miR-30 Family Members Negatively Regulate Osteoblast Differentiation*

Background: microRNAs (miRNAs) are closely related to osteogenesis. Results: miR-30 family members (miR-30a, -30b, -30c, and -30d) mediate the inhibition of osteogenesis by targeting Smad1 and Runx2. Conclusion: miR-30 family members are key negative regulators of BMP-2-mediated osteogenic differentiation. Significance: These findings may provide new insights into understanding the regulatory role of miRNAs in the process of osteogenic differentiation. miRNAs are endogenously expressed 18- to 25-nucleotide RNAs that regulate gene expression through translational repression by binding to a target mRNA. Recently, it has been indicated that miRNAs are closely related to osteogenesis. Our previous data suggested that miR-30 family members might be important regulators during the biomineralization process. However, whether and how they modulate osteogenic differentiation have not been explored. In this study, we demonstrated that miR-30 family members negatively regulate BMP-2-induced osteoblast differentiation by targeting Smad1 and Runx2. Evidentially, overexpression of miR-30 family members led to a decrease of alkaline phosphatase activity, whereas knockdown of them increased the activity. Then bioinformatic analysis identified potential target sites of the miR-30 family located in the 3′ untranslated regions of Smad1 and Runx2. Western blot analysis and quantitative RT-PCR assays demonstrated that miR-30 family members inhibit Smad1 gene expression on the basis of repressing its translation. Furthermore, dual-luciferase reporter assays confirmed that Smad1 is a direct target of miR-30 family members. Rescue experiments that overexpress Smad1 and Runx2 significantly eliminated the inhibitory effect of miR-30 on osteogenic differentiation and provided strong evidence that miR-30 mediates the inhibition of osteogenesis by targeting Smad1 and Runx2. Also, the inhibitory effects of the miR-30 family were validated in mouse bone marrow mesenchymal stem cells. Therefore, our study uncovered that miR-30 family members are key negative regulators of BMP-2-mediated osteogenic differentiation.

miRNAs are endogenously expressed 18-to 25-nucleotide RNAs that regulate gene expression through translational repression by binding to a target mRNA. Recently, it has been indicated that miRNAs are closely related to osteogenesis. Our previous data suggested that miR-30 family members might be important regulators during the biomineralization process. However, whether and how they modulate osteogenic differentiation have not been explored. In this study, we demonstrated that miR-30 family members negatively regulate BMP-2-induced osteoblast differentiation by targeting Smad1 and Runx2. Evidentially, overexpression of miR-30 family members led to a decrease of alkaline phosphatase activity, whereas knockdown of them increased the activity. Then bioinformatic analysis identified potential target sites of the miR-30 family located in the 3 untranslated regions of Smad1 and Runx2. Western blot analysis and quantitative RT-PCR assays demonstrated that miR-30 family members inhibit Smad1 gene expression on the basis of repressing its translation. Furthermore, dual-luciferase reporter assays confirmed that Smad1 is a direct target of miR-30 family members. Rescue experiments that overexpress Smad1 and Runx2 significantly eliminated the inhibitory effect of miR-30 on osteogenic differentiation and provided strong evidence that miR-30 mediates the inhibition of osteogenesis by targeting Smad1 and Runx2. Also, the inhibitory effects of the miR-30 family were validated in mouse bone marrow mesenchymal stem cells. Therefore, our study uncovered that miR-30 family members are key negative regulators of BMP-2-mediated osteogenic differentiation. microRNAs (miRNAs) 3 consisting of 18 -25 nucleotides belong to the single-stranded small non-coding RNA family (1)(2)(3)(4)(5)(6). They bind to the 3Ј UTR of specific target genes and regulate expression of the target genes by promoting the degradation of transcribed mRNAs or by inhibiting their translation (7). Recent studies indicate that miRNAs are important players during the osteogenic differentiation (8 -18).
In a previous study, we investigated the expression profiles of miRNAs in MC3T3-E1 cells treated with Emdogain, a clinical mixture of enamel matrix proteins that can induce biomineralization and osteogenesis (19 -23). The data indicated that the expression levels of some miR-30 family members, such as miR-30a, -30b, -30c, and -30d, were significantly down-regulated during the osteoblast differentiation. Studies by others also found that miR-30a and miR-30d are down-regulated during BMP-2-induced osteogenesis of C2C12 mesenchymal cells (15). Considering that miR-30 family members decreased during osteogenesis induced by different stimuli, it is possible that they may play important roles in the process.
Here we investigated the effects of the miR-30 family on osteoblastic differentiation. We found that miR-30a, -30b, -30c, and -30d expression was down-regulated during BMP-2 stimulation. They were demonstrated to inhibit osteoblast differentiation. Further studies identified Smad1 and Runx2 as common target genes of miR-30 family members. Finally, rescue experiments showed that overexpression of Smad1 and Runx2 significantly eliminated the inhibitory effect of miR-30 on osteogenic differentiation. All these data indicate that miR-30 family members function as negative regulators of osteoblastic differentiation by targeting the master osteogenic transcription factors Smad1 and Runx2. These findings may provide new insights into understanding the regulatory role of miRNAs in the process of osteogenic differentiation.
Cell Culture, Stimulation, and Transfection-MC3T3-E1 cells were obtained from the Chinese Academy of Sciences Cell Bank (Shanghai, China) and were maintained in ␣ modification of Eagle' s minimal essential medium (␣-MEM, Invitrogen) containing 100 units/ml penicillin and 100 g/ml streptomycin. MC3T3-E1 cells (1 ϫ 10 5 /well) were cultured in 6-well plates overnight and treated with or without 200 ng/ml BMP-2 for various setting time points. For some experiment, mouse bone marrow MSCs were prepared from the bone marrow of femurs and tibias harvested from 2-month-old male C57B/L6 mice (24).
MC3T3-E1 (1 ϫ 10 5 ) or mouse bone marrow MSCs (1 ϫ 10 6 ) were cultured overnight in 24-well plates and transfected with 40 nM or 80 nM miR-NC, miR-30 family mimics, oligo-Ctrl, or as-miR-30 family (Genepharma) using Lipofectamine 2000 (Invitrogen). Three days later, these cells were stimulated with or without 200 ng/ml BMP-2 in completed culture medium for varying periods. In addition, the cells were either harvested for protein and mRNA or fixed with 95% ethanol (v/v) for histochemical examination of ALP activity.
Construction of Smad1 and Runx2 Expression Vectors-Complementary DNAs for the mouse Smad1 and Runx2 genes were obtained by an RT-PCR technique using the Prime-Script TM PT reagent kit (TaKaRa). Total RNAs prepared from mouse MC3T3-E1 cells were used for the RT-PCR. The primer sequences are shown in supplemental Table 1. PCR products were digested with BglII and NotI (Promega), purified from agarose gels, and subcloned into pCMV-myc (Clontech). Each cDNA was confirmed by DNA sequencing. The plasmid DNA was transfected into cells using Lipofectamine 2000 (Invitrogen). Mutant primers were used to generate a two-seed region mutant for Smad1 (Smad1 ϩ 3ЈUTR-Mut) and four-seed region mutant for Runx2 (Runx2 ϩ 3ЈUTR-Mut) construct by using KOD-Plus (Toyobo Co.).
ALP Staining and Activity-MC3T3-E1 cells at 3 ϫ 10 5 cells/ well were cultured overnight in 6-well plates. Three days after transfection, the cells were treated with 200 ng/ml BMP-2 for 7 days. Subsequently, the cells were fixed with 95% ethanol (v/v) and then incubated with a substrate solution from an ALP staining kit (Beyotime Institute of Biotechnology, Shanghai, China) in the dark, according to the manufacturer's protocol.

miR-30 Family Members Regulate Osteoblast Differentiation
For ALP activity assays, after incubation, the treated cells were washed twice with PBS, and 200 l of lysis buffer was added to the cell layer and kept on ice for 5 min. The cell lysate was sonicated for 1 min and centrifuged at 1,000 ϫ g at 4°C for 10 min. ALP activity was assayed by a spectrophotometric method using a LabAssayTMALP kit. The absorbance at 405 nm of each well was measured with the microplate reader according to the manufacturer's instruction (15).
Alizarin Red Staining-For detection of calcification during differentiation, BMP-2-treated or untreated mouse bone marrow MSCs were washed twice with PBS and fixed with 500 l of ice-cold 70% ethanol for 10 min. The fixed cells were stained with 500 l of Alizarin red solution (Sigma).
Statistical Analysis-Data are expressed as mean Ϯ S.E. Data were analyzed by one-way or two-way analysis of variance. Multiple comparison between the groups was performed by using the Bonferroni post hoc test method. p Ͻ 0.05 was considered statistically significant. Statistical analysis was carried out using StatView 5.0 software (SAS Institute, Cary, NC) and GraphPad Prism 4.0 software.

Dynamic Changes of miR-30 Family Members during BMP-2-induced Osteoblast Differentiation in MC3T3-E1 Cells-It
has been indicated that some miR-30 family members are down-regulated during osteogenic differentiation (15). Our previous studies also found that more than one member of miR-30 family significantly decreased in response to osteogenesis-related stimuli (data unpublished), suggesting that the miR-30 family may be important for osteogenic differentiation.
Effects of miR-30 Family Members on Osteoblast Differentiation in MC3T3-E1 Cells-It is well known that ALP activity increases in a time-dependent manner in MC3T3-E1 cells after treatment with BMP-2 (25,26). To determine whether miR-30 could affect osteoblast differentiation, miR-30 family mimics (miR-30a, -30b, -30c, and -30d) or inhibitors (as-miR-30a, -30b, -30c, and -30d) were transfected into MC3T3-E1 cells, respectively, followed by BMP-2 treatment for 7 days. Then, the ALP activities in the transfected cells were investigated. As shown in Fig. 2, A and C, the ALP activities in the miR-30 overexpressing cells were significantly suppressed compared with those in the miR-NC transfected cells. On the contrary, knockdown of miR-30 expression increased the ALP activities (Fig. 2, B and D). We also observed that miR-30 inhibited ALP mRNA levels in a dose-dependent manner (Fig. 2, E and F). These results suggested that miR-30 family members act as negative regulators in osteogenesis induced by BMP-2 stimulation.
Smad1 and Runx2 Are Common Targets of the miR-30 Family-To identify the target genes of miR-30 in osteogenesis, we searched for candidate genes using the miRNA target prediction database TargetScan 5.1. Members of the miR-30 family were predicted to target Smad1 and Runx2, which are key downstream mediators of BMP signaling during bone formation (27)(28)(29). There are two and four predicted target sites in the 3ЈUTR of Smad1 and Runx2, respectively. The sequences of these target sites are highly conserved in different vertebrate species (Fig. 3A and supplemental Fig. 1). Runx2 has been demonstrated to be a direct target of miR-30 family members (30,31). However, the function of miR-30 on Smad1 during osteogenic differentiation has not been reported. To test whether Smad1 could be regulated by miR-30, we transfected MC3T3-E1 cells with miR-30 mimics or inhibitors, respectively. The results showed that following transfection, the protein levels of Smad1 decreased when the levels of miR-30a, -30b, -30c, and -30d increased. In contrast, the expression of Smad1 increased after knockdown of miR-30a, -30b, -30c, or -30d. Of note, Smad1 protein level in the as-miR-30a-d mixture (as-miR-30m) cotransfected cells showed nearly a 5-fold increase compared with the control cells, indicating that miR-30 family members are important negative regulators of  MARCH 2, 2012 • VOLUME 287 • NUMBER 10 JOURNAL OF BIOLOGICAL CHEMISTRY 7505 Smad1 (Fig. 3B). Similar changes in Runx2 protein expression were observed (Fig. 3B). These results provide evidence that the miR-30 family negatively regulates Smad1 and Runx2 expression. Furthermore, quantitative RT-PCR assays demonstrated that Smad1 mRNA did not change in miR-30-overexpressing cells (Fig. 3C), indicating that miR-30 family members regulate Smad1 gene expression on the basis of translational repression rather than mRNA degradation.

miR-30 Family Members Regulate Osteoblast Differentiation
Upon binding of the BMP ligand to the type I and type II receptor complexes, the activated type I receptor phosphorylates Smad1/5/8, which then assemble into complexes with Smad4 and translocate into the nucleus to regulate the expression of genes related to osteoblast differentiation, such as ALP and OSC (32,33). To study whether the BMP-2-Smad1 path- B, overexpression or knockdown of miR-30 expression inhibited or enhanced Smad1 and Runx2 expression, respectively. MC3T3-E1 cells were transfected with 40 nM miR-NC, miR-30a, -30b, -30c, -30d, a mixture of miR-30a-d (miR-30m) or oligo-Ctrl, as-miR-30a, -30b, -30c, -30d, and a mixture of as-miR-30a-d (as-miR-30m), respectively. Three days later, the relative expression levels of Smad1 and Runx2 to GAPDH were determined by Western blot assays. C, Smad1 mRNA expression. The relative levels of Smad1 mRNA expression to ␤-actin were determined by quantitative RT-PCR. The cells transfected with control miR-NC or oligo-Ctrl were designated as negative control. Data are representative images or expressed as mean Ϯ S.E. of each group of cells from three separate experiments, and the values of the control cells were designated as 1. MARCH 2, 2012 • VOLUME 287 • NUMBER 10 way could be affected by miR-30, MC3T3-E1 cells were transfected with miR-30 (miR-30a, -30b, -30c, or -30d) or control, oligo-Ctrl, or as-miR-30 (as-miR-30a, -30b, -30c, or -30d) and were then treated with BMP-2 for 30 min. Changes in Smad1, Smad5, and phospho-Smad1/5 (p-Smad1/5) protein levels were determined by Western blot analysis. Similar to Smad1, the expression levels of p-Smad1/5 decreased in miR-30-overexpressing cells, whereas they increased in the miR-30 knockdown cells (Fig. 5B).

miR-30 Family Members Regulate Osteoblast Differentiation
To confirm the speculation that miR-30 family members function through Smad1 and Runx2, miR-30a-d mixture, together with the wild-type Smad1 overexpressing plasmid, 3Ј UTR-mutant Smad1 overexpressing plasmid, wild-type Runx2 overexpressing plasmid, or 3Ј UTR-mutant Runx2-overexpressing plasmid, were cotransfected into MC3T3-E1 cells. It demonstrated that transfection with 3Ј UTR-mutant Smad1and Runx2-expressing plasmids was able to eliminate the difference in Smad1 and Runx2 protein levels between miR-30 transfected and control cells (supplemental Fig. 2). When stimulated with BMP-2, the overexpression of Smad1 and Runx2 significantly abolished the inhibitory effects of miR-30 on ALP expression (Fig. 5C). All of these data provide evidence that miR-30 members mediate the inhibition of osteogenesis by targeting Smad1 and Runx2. FIGURE 4. Smad1 is a direct target of the miR-30 family. A and B, Overexpression or knockdown of miR-30 expression inhibited or enhanced the Renilla luciferase activities. MC3T3-E1 cells were cotransfected with 40 nM miR-NC, miR-30a, -30b, -30c, -30d, a mixture of miR-30a-d (miR-30m) or oligo-Ctrl, as-miR-30a, -30b, -30c, -30d, a mixture of as-miR-30a-d, and 100 ng of reporter plasmid containing the wild-type Smad1 3Ј UTR. 24 h later, Renilla luciferase values, normalized against firefly luciferase, were presented. C, alignment of alterations in the first (Mut1) and/or the second (Mut2) of the seed sites in the psiCHECK-2-Smad1 reporter gene. The four mutated nucleotides are underlined. D, miR-30 family members target the Smad1 seed sites. MC3T3-E1 cells were cotransfected with the luciferase reporter plasmid carrying the wild-type or mutated sites and miR-NC or miR-30a-d mixture (miR-30m), respectively. Effects of miR-30 on the reporter expression were determined 24 h after transfection. Renilla luciferase values, normalized to firefly luciferase, were presented. The cells transfected with control miR-NC or oligo-Ctrl were designated as negative control. Data are expressed as mean Ϯ S.E. of each group of cells from three separate experiments, and the values of the control cells were designated as 1. *, p Ͻ 0.05. Mut, mutant.

miR-30 Family Members Inhibit Osteogenic Differentiation of Primary Mouse Bone Marrow
MSCs-Finally, we addressed the functional activities of miR-30 family members in primary mouse bone marrow MSCs. miR-30a, -30b, -30c, -30d, and miR-30a-d mixture, miR-NC, as-miR-30a, -30b, -30c, -30d, and as-miR-30a-d mixture or oligo-ctrl were transfected into mouse MSCs, respectively. This demonstrated that during the osteogenic differentiation, the miR-30 family member overexpression inhibited the expression levels of ALP, OSC, BSP, and OSX, whereas knockdown of miR-30 family members increased their expression levels (supplemental Table 3). Furthermore, the ALP activities were significantly suppressed or enhanced in the miR-30a-d mixture or as-miR-30a-d mixturetransfected cells, which was compatible with the results of Alizarin red staining (Fig. 6). Both Smad1 and Runx2 were found to be inhibited by miR-30 in MSCs (supplemental Fig. 4). Therefore, these results indicate that miR-30 family members nega-tively regulate the osteogenic differentiation of mouse bone marrow MSCs.

miR-30 Family Members Regulate Osteoblast Differentiation
-30d in MC3T3-E1 cells stimulated by BMP-2 are similar (Fig.  1). This suggests that they may play similar roles in osteoblast differentiation. Here, we provide evidence for the concept that miR-30 family members (miR-30a, -30b, -30c, and -30d) regulate osteoblast differentiation and alter the levels of critical molecule of BMP pathways. In contrast, miR-384 -5p was undetected, and miR-30e did not change in response to BMP-2 stimulation (Fig. 1). Others also found that not all miR-30 family members changed during osteogenic differentiation induced by BMP-2 in C2C12 cells (15). It is possible that the expression of miR-30 family members is regulated differently.
To investigate the effects of miR-30 family members on BMP-2-induced osteogenic differentiation, we first examined the efficiency and specificity of mimics and inhibitors for miR-30 used in subsequent experiments. Overexpression or knockdown of each member barely affected the expression levels of other members (supplemental Fig. 5). miR-30 family overexpression led to decreased ALP activities, whereas knockdown of them increased mRNA and protein levels of ALP compared with the control cells (Fig. 2, A and D). The same effects were also observed in mouse bone marrow MSCs by overexpressing or inhibiting miR-30 ( Fig. 6 and supplemental Fig. 3). These data suggest that miR-30 family members are the negative regulators of osteoblast differentiation induced by BMP-2 in both preosteoblast cell lines and primary cells.
Smad1 is an immediate downstream transducing molecule of the BMP receptor and plays an important role in mediating BMP signaling (34). Osteoblast-specific Smad1 gene knockout mice present with impaired postnatal bone formation (35). So far, several miRNAs have been reported to target Smad1 and regulate its expression in different physiologic conditions. For example, miR-26a regulated osteogenic differentiation of human adipose tissue-derived stem cells by targeting the Smad1 transcription factor (36). miR-199a* was found to adversely regulate early chondrocyte differentiation via directly targeting Smad1 (37). miR-155 targets the 3Ј UTR of multiple components of the BMP signaling cascade, including Smad1 in normal and virus-infected cells (38). Our studies also found new miRNAs regulating Smad1 expression in osteoblast differentiation, indicating that Smad1 could be regulated by different miRNAs under different conditions.
In this study, we found two potential binding sites of miR-30 in the Smad1 3Ј UTR using bioinformatic analysis and provided direct evidence that Smad1 was the common target of miR-30 family members (Figs. 3 and 4). Mutation of both sites partially eliminated the inhibitory effect of exogenous miR-30 (Fig. 5C). It should be highlighted that in the miR-NC-transfected samples, mutation of either or both sites led to obviously higher Renilla luciferase activities. Considering that mutation of binding sites also abolished the inhibitory function of endogenous miR-30, the result further supported the conclusion that both of these two sites are important for regulation of Smad1 expression.
Besides osteogenesis, it would be interesting to investigate the role of miR-30 in osteoclast differentiation as well, or particularly in chondrocyte differentiation, as Smad1 signaling is also involved in chondrogenesis (37). Although we have found that miR-30 family members are associated with osteogenic differentiation of mouse bone marrow MSCs ( Fig. 6 and supplemental Fig. 3), it remains to be determined whether they are differentially expressed in cartilage, bone, or both. Future in vivo experiments in mouse models are necessary to address miR-30 function in depth. In addition to BMP pathways, Smad1 is also a mediator of TGF-␤ pathways in several non-endothelial cell lineages (39), so it is possible that miR-30 family members may be involved in regulating TGF-␤ signaling pathways.
In conclusion, we demonstrated that the members of miR-30 family, in response to BMP-2, act as the negative regulators of early osteoblast differentiation through their suppression of Smad1 and Runx2 transcription factors. This study provides new insights into BMP/Smad signaling regulation in osteoblast differentiation.