
To investigate whether miR-542-3p is directly targeted to SFRP1, we firstly constructed luciferase reporter vectors which contained the predicted miRNA binding site of sFRP1 3'UTR or deleted mutant These results indicate that sFRP1 is the target gene for miR-542-3p.

| MiR-542-3p increased osteogenic differentiation
We first isolated the MSC from the rats and then induced to differentiate into osteoblasts. The expression of miR-542-3p and sFRP1 was measured from 1 to 3 weeks after induction. Osteoblast-induced cells showed a significant increase in the expression level of sFRP1 from 1 to 3 weeks ( Figure 4a). In contrast, the expression level of miR-542-3p was significantly reduced from 1 to 3 weeks, and the expression level of miR-542-3p was the lowest at 3 weeks after induction ( Figure 4b).
Then, we detected the expression of specific markers for

| MiR-542-3p promotes bone formation in vivo
In order to investigate the function of miR-542-3p in vivo, we used ovariectomy (OVX) rats to simulate postmenopausal status. Rats undergoing Sham surgery or OVX were given miR-542-3p inhibitor by FIGURE 1 OVX surgery leads to dramatically decreased bone loss in rats. Histological sections of femur in rats of the sham and OVX groups. Sham: sham control group; OVX: the ovariectomized rats group; Scale bar, 100 μm. All sections were stained with HE  Then a significant decrease in BMD was also detected. The results showed that miR-542-3p inhibitor treated rats exhibited a significant decrease in femur BMD compared to rats treated with miR-542-3p inhibitor mutant or PBS. OVX rats treated with miR-542-3p inhibitor, miR-542-3p inhibitor mutant or PBS also presented reduced femur BMD, whereas miR-542-3p inhibitor treated rats showed minimal BMD ( Figure 5b).
Micro-CT was used to quantify the bone volume/tissue volume ratio (BV/TV). This assay revealed that in Sham rats, a significant decrease in BV/TV was detected in miR-542-3p inhibitor-treated rats compared to rats treated with PBS or miR-542-3p inhibitor mutant (p < 0.05) (Figure 5c). OVX rats treated with PBS or miR-542-3p inhibitor mutant also exhibited a significant decrease in BV/TV, while the lowest in the miR-542-3p inhibitor treated OVX rats (Figure 5c).

| DISCUSSION
Osteoporosis is a metabolic disorder characterized by osteoblast formation and osteoclast resorption imbalance, leading to skeletal fragility and fracture susceptibility. Ovarian hormone deficiency is a major risk factor for postmenopausal women with osteoporosis. It is well known that the OVX rats are the best animal model for the research of postmenopausal osteoporosis in women (Hartke, 1999). In the present study, we indicate that miR-542-3p is a novel miRNA that could ameliorate ovariectomy-induced osteoporosis in rats by directly targeting sFRP1.
There is a growing evidence that miRNAs play key roles in both normal biological processes and the pathogenesis of human diseases through post-transcriptional regulation of gene expression (Lamouille, Subramanyam, Blelloch, & Derynck, 2013). Several decades of miRNAs are considered necessary for bone formation (Lian et al., 2012). In this study, we found a higher level of sFRP1 in MSCs from OVX rats and a lower level of miR-542-3p in serum of OVX rats compared with Sham rats. Additionally, a correlation analysis further confirmed that However, the role of miR-542-3p in osteoporosis is unknown.
Here, we found that miR-542-3p regulates the osteogenesis differentiation of rat MSCs by directly targeting sFRP1. SFRP1 is an antagonist of Wnt signaling, which is an important pathway to maintain bone homeostasis. It has been reported that overexpression of SFRP1 in human osteoblasts accelerates the rate of osteoblast and osteocyte mortality. The lack of SFRP1 in mice leads to reduced osteoblast and osteocyte apoptosis (Bodine et al., 2005). Adult mice deficient in SFRP1 showed resistance to age-related bone loss and improved fracture repair by promoting early bone union (Bodine et al., 2004;Gaur et al., 2009). In addition to osteoblast formation, SFRP1 can directly bind to RANKL and inhibit osteoclast formation (Hausler et al., 2004). Moreover, a small molecule inhibitor (diarylsulfone sulfonamide) that can bind and inhibit SFRP1 was shown to stimulate Wnt/βcatenin signaling to increase bone formation (Bodine et al., 2009). All these confirmed the importance of SFRP1 for bone formation, and suggested that inhibition of SFRP1 may be a potential therapeutic target for increasing bone formation.
Several evidences strongly suggest that SFRP1 is a functional target of miR-542-3p and mediates its regulatory role in osteoblast formation. First, SFRP1 may be a possible target with 9 nt consistent matching site complementary to miR-542-3p in the 3′UTR of sFRP1.
Third, the level of SFRP1 protein was decreased after overexpression of miR-542-3p. Finally, during the process of MSCs differentiation, the SFRP1 protein showed a higher increase in OVX rats with a lower miR-542-3p expression.
Then we observed the in vitro effect of miR-542-3p on osteoblast differentiation using rat MSCs. MiR-542-3p enhanced the expression of osteoblast specific markers. And we further investigated the in vivo effects of miR-542-3p on ovariectomy-induced osteoporosis in rats.
Inhibition of miR-542-3p promoted SFRP1 protein expression, decreased bone mass and bone formation, aggravated bone loss in OVX rats. Whereas expression of miR-542-3p mutant did not inhibit SFRP1 protein expression, bone mass and bone formation in OVX rats.
These data suggested that miR-542-3p influenced bone mass by regulating bone formation in vivo, primarily through its effect on SFRP1.
In conclusion, this study provides evidence that miR-542-3p in rat

| Bone mineral density measurement
Bone mineral density (BMD) of the femur at the end of the experiment by Lunar Prodigy Advance (GE Healthcare, Pittsburgh, PA) using appropriate software specifically for rats (Pastoureau, Chomel, & Bonnet, 1995). Results are given in g/cm 2 .

| Bone histomorphometric analysis
For histological analyses, the femurs were fixed in 70% ethanol, embedded in methyl methacrylate, and serial sectioned into 5-µm by a microtome. The parameters obtained for the bone formation were BFR/BS and Ob.S/BS. The parameters measured for bone resorption were Oc.S/BS and N.Oc/B.Pm.
After euthanasia, the femurs were resected and fixed in 10% formalin for 48 hours, decalcified in 10% ethylenediamine tetraacetic acid (EDTA) (pH 7.0) for 14 days and embedded in paraffin.

| Statistical analysis
Data are presented as mean ± standard deviation (SD). The statistical analysis was performed using Student t test or one-way ANOVA. All experiments were repeated at least three times, and representative experiments are shown. Differences were considered significant at p < 0.05.