Negative Regulator Of Osteogenesis Research Articles

CD34+ synovial fibroblasts exhibit high osteogenic potential in synovial chondromatosis.

Synovial chondromatosis (SC) is a disorder of the synovium characterized by the formation of osteochondral nodules within the synovium. This study aimed to identify the abnormally differentiated progenitor cells and possible pathogenic signaling pathways. Loose bodies and synovium were obtained from patients with SC during knee arthroplasty. Single-cell RNA sequencing was used to identify cell subsets and their gene signatures in SC synovium. Cells derived from osteoarthritis (OA) synovium were used as controls. Multi-differentiation and colony-forming assays were used to identify progenitor cells. The roles of transcription factors and signaling pathways were investigated through computational analysis and experimental verification. We identified an increased proportion of CD34+ sublining fibroblasts in SC synovium. CD34+CD31- cells and CD34-CD31- cells were sorted from SC synovium. Compared with CD34- cells, CD34+ cells had larger alkaline phosphatase (ALP)-stained area and calcified area after osteogenic induction. In addition, CD34+ cells exhibited a stronger tube formation ability than CD34- cells. Our bioinformatic analysis suggested the expression of TWIST1, a negative regulator of osteogenesis, in CD34- sublining fibroblasts and was regulated by the TGF-β signaling pathway. The experiment showed that CD34+ cells acquired the TWIST1 expression during culture and the combination of TGF-β1 and harmine, an inhibitor of Twist1, could further stimulate the osteogenesis of CD34+ cells. Overall, CD34+ synovial fibroblasts in SC synovium have multiple differentiation potentials, especially osteogenic differentiation potential, and might be responsible for the pathogenesis of SC.

Atf7ip Inhibits Osteoblast Differentiation via Negative Regulation of the Sp7 Transcription Factor

Epigenetic modifications are critical for cell differentiation and growth. As a regulator of H3K9 methylation, Setdb1 is implicated in osteoblast proliferation and differentiation. The activity and nucleus localization of Setdb1 are regulated by its binding partner, Atf7ip. However, whether Atf7ip is involved in the regulation of osteoblast differentiation remains largely unclear. In the present study, we found that Atf7ip expression was upregulated during the osteogenesis of primary bone marrow stromal cells and MC3T3-E1 cells, and was induced in PTH-treated cells. The overexpression of Atf7ip impaired osteoblast differentiation in MC3T3-E1 cells regardless of PTH treatment, as measured by the expression of osteoblast differentiation markers, Alp-positive cells, Alp activity, and calcium deposition. Conversely, the depletion of Atf7ip in MC3T3-E1 cells promoted osteoblast differentiation. Compared with the control mice, animals with Atf7ip deletion in the osteoblasts (Oc-Cre;Atf7ipf/f) showed more bone formation and a significant increase in the bone trabeculae microarchitecture, as reflected by μ-CT and bone histomorphometry. Mechanistically, Atf7ip contributed to the nucleus localization of Setdb1 in MC3T3-E1, but did not affect Setdb1 expression. Atf7ip negatively regulated Sp7 expression, and through specific siRNA, Sp7 knockdown attenuated the enhancing role of Atf7ip deletion in osteoblast differentiation. Through these data, we identified Atf7ip as a novel negative regulator of osteogenesis, possibly via its epigenetic regulation of Sp7 expression, and demonstrated that Atf7ip inhibition is a potential therapeutic measure for enhancing bone formation.

DNMT1 is a negative regulator of osteogenesis.

ABSTRACTThe role and underlying mechanisms of DNA methylation in osteogenesis/chondrogenesis remain poorly understood. We here reveal DNA methyltransferase 1 (DNMT1), which is responsible for copying DNA methylation onto the newly synthesized DNA strand after DNA replication, is overexpressed in sponge bone of people and mice with senile osteoporosis and required for suppression of osteoblast (OB) differentiation of mesenchymal stem cells (MSCs) and osteoprogenitors. Depletion of DNMT1 results in demethylation at the promoters of key osteogenic genes such as RORA and Fgfr2, and consequent upregulation of their transcription in vitro. Mechanistically, DNMT1 binds exactly to the promoters of these genes and are responsible for their 5-mc methylation. Conversely, simultaneous depletion of RORA or Fgfr2 blunts the effects of DNMT1 silencing on OB differentiation, suggesting RORA or Fgfr2 may be crucial for modulating osteogenic differentiation downstream of DNMT1. Collectively, these results reveal DNMT1 as a key repressor of OB differentiation and bone formation while providing us a new rationale for specific inhibition of DNMT1 as a potential therapeutic strategy to treat age-related bone loss.

Identification of a novel variant of the ciliopathic gene FUZZY associated with craniosynostosis

Craniosynostosis is a birth defect occurring in approximately one in 2000 live births, where premature fusion of the cranial bones inhibits growth of the skull during critical periods of brain development. The resulting changes in skull shape can lead to compression of the brain, causing severe complications. While we have some understanding of the molecular pathology of craniosynostosis, a large proportion of cases are of unknown genetic aetiology. Based on studies in mouse, we previously proposed that the ciliopathy gene Fuz should be considered a candidate craniosynostosis gene. Here, we report a novel variant of FUZ (c.851 G > C, p.(Arg284Pro)) found in monozygotic twins presenting with craniosynostosis. To investigate whether Fuz has a direct role in regulating osteogenic fate and mineralisation, we cultured primary osteoblasts and mouse embryonic fibroblasts (MEFs) from Fuz mutant mice. Loss of Fuz resulted in increased osteoblastic mineralisation. This suggests that FUZ protein normally acts as a negative regulator of osteogenesis. We then used Fuz mutant MEFs, which lose functional primary cilia, to test whether the FUZ p.(Arg284Pro) variant could restore FUZ function during ciliogenesis. We found that expression of the FUZ p.(Arg284Pro) variant was sufficient to partially restore cilia numbers, but did not mediate a comparable response to Hedgehog pathway activation. Together, this suggests the osteogenic effects of FUZ p.(Arg284Pro) do not depend upon initiation of ciliogenesis.

Aldo-keto reductase family 1 member C1 regulates the osteogenic differentiation of human ASCs by targeting the progesterone receptor

BackgroundAs a promising way to repair bone defect, bone tissue engineering has attracted a lot of attentions from researchers in recent years. Searching for new molecular target to modify the seed cells and enhance their osteogenesis capacity is one of the hot topics in this field. As a member of aldo-keto reductase family, aldo-keto reductase family 1 member C1 (AKR1C1) is reported to associate with various tumors. However, whether AKR1C1 takes part in regulating differentiation of adipose-derived mesenchymal stromal/stem cells (ASCs) and its relationship with progesterone receptor (PGR) remain unclear.MethodsLost-and-gain-of-function experiments were performed using knockdown and overexpression of AKR1C1 to identify its role in regulating osteogenic and adipogenic differentiation of hASCs in vitro. Heterotypic bone and adipose tissue formation assay in nude mice were used to conduct the in vivo experiment. Plasmid and siRNA of PGR, as well as western blot, were used to clarify the mechanism AKR1C1 regulating osteogenesis.ResultsOur results demonstrated that AKR1C1 acted as a negative regulator of osteogenesis and a positive regulator of adipogenesis of hASCs via its enzyme activity both in vitro and in vivo. Mechanistically, PGR mediated the regulation of AKR1C1 on osteogenesis.ConclusionsCollectively, our study suggested that AKR1C1 could serve as a regulator of osteogenic differentiation via targeting PGR and be used as a new molecular target for ASCs modification in bone tissue engineering.

Antidiuretic hormone inhibits osteogenic differentiation of dental follicle stem cells via V1a receptors and the PLC-IP3 pathway

ObjectiveThe aim of this study was to elucidate the molecular mechanism by which antidiuretic hormone (ADH) inhibited osteogenesis in dental follicle stem cells. DesignRat dental follicle stem cells were cultured in osteogenic differentiation medium supplemented with ADH. Alkaline phosphatase enzyme activity, Alizarin Red S staining, MTT assay and RT-qPCR was used to examine ADH’s impact on cell mineralization, viability, and osteogenic gene expression. Real-time calcium imaging analysis was performed to identify the ADH receptor and its mechanism of action. ResultsADH supplementation to the osteogenic differentiation medium inhibited cell mineralization without compromising cell viability and downregulated the expression of key osteogenic genes: DCN (Decorin), RUNX2 (Runt-related transcription factor 2) and BSP (Bone sialoprotein). Real-time calcium imaging analysis revealed that ADH (1−1000 nM) increased intracellular calcium in a concentration-dependent manner. Pretreatment of cells with V2255, a V1a receptor blocker, inhibited the calcium signals, but not with the V1b (Nelivaptan) or V2 (Tolvaptan). V2255 also reversed the inhibitory effect of ADH on osteogenesis. Furthermore, U73122, a Phospholipase C (PLC) inhibitor, 2-APB, an Inositol Triphosphate (IP3) receptor blocker, and depletion of endoplasmic reticulum calcium stores abolished the calcium signals by ADH. ConclusionsOur results demonstrated that ADH activates V1a receptors and the PLC-IP3 pathway to stimulate intracellular calcium signals, which inhibits cell mineralization and osteogenic gene expression. These findings uncovered a novel function for ADH as a negative regulator of osteogenesis in dental follicle stem cells. The role of ADH in the pathogenesis of bone diseases remains to be determined.

The construction of a novel xenograft bovine bone scaffold, (DSS)6-liposome/CKIP-1 siRNA/calcine bone and its osteogenesis evaluation on skull defect in rats.

BackgroundXenograft bone scaffolds have advantages such as mechanical strength, sufficient source and safety. Combined with siRNA properly targeting CKIP-1, a negative regulator of osteogenesis, may contribute to the repair result of calcine bone alone.MethodsHerein, we constructed a novel xenograft bovine bone scaffold namely (DSS)6-liposome/CKIP-1 siRNA/calcine bone, the characteristics of which were investigated by confirming the effect of (DSS)6-liposome, observing the appearance and testing mechanical strength of calcine bone, and observing the combined result of CKIP-1 siRNA by FAM immunofluorescence. In addition, cytotoxicity by CCK-8 and LDH activity of L929 ​cells and MC3T3-E1 osteoblasts cultured with the scaffold were tested in vitro, primary osteoblasts proliferation, the mRNA expressions of CKIP-1, ALP, COL1-α and OCN, the protein expressions of CKIP-1, BMP-2, COL-1 and Runx2 and calcium nodules were also determined by CCK-8, RT-qPCR, western-blot and Alizarin Red staining in vitro. Then, we successively established the skull defect model for evaluating the repair result of the novel scaffold by HE staining of 2, 4, 8 and 12 weeks, immumohistochemical stainings of 2, 4, 8 and 12 weeks such as ALP, COL-1α and OCN, Mirco-CT scanning of 4 and 12 weeks and the relative parameters and so on in vivo.ResultsIt indicated that (DSS)6-liposome/CKIP-1 siRNA/calcine bone could successfully knock down the CKIP-1 mRNA and protein expressions, promote osteoblasts proliferation with the little cytotoxicity in vitro, increase the protein expressions of BMP-2, COL-1 and Runx2 in vitro, increase mRNA expressions of ALP, COL-1α and OCN in vitro and in vivo, and have a better bone defect repair effect with few side effects in rats after 12 weeks.ConclusionOur research indicates (DSS)6-liposome/CKIP-1 siRNA/calcine bone could repair skull defects well in rats, and it may lay the foundation of applicating the novel xenograft bone scaffold in the clinical.The Translational potential of this articleThese findings provide evidence that (DSS)6- liposome/CKIP-1 siRNA/calcine bone could be used as a novel xenograft bone scaffold for osteogenesis with the good safety.

The Role of Melanotransferrin (CD228) in the regulation of the differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells (hBM-MSC).

Melanotransferrin (CD228), firstly reported as a melanoma-associated antigen, is a membrane-bound glycoprotein of an iron-binding transferrin homolog. CD228 was found to be expressed significantly higher in human bone marrow-derived mesenchymal stem cells (hBM-MSC) than in human embryonic fibroblasts (FB) by RT-PCR, western blotting and flow cytometry. The expression of CD228 declined in aged hBM-MSC as osteogenesis-related genes did. We examined a possible role for CD228 in the regulation of osteogenesis and adipogenesis of hBM-MSC. Surprisingly, siRNA-mediated CD228 knockdown increased the expression of the transcription factor DLX5 and enhanced osteogenesis of hBM-MSC evidenced by an increased expression of the runt-related transcription factor 2 (RUNX2), osterix (Osx), and osteocalcin (OC), as well as higher alkaline phosphatase (ALP) activity and extracellular calcium deposition. Interestingly, hBM-MSC transfected with CD228 siRNA also showed an increase in intracellular lipid level during adipogenesis, indicated by oil red O staining of differentiated adipocytes. Overall, our study unveils CD228 as a cell surface molecule expressed by young hBM-MSC, but not by FB. It also provides evidence to suggest a role for CD228 as a negative regulator of osteogenesis and of lipid accumulation during adipogenesis in hBM-MSC in vitro.

MiR-137 knockdown promotes the osteogenic differentiation of human adipose-derived stem cells via the LSD1/BMP2/SMAD4 signaling network.

MicroRNAs are a group of endogenous regulators that participate in several cellular physiological processes. However, the role of miR-137 in the osteogenic differentiation of human adipose-derived stem cells (hASCs) has not been reported. This study verified a general downward trend in miR-137 expression during the osteogenic differentiation of hASCs. MiR-137 knockdown promoted the osteogenesis of hASCs in vitro and in vivo. Mechanistically, inhibition of miR-137 activated the bone morphogenetic protein 2 (BMP2)-mothers against the decapentaplegic homolog 4 (SMAD4) pathway, whereas repressed lysine-specific histone demethylase 1 (LSD1), which was confirmed as a negative regulator of osteogenesis in our previous studies. Furthermore, LSD1 knockdown enhanced the expression of BMP2 and SMAD4, suggesting the coordination of LSD1 in the osteogenic regulation of miR-137. This study indicated that miR-137 negatively regulated the osteogenic differentiation of hASCs via the LSD1/BMP2/SMAD4 signaling network, revealing a new potential therapeutic target of hASC-based bone tissue engineering.

MiR-384-5p Targets Gli2 and Negatively Regulates Age-Related Osteogenic Differentiation of Rat Bone Marrow Mesenchymal Stem Cells.

Aberrant microRNA expression correlates with age-related osteoporosis, which impairs bone formation by regulating osteoblastic activity, thus leading to age-related bone loss. In this study, we observed that miR-384-5p was significantly upregulated in bone marrow mesenchymal stem cells (BMSCs) from aged rats compared with BMSCs from young rats. In vitro functional assays revealed that overexpression of miR-384-5p in young BMSCs inhibited osteogenic differentiation and accelerated senescence, whereas knockdown of miR-384-5p in aged BMSCs had the opposite effects. Furthermore, we demonstrated that miR-384-5p inhibited the expression of Gli2 at both the mRNA and protein levels by directly binding to the 3' untranslated region of Gli2 mRNA. The osteogenic capacity of Gli2-knockdown BMSCs was rejuvenated by miR-384-5p inhibition. Finally, in vivo assays showed that the inhibition of miR-384-5p prevented bone loss and increased the osteogenic capacity in aged rats. Overall, our study suggests that miR-384-5p functions as a negative regulator of osteogenesis, indicating that the inhibition of miR-384-5p may be a therapeutic strategy against age-related bone loss.

MiR-877-3p promotes TGF-β1-induced osteoblast differentiation of MC3T3-E1 cells by targeting Smad7.

MicroRNAs (miRNAs) are emerging as important regulators of various physiological and pathological processes and may serve key roles in the maintenance of bone homeostasis via effects on osteoblast differentiation. The aim of the present study was to define the role of miR-877-3p in osteoblast differentiation using MC3T3-E1 cells, an osteoblast precursor cell line. It was demonstrated using RT-qPCR analysis that miR-877-3p was gradually increased in MC3T3-E1 cells during the osteoblastic differentiation induced by transforming growth factor (TGF)-β1. Gain-of-function and loss-of-function experiments revealed that the overexpression of miR-877-3p promoted the osteoblastic differentiation of MC3T3-E1 cells, whereas depletion of miR-877-3p inhibited this process in vitro and in vivo. Bioinformatics analysis and validation experiments demonstrated that Smad7, which acts as a negative regulator of osteogenesis, was a target of miR-877-3p. Furthermore, the overexpression of Smad7 partially reversed the osteoblastic differentiation of MC3T3-E1 cells induced by miR-877-3p. In conclusion, the results of the present study suggest that the miR-877-3p/Smad7 axis is associated with the osteoblastic differentiation of MC3T3-E1 cells and may indicate a potential therapeutic approach for osteogenesis disorders.

MicroRNA-23a inhibits osteogenesis of periodontal mesenchymal stem cells by targeting bone morphogenetic protein signaling

ObjectivesTo investigate the role of microRNA-23a (miR-23a) in the osteogenesis of periodontal mesenchymal stem cells (PDLSCs) in periodontitis. Materials and methodsGingival crevicular fluid samples were collected from 21 control subjects and 29 patients with chronic periodontitis. MiR-23a was determined by quantitative real-time PCR. PDLSCs were transfected with miR-23a overexpressing lentiviruses. Subsequently, PDLSCs were induced with osteogenic differentiation media. Osteogenic differentiation of PDLSCs was assessed by alkaline phosphatase activity assay, Alizarin red staining, and qRT-PCT detection of osteogenic gene expression. Western blot was performed to detect the protein levels of the SMAD family member 1/5/9 (Smad1/5/9) and their phosphorylation level. TargetScan was used to predict the target gene of miR-23a. Cotransfections of bone morphogenetic protein receptor type 1B (BMPR1B) and miR-23a applied to explore the relationship between BMPR1B and miR-23a. ResultsMiR-23a was significantly increased in PDLSCs and gingival crevicular fluid of periodontitis patients. Patients with gingival crevicular fluid miR-23a levels above a threshold showed more clinical indicators of periodontitis. After periodontal therapy, miR-23a levels significantly decreased. Overexpression miR-23a inhibited osteogenesis of PDLSCs, which was evidenced by reduced Alizarin Red S and osteogenic gene expressions. In addition, miR-23a inhibited the phosphorylation of Smad1/5/9. TargetScan predicted that BMPR1B is a target gene of miR-23a. Overexpression of BMPR1B abolished the effects caused by overexpression of miR-23a. ConclusionOur study provides novel evidence that miR-23a acts as a negative regulator of osteogenesis in periodontitis patients’PDLSCs and that miR-23a may serve as a biomarker and potential target of periodontitis.

MicroRNA‑223 promotes osteoblast differentiation of MC3T3‑E1 cells by targeting histone deacetylase 2.

MicroRNAs (miRNAs) have emerged as pivotal regulators in various physiological and pathological processes at the post‑transcriptional level, and may serve important roles in osteogenic differentiation. However, their roles and functions are not fully understood. In the present study, miR‑223‑5p was identified as a modulator of osteoblastic differentiation in MC3T3‑E1 cells. Reverse transcription‑quantitative polymerase chain reaction and western blotting demonstrated that miR‑223‑5p was significantly upregulated in MC3T3‑E1 cells following the induction of osteoblast differentiation. Overexpression of miR‑223‑5p promoted osteogenic differentiation both invitro and vivo. Expression of histone deacetylase2 (HDAC2), which acts as a negative regulator of osteogenesis, was regulated by miR‑223‑5p. Collectively, the results of the present study revealed a novel miR‑223‑5p/HDAC2 axis that regulates osteoblast differentiation, and may serve as a potential target for enhancing bone formation invivo.

The Forkhead Box C1, a Novel Negative Regulator of Osteogenesis, Plays a Crucial Role in Odontogenic Differentiation of Dental Pulp Stem Cells.

The forkhead box C1 (Foxc1) protein, a member of the forkhead/winged helix transcription factor family, is required in stem cell developmental processes. Recently, multiple studies have indicated the crucial role of Foxc1 in mesenchymal stem cell differentiation, but the precise effects and mechanisms on dental pulp stem cells (DPSCs) remain unclear. In this study, we evaluate the role of Foxc1 on the odontogenic differentiation and proliferation of DPSCs. Our results show that Foxc1 decreases time dependently in odontogenic differentiation of DPSCs. Meanwhile, overexpression of Foxc1 could significantly inhibit the mineralization of DPSCs and the expression of odontogenic-related genes, such as runt-related transcription factor 2 (Runx2), dentin sialophosphoprote (DSPP), and dentin matrix acidic phosphoprotein 1 (DMP-1). Foxc1 overexpression does not significantly alter the proliferation of DPSCs. In addition, Foxc1 reduces the expression of p-Smad1/5, an important modulator of bone morphogenetic protein (BMP)/Smad signaling pathway, inhibiting BMP/Smad signaling pathway. In conclusion, our data demonstrated that Foxc1 inhibits odontogenic differentiation of DPSCs and odontogenic-related gene expression through the BMP/Smad signaling pathway which may be useful for the dental regeneration and repair.

Nanotopographical cues of electrospun PLLA efficiently modulate non-coding RNA network to osteogenic differentiation of mesenchymal stem cells during BMP signaling pathway

Application of stem cells in combination with nanofibrous substrates is an interesting biomimetic approach for enhanced regeneration of damaged tissues such as bone and cartilage. The investigation of the complex interplay between nanotopographical cues of niche and noncoding RNAs in stem cells fate is an effective tool to find a new strategy for enhancing the induction of osteogenesis. In this study, we investigated the effects of aligned and random orientations of nanofibers as a natural ECM-mimicking environment on the network of noncoding RNA in mesenchymal stem cells. Aligned and randomly oriented Ploy (L-lactide) PLLA scaffolds were fabricated via electrospinning. Human Adipose Tissue-Derived Mesenchymal Stem Cells (hASCs) were isolated from adipose tissue and were cultured on surfaces of these scaffolds. Their capacity to support hMSCs proliferation was also investigated by MTT assay and the expression of c-Myc gene. Then, after 7, 14 and 21 days, the osteogenic commitment of hMSCs and the miRNA regulatory network in BMP signaling pathway were evaluated by measuring alkaline phosphatase (ALP) activity, extracellular calcium deposition, and bone-related gene activation by Real-Time PCR. Furthermore, osteogenic differentiation was evaluated with regard to their noncoding RNA network. Our results for the first time showed an interaction between nanotopographical cues and miRNA activity in hMSCs. We found that the nanotopographical cues could be used to influence the osteogenic differentiation process of hMSCs through the modulation of lncRNAs and miR-125b as negative regulators of osteogenesis as well as the H19 modulator BMP signaling pathway that acts as a miRNA sponge. Moreover, we also demonstrated for the first time that MEG3 as a long noncoding RNA is controlled by miR-125b and microRNA-triggered lncRNA decay mechanism. This strategy seems to be an important tool for controlling stem cell fate in engineered tissues and provide new insights into most biocompatible scaffolds for bone-graft substitutes.

Linc-ROR Promotes Osteogenic Differentiation of Mesenchymal Stem Cells by Functioning as a Competing Endogenous RNA for miR-138 and miR-145

Long noncoding RNAs (lncRNAs), which serve as important and powerful regulators of various biological activities, have gained widespread attention in recent years. Emerging evidence has shown that some lncRNAs play important regulatory roles in osteoblast differentiation of mesenchymal stem cells (MSCs), suggesting a potential therapeutic strategy for bone fracture. As a recently identified lncRNA, linc-ROR was reported to mediate the reprogramming ability of differentiated cells into induced pluripotent stem cells (iPSCs) and human embryonic stem cells (ESCs) self-renewal. However, other functions of linc-ROR remain elusive. In this study, linc-ROR was found to be upregulated during osteogenesis of human bone-marrow-derived MSCs. Ectopic expression of linc-ROR significantly accelerated, whereas knockdown of linc-ROR suppressed, osteoblast differentiation. Using bioinformatic prediction and luciferase reporter assays, we demonstrated that linc-ROR functioned as a microRNA (miRNA) sponge for miR-138 and miR-145, both of which were negative regulators of osteogenesis. Further investigations revealed that linc-ROR antagonized the functions of these two miRNAs and led to the de-repression of their shared target ZEB2, which eventually activated Wnt/β-catenin pathway and hence potentiated osteogenesis. Taken together, linc-ROR modulated osteoblast differentiation by acting as a competing endogenous RNA (ceRNA), which may shed light on the functional characterization of lncRNAs in coordinating osteogenesis.

Identification of novel microRNA inhibiting actin cytoskeletal rearrangement thereby suppressing osteoblast differentiation.

We report the role of miR-1187 in regulation of osteoblast functions. Over-expression of miR-1187 inhibited osteoblast differentiation. Target prediction analysis tools and experimental validation by luciferase 3' UTR reporter assay identified BMPR-II and ArhGEF-9 as direct targets of miR-1187. ArhGEF-9 activates Cdc42 which has a major role in actin reorganization. BMP-2 also induces actin polymerization. Role of miR-1187 in actin reorganization was determined by western blotting, immunofluorescence, and in vivo gene silencing studies. Reduced protein levels of BMPR-II, activated Cdc42, and downstream signaling molecules were observed in miR-1187-transfected osteoblasts. miR-1187 over-expression resulted in decreased actin polymerization. Additionally, P-cofilin, which does not bind F-actin, was decreased in miR-1187-transfected cells. These results were corroborated by administration of BMPR-II exogenously in miR-1187-transfected osteoblasts. Silencing of miR-1187 in neonatal mice mitigated all the inhibitory effects of miR-1187 on actin cytoskeletal rearrangement. Importantly, in vivo treatment of miR-1187 inhibitor to ovariectomized BALB/c mice led to significant improvement in trabecular bone microarchitecture. Overall, miR-1187 functions as a negative regulator of osteogenesis by repressing BMPR-II and ArhGEF-9 expression thus suppressing non-Smad BMP2/Cdc42 signaling pathway and inhibiting actin reorganization. miR-1187 functions as a negative regulator of osteogenesis by repressing BMPR-II expression, which in turn, suppresses non-Smad BMP2/Cdc42 signaling pathway, thus inhibiting actin cytoskeletal rearrangement. Silencing of miR-1187 significantly improves trabecular bone microarchitecture. As miR-1187 exerts a negative regulatory role in osteoblasts function, hence, we propose that therapeutic approaches targeting miR-1187 could be useful in enhancing the bone formation and treatment of pathological conditions of bone loss.

MicroRNA Regulation in Osteogenic and Adipogenic Differentiation of Bone Mesenchymal Stem Cells and its Application in Bone Regeneration.

Bone mesenchymal stem cells (BMSCs) are multipotent stromal cells providing a useful cell source for treating bone diseases and metabolic disorders. BMSCs fate determination and lineage progression are controlled by multiple cytokines, transcriptional factors, signaling pathways, and microRNAs (miRNAs). MiRNAs are small non-coding RNAs that inhibit the posttranscriptional gene expression or degrade their targets. They are closely involved in controlling the key steps of osteogenesis and adipogenesis of BMSCs. We aim to summarize the roles of miRNAs and their pathways in regulating osteogenic and adipogenic differentiation of BMSCs, and sketch its preliminary applications in bone regeneration. We reviewed the published literature about the microRNA regulation in osteogenic and adipogenic differentiation of BMSCs. Most of miRNAs are expressed in BMSCs, perform as negative regulators of osteogenesis and have bidirectional effects on adipogenesis. Runx2 and PPARγ are two key transcriptional factors in osteogenesis and adipogenesis, respectively. Anti-miRNAs or miRNA mimics is potential therapeutic strategy to repress pathological miRNAs for cellular therapies to bone diseases. The preliminary applications of miRNAs in BMSCs strongly suggested their bright future in bone regeneration.

MiR-503 Promotes Bone Formation in Distraction Osteogenesis through Suppressing Smurf1 Expression

Distraction osteogenesis (DO) is a unique technique for promoting bone formation in clinical practice. However the underlying mechanism remains elusive. As epigenetic mediators, microRNAs have been reported to play important roles in regulating osteogenesis. In this study, after successfully established the DO model of rats, a microRNA microarray was performed to find molecular targets for DO. Total 100 microRNAs were identified as differently expressed, with miR-503 being one of the most significantly up-regulated miRNAs in DO. The further investigation also showed that miR-503 was upregulated during osteogenesis in mesenchymal stem cells of rats, and overexpression of miR-503 significantly promoted osteogenesis in vitro and accelerated mineralization in DO process in vivo. By using bioinformatic investigations and luciferase activities, we successfully demonstrated that Smurf1, a negative regulator of osteogenesis, was a real target of miR-503. Furthermore, Smurf1 knockdown promoted osteogenesis and antagomir-503 abolished the promotive effect, suggesting that miR-503 mediated osteogenic differentiation via suppressing Smurf1 expression. To sum up, these findings indicated that miR-503 promoted osteogenesis and accelerated bone formation, which may shed light on the development for a potential therapeutic target for bone repair.

MiR-5100 promotes osteogenic differentiation by targeting Tob2.

MicroRNAs have emerged as pivotal regulators in various physiological and pathological processes, including osteogenesis. Here we discuss the contribution of miR-5100 to osteoblast differentiation and mineralization. We found that miR-5100 was upregulated during osteoblast differentiation in ST2 and MC3T3-E1 cells. Next, we verified that miR-5100 can promote osteogenic differentiation with gain-of-function and loss-of-function experiments. Target prediction analysis and experimental validation demonstrated that Tob2, which acts as a negative regulator of osteogenesis, was negatively regulated by miR-5100. Furthermore, we confirmed that the important bone-related transcription factor osterix, which can be degraded by binding to Tob2, was influenced by miR-5100 during osteoblast differentiation. Collectively, our results revealed a new molecular mechanism that fine-tunes osteoblast differentiation through miR-5100/Tob2/osterix networks.