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Keywords:

  • Sp7;
  • miRNA;
  • OSTEOBLAST;
  • MINERALIZATION;
  • FEEDBACK LOOP

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

microRNAs (miRNAs) play pivotal roles in osteoblast differentiation. However, the mechanisms of miRNAs regulating osteoblast mineralization still need further investigation. Here, we performed miRNA profiling and identified that miR-93 was the most significantly downregulated miRNA during osteoblast mineralization. Overexpression of miR-93 in cultured primary mouse osteoblasts attenuated osteoblast mineralization. Expression of the Sp7 transcription factor 7 (Sp7, Osterix), a zinc finger transcription factor and critical regulator of osteoblast mineralization, was found to be inversely correlated with miR-93. Then Sp7 was confirmed to be a target of miR-93. Overexpression of miR-93 in cultured osteoblasts reduced Sp7 protein expression without affecting its mRNA level. Luciferase reporter assay showed that miR-93 directly targeted Sp7 by specifically binding to the target coding sequence region (CDS) of Sp7. Experiments such as electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP), and promoter luciferase reporter assay confirmed that Sp7 bound to the promoter of miR-93. Furthermore, overexpression of Sp7 reduced miR-93 transcription, whereas blocking the expression of Sp7 promoted miR-93 transcription. Our study showed that miR-93 was an important regulator in osteoblast mineralization and miR-93 carried out its function through a novel miR-93/Sp7 regulatory feedback loop. Our findings provide new insights into the roles of miRNAs in osteoblast mineralization. © 2012 American Society for Bone and Mineral Research.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

microRNAs (miRNAs) are small, functional, highly conserved, noncoding RNAs of 19 to 23 nucleotides that regulate the transcription of mRNAs in proteins.1, 2 miRNAs negatively regulate translation of specific mRNAs by base-pairing with partially or fully complementary sequences in target mRNAs to modulate diverse biological and cellular processes.3, 4

Recent studies have suggested that miRNAs play crucial roles in osteoblast differentiation.5 miR-133 and miR-135 impeded osteoblast differentiation by inhibiting the runt-related transcription factor 2 (Runx2) and SMAD family member 5 (Smad5) pathways that synergistically contributed to bone formation.6 miR-125b was reported to functionally inhibit proliferation of osteoblasts by attenuating v-erb-b2 erythroblastic leukemia viral oncogene homolog 2 (ErbB2) receptor tyrosine kinase.7 A network connecting Runx2, special AT-rich sequence binding protein 2 (SATB2), and the miR-23a∼27a∼24-2 cluster were verified to regulate the osteoblast differentiation program.8 Our previous studies also identified two new miRNAs (miR-2861/miR-3960 cluster) in mouse osteoblasts that promoted osteoblast differentiation by repressing histone deacetylase 5 (HDAC5) and homeobox A2 (Hoxa2) expression at the posttranscriptional level.9, 10 However, the functions of miRNAs in osteoblast mineralization need further investigation.

Matrix mineralization is a key stage in bone formation, which is a tightly regulated process. Bone-specific genes and signaling pathways are involved in the regulation of osteoblast mineralization.11, 12 Primary osteoblasts are able to form mineralized nodules in the process of terminal osteoblast differentiation.13 The present study was undertaken to investigate the role of target miRNA in the process of osteoblast mineralization in vitro.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

Cell cultures

We established primary mouse osteoblasts from calvaria of C57BL/6 mice as described.14 Mouse calvarial osteoblast cultures were grown in α-minimum essential medium (α-MEM; Invitrogen, Carlsbad, CA, USA) supplemented with 5% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 µg/mL streptomycin. For the induction of mineralization, osteoblasts were cultured in mineralization-inducing medium, α-MEM supplemented with 50 mg/L ascorbic acid (Invitrogen) and 10 mM β-glycerophosphate (β-GP) (Sigma Chemical Corp., St. Louis, MO, USA) for 21 days in a humidified 5% CO2 atmosphere at 37°C. The culture medium was changed every 2 days.

Microarray analysis of miRNAs

We used osteoblasts cultured in mineralization-inducing medium for 3, 14, and 21 days for miRNA array analysis. Small RNA was isolated using the mirVana miRNA Isolation Kit (Ambion, Inc., Austin, TX, USA) and sent to LC Sciences (http://www.lcsciences.com; Houston, TX, USA) for miRNA microarray screening, which contained probe sequences based on Sanger miRBase database release 16.0 (http://www.mirbase.org). Hybridization was detected by fluorescence labeling with tag-specific Cy3 and Cy5 dyes. Microarray procedures and data analysis were performed as described.15 For miRNA quantitation, the fold change at each time point was obtained by normalizing log2 fluorescence with log2 fluorescence of day 3. The normalized fold changes were analyzed by dChip software (http://dchip-surv.chenglilab.org/; Boston, MA, USA). A selected subset of miRNAs decreased more than twofold was selected for further analysis and the result was confirmed by real-time qPCR.

qRT-PCR analysis

See Supplemental Methods.

Northern blot

See Supplemental Methods.

Western blot

See Supplemental Methods.

Alizarin Red S staining

See Supplemental Methods.

Bioinformatics analysis

We used Rna22 (http://cbcsrv.watson.ibm.com/rna22.html) to predict sp7 coding sequence region (CDS) combined by miR-93. TargetScan (http://www.targetscan.org) was used to predict target genes of miR-93. TFSearch (http://www.cbrc.jp/research/db/TFSEARCH.html) was used to find transcription factors which can combine with the promoter of miR-93.

Lentiviral transduction

See Supplemental Methods.

Luciferase reporter assay

See Supplemental Methods.

Electrophoretic mobility shift assay (EMSA)

See Supplemental Methods.

Chromatin immunoprecipitation (ChIP)

See Supplemental Methods.

Statistical analyses

Data are presented as mean ± SD. Comparisons were made using a one-way analysis of variance. All experiments were repeated at least three times, and representative experiments are shown.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

Downregulation of microRNAs during osteoblast differentiation and mineralization

Osteoblasts were cultured in medium supplemented with 50 mg/L ascorbic acid and 10 mM β-glycerophosphate. To understand potential involved miRNAs in osteoblast mineralization, we analyzed the expression of miRNAs using an established microarray platform that contained probe sequences for 1040 mature mouse miRNAs.16. Fifty-six miRNAs were detected; five of them were upregulated obviously and eight of them were downregulated notably during the mineralization stage (Fig. 1A). Forty-three miRNAs showed changes below the set threshold. In the group of the miRNAs that were significantly expressed and statistically changed, miR-93 was found to be changed most obviously. qRT-PCR was used to verify the microarray result. Expression of mature miR-93 decreased about 4.5-fold from high levels on day 3 to minimum level on day 14 in the process, with a continuous low level during the mineralization process (Fig. 1B).

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Figure 1. miR-93 decreased most obviously during osteoblast mineralization. (A) A representative heat map of one sample of the repeated microarray expression analysis of genes significantly altered (n = 3; p < 0.01) in the process of osteoblast mineralization. Small RNA of osteoblasts during differentiation (3, 14, and 21 days) was used for miRNA microarray analysis. The color bar was extracted to show the color contrast level of the heat map. Red and green indicate high expression levels and low expression levels, respectively. For each capture probe the median of four background corrected replicas ± SD was calculated. Every experiment was normalized to the average of total signal intensity on each array. For comparative analysis, technical and biological replicas were averaged after normalization. Shown are parts of the miRNAs regulated more than twofold. (B) qRT-PCR analysis showed the time-dependent expression of miR-93 during osteoblasts differentiation and mineralization. miR-93 decreased about 4.5-fold from high levels at day 3 to minimum level at day 14 in osteoblast differentiation, with a continuous low level during the mineralization process. The level of miR-93 mRNA was normalized to U6 (n = 5).

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miR-93 overexpression inhibited osteoblast mineralization

The role of miR-93 in osteoblast mineralization was determined by overexpressing experiment. Osteoblasts were transfected with lentiviral pre-miR-93 on day 3 in the differentiation medium, and then Northern blot was performed to evaluate the level of miR-93 (Fig. 2A). Empty vector (miR-C) was used as a control to transfect osteoblasts. Mineralized matrix formation in osteoblasts were evaluated by Alizarin Red S staining after cultured for 14 and 21 days in differentiation medium (Fig. 2B). We found miR-93 overexpression rather than the control reduced the mineralized nodules of osteoblasts. These results indicated that overexpression of miR-93 inhibited mineralization of osteoblasts.

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Figure 2. Effects of miR-93 on osteoblast mineralization are shown. (A) Northern blot to verify the overexpression of miR-93 by lentivirus infection. U6 snRNA was used as loading control (n = 3). (B) Microscopic view (left) of effects of miR-93 overexpression on mineralization of the matrix. Osteoblasts infected by lentivirus were cultured in medium with 50 mg/L ascorbic acid and 10 mM β-glycerophosphate for 14 or 21 days. Mineralization of the matrix was determined by Alizarin Red S staining. Shown is a representative microscopic view at a magnification of ×200 for control and cells treated with lentivirus infection in cultures of 14 and 21 days. Quantification of Alizarin Red S stains (right) via extraction with cetyl-pyridinium chloride. The amount of released dye was quantified by spectrophotometry at 540 nm. The bars represent the mean ± SD (n = 5; *p < 0.05 versus control).

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The target mRNAs of miR-93

Hundreds of mRNAs were found to be putative targets of miR-93 by bioinformatics, such as Sp7, Eph receptor A4 (EPHA4), SMAD7, SACS, zinc finger protein of the cerebellum 1 (ZIC1), coagulation factor II (F2R), and family with sequence similarity 3, member C (FAM3C) (Supplemental Table 1). Among them, Sp7 has been reported to be an important regulator of osteoblast mineralization.

To understand the action of miR-93, we observed protein levels of Sp7 along with some representative targets such as SMAD7 and EPHA4 in osteoblasts with miR-93 transduction. Our results showed that overexpression of miR-93 reduced Sp7 protein levels, but not SMAD7 and EPHA4 protein levels (Fig. 3C).

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Figure 3. miR-93 directly targeted Sp7. (A) Schematic representing the putative target site of miR-93 in mouse Sp7 CDS and the base-pairing of miR-93 sequences with wild-type (WT) and mutant (MUT) CDS regions of Sp7. Three mutations are underlined. (B) miR-93 targeted the Sp7 CDS. Osteoblasts were cotransfected with the luciferase reporters carrying wild-type Sp7 CDS (WT-Sp7-CDS) or mutated Sp7 CDS (MUT-Sp7-CDS) and pre-miR-93 or miR-C for 48 hours. On the left, Northern blot analysis shows that miR-93 was overexpressed in osteoblasts infected with pre-miR-93 lentivirus. On the right, the bar chart shows the luciferase activities. Concurrent transfection of pre-miR-93 decreased the reporter activity of WT-Sp7-CDS but not the reporter activity of MUT-Sp7-CDS. The error bars represent the mean ± SD (*p < 0.05 versus MUT-Sp7-CDS; n = 5). (C) miR-93 repressed Sp7 expression posttranscriptionally. Osteoblasts were infected by pre-miR-93 or miR-C lentivirus and protein was extracted after culture for 48 hours. Western blot showed only Sp7 protein expression was reduced in the group by miR-93 overexpression. miR-93 overexpression resulted in an about threefold decrease in the amount of Sp7 protein. Results were indicated as the ratio of Sp7/β-actin. (D) Quantitative RT-PCR was used to determine the levels of Sp7 mRNA. There is no significant change in Sp7 mRNA level in response to miR-93 overexpression. The level of Sp7 mRNA was normalized to GAPDH. The error bars represent the mean ± SD (*p < 0.05 versus miR-C; n = 5).

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Levels of miR-93 inversely correlated with Sp7 expression during osteoblast differentiation and mineralization

Expression of Sp7 protein in osteoblasts increased gradually to the peak on day 14. Then the levels were maintained stably (Supplemental Fig. 1A, C). Compared with Sp7 protein level, Sp7 mRNA expression changed in a smaller extent (Supplemental Fig. 1B).

Results revealed that miR-93 was expressed at a relative high level on day 3, then decreased throughout osteoblast differentiation and mineralization (Fig. 1B). These results showed that the expression profile of miR-93 was reverse to that of Sp7 protein.

These results indicated that miR-93 was negatively correlated with Sp7 during osteoblast mineralization.

Sp7 is the target gene of miR-93

The computational algorithms, Rna22, have been developed to predict the putative miRNA targets.17 Rna22 can identify putative miRNA binding sites along the length of the entire mRNA transcripts and allow the G:U pair. One putative target site of the miR-93 seed sequence was predicted in the CDS of Sp7 (Fig. 3A). Therefore, we presumed that Sp7 was the target gene of miR-93.

To experimentally validate the assumption, we produced a luciferase reporter plasmid. The wild-type or mutated CDS of Sp7 was cloned into the 3′ untranslated region (3′-UTR) of the luciferase gene (Fig. 3A). Osteoblasts were cotransfected with the Sp7 luciferase expression vector (WT-pGL3-Sp7) or mutant CDS of Sp7 luciferase expression vector (MUT-pGL3-Sp7) and pre-miR-93 or miR-C. We found that the luciferase activity of the wild-type Sp7 CDS construct was significantly repressed by ectopic expression of miR-93 (Fig. 3B). A three-nucleotide mutation of the putative binding site in the Sp7 CDS (MUT-pGL3-Sp7) abolished this repression (Fig. 3B). Thus, these results suggested that miR-93 directly targeted Sp7 by specifically binding with the target CDS of Sp7.

Additionally, pre-miR-93 was introduced into osteoblasts to identify the function of miR-93 on Sp7 directly. Transfection of the pre-miR-93, rather than the control, resulted in a decreased Sp7 protein levels after 48 hours (Fig. 3C). However, the Sp7 mRNA levels were not affected (Fig. 3D). These results revealed that miR-93 posttranscriptionally repressed Sp7 protein expression.

We next cotransfected the WT or mutant Sp7 CDS construct with pre-miR-93 into osteoblasts to determine whether the biological effects of miR-93 could be rescued by the mutant Sp7 CDS construct. Osteoblasts were cotransfected with the WT or mutant Sp7 CDS construct and pre-miR-93 or miR-C. Western blotting showed that the mutant Sp7 CDS construct was able to rescue the pre-miR-93–induced downregulation of Sp7 protein (Fig. 4A). And results showed that the mutant Sp7 CDS construct was also able to rescue the pre-miR-93–induced suppression of osteoblast mineralization (Fig. 4B). This showed that miR-93 regulated osteoblast mineralization by targeting Sp7 and Sp7 was the very important target gene of miR-93 in osteoblast mineralization.

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Figure 4. The mutant Sp7 CDS construct rescued miR-93–induced suppression of osteoblast mineralization. Osteoblasts were cotransfected with the wild-type (WT) or mutant Sp7 CDS construct and pre-miR-93 or miR-C for 21 days. (A) Transfection of the mutant Sp7 into osteoblasts rescued the pre-miR-93–induced downregulation of Sp7 protein. (B) Transfection of mutant Sp7 into osteoblasts rescued the pre-miR-93–induced decrease of mineralized nodules. Data are shown as means ± SD (*p < 0.05 versus pre-miR-93 + WT-Sp7, n = 3).

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Sp7 binds to miR-93 promoter and inhibits transcription of miR-93

We identified the transcription start sites (TSSs) of miR-93 by examining the data established by Marson and colleagues.18 They identified TSSs using a published method,19 which was based on the knowledge that H3K4me3-modified nucleosomes occur near the TSSs of actively transcribed genes. They used ChIP coupled to DNA microarray analysis (ChIP-chip) to determine how nucleosomes with H3K4me3 are distributed across the entire genome, and find the TSS for miRNA. Thus, a library of TSS for mouse miRNA was established, and the miR-93 gene TSS was identified. miR-93 TSS was located on mouse chromosome 5 (Genbank: NC_000071.5). Then a potential binding site for Sp7 was found located just 1432 nucleotides upstream from the TSS of miR-93 by examining the regions 2 kb upstream of the miR-93 TSS using the TFSearch prediction program. The sequence CCCGCCCCCA was the binding site for Sp7 (Fig. 5A).

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Figure 5. Analysis of Sp7 binding to promoter of miR-93. (A) A Sp7 binding site was located upstream of miR-93. (B) EMSA for Sp7 binding to the promoter of the miR-93. EMSAs were performed using labeled oligonucleotide probes derived from the promoter of miR-93 of induced osteoblasts. The labeled oligonucleotide probes (WT Oligo 1) were incubated alone (lane 1), in combination with nuclear extracts (NE; lane 2), in the presence of 10- or 100-fold molar excess of specific unlabeled competitor probe (WT Oligo1) (comp; lanes 3 and 4) or unlabeled mutant (Mt) competitor probe (comp; mutant Oligo 1) (lanes 5 and 6), and in the presence of the Sp7 antibody (anti-Sp7) (Ab; lane 7) or IgG control antibody (Ab; lane 8) (n = 3). (C) Schematic representation of the promoter region of miR-93. The positions of the Sp7 binding site and primer sites for ChIP assays are indicated. (D) ChIP assay showed Sp7 binding to miR-93 promoter through the putative Sp7 binding site. ChIP assays were performed using no antibodies (input; lanes 1, 2, and 3), Sp7 antibodies (lanes 4, 5, and 6), and control IgG antibody (lane 7). Primer-B (−1948/−1789; lanes 2 and 5) and primer-C (−314/−155, lanes 3 and 6) were used as negative controls for PCR. (E) Functional activity of the Sp7 site in the −1432-bp mouse miR-93 promoter and its mutant constructs with luciferase reporter. The reporter constructs were cotransfected with control vector (gray bars) or Sp7 expression construct (solid black bars) in osteoblast cells. The error bars represent the mean ± S.D (*p < 0.05; n = 5).

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To validate whether Sp7 could physically bind with the putative binding sites, we carried out EMSA using nuclear extracts prepared from osteoblasts cultured with ascorbic acid and β-GP. Double-stranded oligonucleotides bearing the core sequence plus surrounding nucleotides (wild-type oligo D1) were synthesized, labeled, and used in EMSA (Fig. 5B). The EMSA experiment showed that the wild-type probes clearly formed a binding complex with Sp7 protein (Fig. 5B, lane 2). Furthermore, the specificity binding between the labeled wild-type D1 probe and Sp7 was not competed by a 1:10 or 1:100 excess of unlabeled mutant D1 probe (Fig. 5B, lanes 5 and 6). But an unlabeled wild-type probe competed with the binding complex in a dose-dependent manner (Fig. 5B, lanes 3 and 4). The supershift confirms the existence of Sp7 in the binding complex. It was shown by the formation of a supershift with specific anti-Sp7 antibody (Fig. 5B, lane 7), whereas immunoglobulin G (IgG) antibodies as a control had no such effect (Fig. 5B, lane 8).

ChIP assays were also performed to verify the possible association of the Sp7 protein with the promoter of the miR-93 in osteoblasts. Cross-linked and fragmented DNA-protein complexes were immunoprecipitated with Sp7 antibody, no antibody, or control IgG antibody. After immunoprecipitation, PCR analysis on purified DNA was performed with primer-A that spans Sp7 potential binding sites (−1441/−1432) in the promoter of miR-93. Primer-B and primer-C, which span the 5′- and 3′-distal regions of putative Sp7 binding sites, were used as negative controls in the PCR assays (Fig. 5C, D). The antibodies against Sp7 could specifically immunoprecipitate the DNA fragment containing the potential Sp7 binding sites (Fig. 5D, lane 4). These findings further confirmed that Sp7 bound to the putative binding sites of the miR-93 promoter.

To establish a direct link between Sp7 and transcriptional control of miR-93 expression, we cloned the promoter regions of miR-93 that contained the Sp7 binding site or Sp7 binding site mutation (Fig. 5E) into luciferase reporter and then cotransfected with full-length Sp7 or empty vector for promoter activity assays. As shown in Fig. 5E, we found that miR-93 promoter activities were significantly reduced by Sp7 when compared to empty vector control. In contrast, there was no significant difference in the promoter activities between the Sp7 binding site mutation-transfected and control cells (Fig. 5E). Altogether, these data suggested that Sp7 directly repressed transcription via physical binding resulting in decreased expression of miR-93.

To further study the effects of Sp7 levels on miR-93 gene expression, we changed the functional levels of Sp7 in osteoblasts by means of full-length Sp7 or short hairpin RNA (shRNA) against Sp7 lentivirus expression vectors. Empty vector or shRNA control was used as a control. It showed that miR-93 expression was reduced in the Sp7 overexpression group but increased in the shRNA-Sp7 group in osteoblasts (Fig. 6A, B). The overexpression or downregulation of Sp7 protein was confirmed by Western blot analysis compared with the control (Fig. 6A, B).

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Figure 6. Effects of Sp7 overexpression or knockdown on miR-93 expression. (A) Overexpression of Sp7 attenuated miR-93 expression. Osteoblasts were infected with Sp7 lentivirus (PAJ-Ubi-Sp7) or PAJ-C. (Top) Western blot showed that Sp7 protein levels were elevated after infection for 48 hours. Northern blot represented miR-93 expression levels in control or Sp7-overexpressing cells at 48 hours after infection. (Bottom) qRT-PCR 48 hours after Sp7 lentivirus transduction and the 3, 5, 7, 14, and 21 day of Sp7-overexpressing cells showed miR-93 decreased (*p < 0.05 versus miR-C; n = 5). (B) Knockdown of Sp7 promoted miR-93 expression. Osteoblasts were infected with sh-Sp7 lentivirus (PAJ-sh-Sp7) or shRNA control (sh-C) for 48 hours. (Top) Sp7 protein levels were detected using Western blot. Northern blot revealed miR-93 expression using total RNA isolated from osteoblasts at 48 hours after transfection. (Bottom) qRT-PCR 48 hours after sh-Sp7 lentivirus transduction and the 3, 5, 7, 14, and 21 day of Sp7-knockdown cells showed miR-93 increased (*p < 0.05 versus miR-C; n = 5).

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These results indicated that Sp7 could directly block the expression of miR-93 by binding to the putative binding site of the miR-93 promoter.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

Previous studies have identified plentiful miRNAs and factors participated in osteoblast differentiation, proliferation, and mineralization. Our study showed that miR-93 was involved in osteoblast mineralization and exerted its roles through interaction with the bone-specific factor Sp7.

miRNAs made contributions in osteoblast differentiation,20 but the mechanisms of their actions in osteoblast mineralization have not been clearly shown. In the present study, microarray analysis revealed that miR-93 exhibited maximal change during osteoblast mineralization. We then showed that the expression of miR-93 gradually decreased during osteoblast mineralization. It suggested that miR-93 played an important role in osteoblast mineralization. The functional role of miR-93 was further confirmed by overexpression experiments. Repression of mineralization occurred in the miR-93 overexpression group compared with the control. These results indicated that miR-93 regulated osteoblast mineralization, and then our study revealed the mechanism of this action.

Hundreds of mRNAs were found to be putative targets of miR-93 through bioinformatics. We briefly analyzed some of those with a better-known function and directly related to osteoblast differentiation and mineralization. Among them, Sp7 has been reported to be associated with osteoblast mineralization. Furthermore, our data showed that Sp7 decreased during osteoblast mineralization. Meanwhile, the CDS of Sp7 harbors a binding site for miR-93, suggesting that Sp7 might be a direct target of miR-93. We therefore evaluated the effect of miR-93 on Sp7.

Sp7 is a transcription factor playing a crucial role in osteoblast mineralization. As a member of the Sp family of zinc finger–containing transcription factors, Sp7 has been shown to be indispensable for the mineralization of osteoblasts in vivo.21–23 Sp7-deficient mice only form cartilage without bone formation.24 Moreover, Mikami and colleagues25 have reported Sp7 expression level was consistent with the degree of mineralization. Many pivotal genes in mineralization of the bone matrix such as collagen 1 (α1), bone sialoprotein, osteopontin, and osteocalcin were regulated by Sp7.26, 27 Furthermore, Baek and colleagues28 have concluded that Sp7 continues to be required for bone formation in growing and adult bones. Due to this critical regulation of the osteoblast phenotype by Sp7, identifying upstream regulators of this gene is the key to our understanding of osteoblast mineralization.

Our results showed that during osteoblast mineralization the level of miR-93 gradually reduced, which was reverse to that of Sp7. miRNAs carry out functions by negatively regulating translation of specific mRNAs.29 We explored the effects of miR-93 on Sp7. As a result, overexpression of miR-93 only decreased the expression of Sp7 protein but not mRNA, which suggested that miR-93 regulated osteoblast mineralization via suppressing Sp7 at the posttranscriptional level.

miRNAs perform the posttranscriptional inhibition of their target mRNAs mainly by base-pairing to the complementary sites in the 3′-UTR of the target mRNA.30 But recent studies have showed that miRNAs could exercise their control on mRNA by binding to the CDS region of transcription factors.31, 32 The CDS of Sp7 harbored a potential target of miR-93, which was predicted by Rna22. The repressive effect of miR-93 on the targeted site of Sp7 CDS was proven by luciferase reporter assay, and mutations of the target site in the Sp7 CDS abolished this repression. Besides, transfection of mutant Sp7 rescued the decreased mineralized nodules and the Sp7 protein level of osteoblasts induced by pre-miR-93. It confirmed that Sp7 was the targeted mRNA of miR-93.

Recently, Marson and colleagues18 predicted promoter sites and TSS for miRNA by use of the genomic coordinates of the H3K4me3-enriched loci derived from multiple cell types and bioinformatics analysis. The authors created a library of candidate promoters and TSS for miRNA in both the human and mouse genomes. In the present study, we predicted the promoter and TSS of miR-93 based on these published data.

We then examined the promoter region using the TFSearch prediction program and found a potential binding site (CCCGCCCCCA)33 for Sp7 just upstream of the miR-93 TSS. In our studies, osteoblasts transfected with Sp7 vector showed decreased miR-93 expression level, whereas block of Sp7 expression promoted miR-93 transcription. It verified that Sp7 acted as an inhibitor of miR-93 expression in osteoblast mineralization. Furthermore, some experiments, such as EMSA, ChIP, and promoter luciferase reporter assay confirmed that Sp7 reduced miR-93 expression through binding to the promoter regions of the miR-93. Therefore, we revealed that Sp7 protein levels were maintained in osteoblast mineralization via the combination between Sp7 and miR-93 promoter, which transcriptionally reduced the expression of miR-93.

These data allowed us to develop a model to show the miR-93 and Sp7 function loop (Fig. 7), which was a part of the complex regulatory network in osteoblast. During the process of osteoblast mineralization, Sp7 was a crucial factor that promoted osteoblast mineralization. First, Sp7 repressed the expression of miR-93, and then the downregulated miR-93 expression maintained the stable Sp7 protein levels by attenuating the posttranscriptional inhibition of miR-93 on Sp7. It suggested that a unique autoregulatory feedback loop existed between Sp7 and miR-93. Many miRNAs had been shown to mediate osteoblast metabolism. For instance, miR-133 and miR-135 target osteogenic transcription factors (Runx2) and Smad5, respectively, during bone morphogenic protein 2 (BMP2)-induced osteogenesis of C2C12 mesenchymal cells.8 miR-26a negatively regulated osteoblast differentiation by targeting the SMAD1 transcription factor.34 miR-206 regulated osteoblast differentiation by targeting Cx43.35 miR-93 and Sp7 worked along with those regulatory factors, and played their roles in osteoblast differentiation and mineralization.

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Figure 7. Graphic representation of autoregulatory feedback loop between Sp7 and miR-93. Sp7 was a crucial factor that promoted osteoblast mineralization. miR-93 represses the protein levels of Sp7. In turn, Sp7 transinactivates miR-93. They keep a relative balance in osteoblast mineralization.

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In conclusion, the present study provided evidence that the miR-93 played an important role in osteoblast mineralization through a novel miR-93/Sp7 regulatory feedback loop. Our findings also provide new insights into the roles and regulatory mechanisms of miRNAs in osteoblast mineralization.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

This work was supported by China National Funds for Distinguished Young Scientists grant 81125006, National Natural Science Foundation grant 30870925 and 81000122 from China, and Specialized Research Fund for the Doctoral program of High Education grant 20110162110038 from China.

Authors' roles: X-HL designed this experiment and wrote the paper. LY carried out it, analyzed the results, and wrote the paper. PC performed real-time PCR experiments. CC and H-BH carried out part of the bioinformatics analysis. G-QX helped with analysis of results. H-DZ instructed the cell cultures. Prof. Hui Xie and Xian-Ping Wu directed the luciferase reporter assay experiment and lentiviral transduction experiment.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgements
  9. References
  10. Supporting Information

Additional Supporting Information may be found in the online version of this article.

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jbmr_1621_sm_SupplMaterials.pdf292KSupplementary Materials

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