Regulation Of Osteogenic Differentiation Research Articles

Construction of an interactome network among circRNA-miRNA-mRNA reveals new biomarkers in hBMSCs osteogenic differentiation

Human bone marrow mesenchymal stem cells (hBMSCs) are adult stem cells residing in the bone marrow, characterized by their capacity for multi-directional differentiation, self-renewal, migration, and engraftment. Serving as seed cells, BMSCs play a pivotal role in the regeneration of bone defects. Hence, investigating the transcription factors and signaling pathways involved in the regulation of osteogenic differentiation in BMSCs holds significant importance. Recent research has unveiled that certain circular RNAs (circRNAs) can function as molecular sponges, influencing the osteogenic differentiation process of mesenchymal stem cells. However, many circRNAs remain undiscovered, and their precise mechanisms remain elusive. Therefore, the objective of this study is to construct an osteogenic differentiation-related circRNA-miRNA-mRNA network in hBMSCs. Subsequently, through bioinformatics analysis, we constructed a ceRNA network related to the osteogenic differentiation ability of hBMSCs, comprising 22 circRNAs, 17 miRNAs, and 15 mRNAs. The potential circRNA-miRNA-mRNA axes, including the role of hsa_circ_0001600 in promoting the osteogenic differentiation of hBMSCs through the targeted regulation of hsa-miR-542-3p, were validated through in vitro experiments.

Influence of budesonide and fluticasone propionate in the anti-osteoporotic potential in human bone marrow-derived mesenchymal stem cells via stimulation of osteogenic differentiation

Osteoporosis is a prevalent bone condition with adverse effects observed in patients undergoing long-term glucocorticoid therapy, resulting in bone demineralization and tissue loss. There has been limited studies on the global response to dexamethasone in terms of comparing its expression profile to other common glucocorticoids during osteogenic differentiation. This study focused on the downregulated gene expression profile of glucocorticoid compounds; dexamethasone, budesonide, and fluticasone propionate, during osteogenic differentiation to elucidate the related target genes and pathways associated with the anti-osteoporotic potential of telomerase-immortalized human bone marrow-derived mesenchymal stem cells using a bioinformatics approach. Based on gene expression microarrays experiments and bioinformatics analysis, several key genes involved in the regulation of osteogenic differentiation and osteoporosis development in mesenchymal stem cells that were targeted by these specific glucocorticoids were determined. Network analysis using GeneCards, OMIM, and CTD databases were performed and osteoporosis-related genes were identified. LIMMA and moderated Welch test R packages were performed to determine significant downregulated differentially expressed genes for each glucocorticoid treatment. A total of 479 (dexamethasone), 84 (budesonide), and 889 (fluticasone propionate) differentially expressed genes were identified for each glucocorticoid, of which 35 common genes overlapped. Enrichment pathway analysis was conducted using Metascape, and protein-protein interaction networks were constructed using the STRING database and Cytoscape software to determine potential target genes involved with osteoporosis. Enrichment pathway analysis revealed genes involved in 3 Reactome pathways namely cytokine signaling in immune system, immune system and the interferon alpha/beta signaling pathways and identified 10 hub genes based on the PPI network to determine potential target pathways associated with osteoporosis. These findings provide preliminary insights into the relationship between the key target genes of dexamethasone, budesonide, and fluticasone propionate, and the pathways associated with regulated osteoporosis metabolism during osteogenic differentiation.

Oncostatin M promotes osteogenic differentiation of tendon-derived stem cells through the JAK2/STAT3 signalling pathway

PurposeOncostatin M (OSM) is involved in the regulation of osteogenic differentiation and has a major role in the development of heterotopic ossification. The role of OSM in osteogenic differentiation of tendon-derived stem cells (TDSCs) and its mechanism have not been reported. This study aim to investigate the role of OSM in osteogenic differentiation of TDSCs and study the mechanism.MethodsTDSCs were differentiated in osteogenic differentiation medium for 7 days. Recombinant OSM was added to the osteogenic differentiation medium for 7 and 14 days. The effect of Janus kinase 2 (JAK2) inhibitor AZD1480 and signal transducer and activator of transcription 3 (STAT3) inhibitor stattic in the presence of recombinant OSM on osteogenic differentiation of TDSCs was examined after differentiation for 7 and 14 days. Alkaline phosphatase and alizarin red staining were used to assess the effects on early and mid-stage osteogenic differentiation, respectively. Western blotting and qPCR were used to assess the expression of receptor and signalling pathway-related proteins and osteogenic marker genes, respectively.ResultsTDSCs were successfully induced to differentiate into osteoblasts. Recombinant OSM promoted osteogenic differentiation of TDSCs to early and mid-stages. After addition of AZD1480 or stattic, decreased alkaline phosphatase and alizarin red staining were observed in the early and mid-stages of osteogenic differentiation. Additionally, decreased expression of receptor and pathway-related proteins, and osteogenic genes was found by western blotting and qPCR, respectively.ConclusionOSM promotes osteogenic differentiation of TDSCs and the JAK2/STAT3 signalling pathway plays an important role.

KLF9 regulates osteogenic differentiation of mesenchymal stem cells.

Osteoporosis is a progressive skeletal disease which is characterized by reduced bone mass and degradation of bone microstructure. Mesenchymal stem cells (MSCs) have the potential to inhibit osteoporosis since they are multipotent stem cells that can differentiate into multiple types of cells including osteoblasts. Hence the mechanism of osteogenic differentiation of MSCs deserves comprehensive study. Here we report that KLF9 is a novel regulator in osteogenic differentiation of MSCs. We observed that depletion of KLF9 can largely compromise the osteogenic differentiation ability of MSCs. In addition, we revealed that inhibition of the PI3K-Akt pathway could also affect osteogenic differentiation since KLF9 depletion inhibits PI3K expression. Finally, we discovered that KLF9 expression can be induced by dexamethasone which is an essential component in osteogenic induction medium. Taken together, our study provides new insights into the regulatory role of KLF9 in osteogenic differentiation of MSCs.

Enhancing mechanical property, osteogenesis and angiogenesis of 3D-plotted β-tricalcium phosphate bioceramic scaffolds incorporated with magnesium silicate: In Vitro and In Vivo study

The balance between porosity and compressive strength, as well as the pro-osteogenic and pro-angiogenesis effects of β-tricalcium phosphate (β-TCP) are not satisfactory in the clinical application even though it has good biocompatibility, osseointegration and osteoconductivity. Herein, different contents (10, 20 and 30 wt%) of magnesium silicate (Mg2SiO4, MS) and β-TCP composite bioceramic scaffolds (MS/β-TCP) were fabricated by three-dimensional fiber deposition (3DF) technology. The physicochemical, in vitro osteogenic and angiogenic properties, the early (6 weeks) cranium defect repair effects in rats and the underlying molecular mechanism on accelerating osteogenic differentiation of MS/β-TCP were systematically investigated. The results showed that MS compounding not only promoted the sintering of β-TCP and significantly enhanced the compressive strength, but also greatly improved its osteogenic and angiogenic performances, such as the viability, adhesion, and proliferation of mouse bone marrow mesenchymal stem cells (mBMSCs) and human umbilical vein endothelial cells (HUVECs), ALP activity and the expression of adhesion-related, osteogenesis-related and angiogenesis-related genes. The in vivo experiments presented that 10MS/β-TCP had more new bone formation and better angiogenesis compared to β-TCP and Blank. Analysis of the transcriptome sequencing results confirmed that the Wnt signaling pathway was involved in the regulation of osteogenic differentiation in mBMSCs by the MS/β-TCP composite bioceramic scaffold. The MS/β-TCP holds great potential in bone repair and regeneration.

Marginal effect of hydrostatic pressure on osteogenic differentiation of mesenchymal stem cells and its mechanism

Hydrostatic pressure (HP) is one of the important mechanical stimuli to maintain the structure and function of bone tissue. Under non-physiological conditions, abnormal increases or decreases in HP can lead to loss of bone tissue function. Although HP has been proven to affect osteogenic differentiation, the rules and mechanisms of its regulatory effect are still unclear. In this study, the HP showed a marginal effect on promoting osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). The regulation of osteogenic differentiation by HP is closely related to the Piezo1 ion channel. On the one hand, the activity of mTOR is inhibited by HP through the CaMKK2/AMPKα/mTOR signaling axis, thereby promoting the osteogenic differentiation. On the other hand, the cell morphology is also regulated by HP and tends to be round, which is not conducive to osteogenesis. The two regulatory modes jointly control the ultimate osteogenic differentiation ability of BMSCs in a complex way. This study provides new insights into the mechanism by which HP regulates osteogenic differentiation, suggesting the need to consider the role of signaling cascades and physical changes simultaneously.

Effects of tension on mitochondrial autophagy and osteogenic differentiation of periodontal ligament stem cells.

This study aimed to explore the osteogenic ability and mitochondrial autophagy of periodontal ligament stem cells (PDLSCs) under cyclic tensile stress (CTS). Primary PDLSCs were isolated from the periodontal membrane and cultured by passage. Alizarin red staining, alkaline phosphatase detection, reverse transcription polymerase chain reaction (RT-PCR), and Western blotting were used to detect the osteogenic differentiation level of PDLSCs. Mitochondrial autophagy in PDLSCs after CTS was measured using a mitochondrial autophagy detection kit, and the expression levels of autophagy-related proteins LC3B, LAMP1 and Beclin1 were measured using cellular immunofluorescence technology, RT-PCR and Western blot. After applying CTS, the osteogenic differentiation ability of PDLSCs was significantly improved, and the expression of alkaline phosphatase on the surface of the cell membrane and the formation of calcium nodules in PDLSCs were significantly increased respectively. We also studied the relevant mechanism of action and found that applying CTS can promote the osteogenic differentiation of PDLSCs and is related to the activation of mitochondrial autophagy. This study provides new insights into the mechanism of increased osteogenic differentiation on the tension side of orthodontic teeth and provides new experimental evidence for the involvement of mitochondrial autophagy in the regulation of osteogenic differentiation.

CK1ε drives osteogenic differentiation of bone marrow mesenchymal stem cells via activating Wnt/β-catenin pathway.

The treatment of bone defects is a difficult problem in orthopedics. At present, the treatment mainly relies on autologous or allogeneic bone transplantation, which may lead to some complications such as foreign body rejection, local infection, pain, or numbness at the bone donor site. Local injection of conservative therapy to treat bone defects is one of the research hotspots at present. Bone marrow mesenchymal stem cells (BMSCs) can self-renew, significantly proliferate, and differentiate into various types of cells. Although it has been reported that CK1ε could mediate the Wnt/β-catenin pathway, leading to the development of the diseases, whether CK1ε plays a role in bone regeneration through the Wnt/β-catenin pathway has rarely been reported. The purpose of this study was to investigate whether CK1ε was involved in the osteogenic differentiation (OD) of BMSCs through the Wnt/β-catenin pathway and explore the mechanism. We used quantitative reverse transcription-polymerase chain reaction (qRT-qPCR), Western blots, immunofluorescence, alkaline phosphatase, and alizarin red staining to detect the effect of CK1ε on the OD of BMSCs and the Wnt/β-catenin signaling pathway. CK1ε was highly expressed in BMSCs with OD, and our study further demonstrated that CK1ε might promote the OD of BMSCs by activating DLV2 phosphorylation, initiating Wnt signaling downstream, and activating β-catenin nuclear transfer. In addition, by locally injecting a CK1ε-carrying adeno-associated virus (AAV5- CK1ε) into a femoral condyle defect rat model, the overexpression of CK1ε significantly promoted bone repair. Our data show that CK1ε was involved in the regulation of OD by mediating Wnt/β-catenin. This may provide a new strategy for the treatment of bone defects.

Circ_0114581 promotes osteogenic differentiation of BMSCs via the MiR-155-5p/HNRNPA3 axis

Osteoporosis (OP) is a common metabolic bone disease characterized by deterioration of bone tissue structure, reduction of bone mass, and susceptibility to fracture. More and new suitable therapeutic targets need to be discovered. The purpose of this study was to explore the ceRNA mechanisms of circRNAs involved in osteoporosis. In this study, a competing endogenous RNA (ceRNA) regulatory network was obtained through the application of OP-related high throughput data sets. Our results provided evidence that HNRNPA3 was involved in the regulation of osteogenic differentiation in BMSCs. Testing of human bone tissues and ovariectomized mice bones proved that its expression level was negatively correlated with OP. The utilization of miRNA mimic or inhibitor proved that miR-155-5p could negatively regulate the expression of HNRNPA3, while overexpression of hsa_circ_0114581 with a circRNA overexpression vector proved that hsa_circ_0114581 could indirectly promoted HNRNPA3 expression and osteogenic differentiation by sponging hsa-miR-155-5p. A serious of luciferase reporter assay experiments further verified the binding site between miR-155-5p and HNRNPA3 and the binding site between miR-155-5p and hsa_circ_0114581. This study proved that the hsa_circ_0114581/hsa-miR-155-5p/HNRNPA3 axis was related with OP. The results reveal valuable insights into the pathogenesis of OP and noncoding RNA markers that may have a treatment role and will help to provide hypotheses for future studies.

Long non-coding RNA CASC2 regulates osteoblasts matrix mineralization.

Long non-coding RNAs (lncRNAs) are master regulators of gene expression and have recently emerged as potential innovative therapeutic targets. The deregulation of lncRNA expression patterns has been associated with age-related and noncommunicable diseases in the bone tissue, including osteoporosis and tumors. However, the specific role of lncRNAs in physiological or pathological conditions in the bone tissue still needs to be further clarified, for their exploitation as therapeutic tools. In the present study, we evaluate the potential of the lncRNA CASC2 as a regulator of osteogenic differentiation and mineralization. Results show that CASC2 expression is decreased during osteogenic differentiation of human bone marrow-derived Mesenchymal Stem/Stromal cells (hMSCs). CASC2 knockdown, using small interfering RNA against CASC2 (siCASC2), increases the expression of the late osteogenic marker Bone Sialoprotein (BSP), but does not impact ALP staining level nor the expression of early osteogenic transcripts, including RUNX2 and OPG. Although siCASC2 does not impact hMSC proliferation nor apoptosis, it promotes the mineralization of hMSC cultured under osteogenic-inducing conditions, as shown by the increase of calcium deposits. Mass spectrometry-based proteomic analysis revealed that 89 proteins are regulated by CASC2 at late osteogenic stages, including proteins associated with bone diseases or anthropometric and musculoskeletal traits. Specifically, the Cartilage Oligomeric Matrix Protein (COMP) is highly enhanced by CASC2 knockdown at late stages of osteogenic differentiation, at both transcriptional and protein level. On the other hand, inhibition of COMP impairs osteoblasts mineralization as well as the expression of BSP. The results indicate that lncRNA CASC2 regulates late osteogenic differentiation and mineralization in hMSC via COMP and BSP. In conclusion, this study suggests that targeting lncRNA CASC2 could be a potential approach for modulating bone mineralization.

Mesenchymal Stem Cell-Derived from Dental Tissues-Related lncRNAs: A New Regulator in Osteogenic Differentiation

Odontogenic stem cells are mesenchymal stem cells (MSCs) with multipotential differentiation potential from different dental tissues. Their osteogenic differentiation is of great significance in bone tissue engineering. In recent years, it has been found that long noncoding RNAs (lncRNAs) participate in regulating the osteoblastic differentiation of stem cells at the epigenetic level, transcriptional level, and posttranscriptional level. We reviewed the existing lncRNA related to the osteogenic differentiation of odontogenic stem cells and emphasized the critical mechanism of lncRNA in the osteogenic differentiation of odontogenic stem cells. These findings are expected to be an important target for promoting osteoblastic differentiation of odontogenic stem cells in bone regeneration therapy with lncRNA.

HSPB7 oppositely regulates human mesenchymal stromal cell-derived osteogenesis and adipogenesis

BackgroundRecent evidence suggests that accumulation of marrow adipose tissue induced by aberrant lineage allocation of bone marrow-derived mesenchymal stromal cells (BMSCs) contributes to the pathophysiologic processes of osteoporosis. Although master regulators of lineage commitment have been well documented, molecular switches between osteogenesis and adipogenesis are largely unknown.MethodsHSPB7 gene expression during osteogenic and adipogenic differentiation of BMSCs was evaluated by qPCR and Western blot analyses. Lentiviral-mediated knockdown or overexpression of HSPB7 and its deletion constructs were used to assess its function. The organization of cytoskeleton was examined by immunofluorescent staining. ALP activity, calcium assay, Alizarin Red S staining and Oil Red O staining were performed in vitro during osteoblast or adipocyte differentiation. SB431542 and Activin A antibody were used to identify the mechanism of Activin A in the regulation of osteogenic differentiation in BMSCs.ResultsIn this study, we identified HSPB7 capable of oppositely regulating osteogenic and adipogenic differentiation of BMSCs. HSPB7 silencing promoted adipogenesis while reducing osteogenic differentiation and mineralization. Conversely, overexpression of HSPB7 strongly enhanced osteogenesis, but no effect was observed on adipogenic differentiation. Deletion of the N-terminal or C-terminal domain of HSPB7 led to decreased osteoblastic potency and mineralization. Mechanistically, our data showed that Activin A is a downstream target participating in HSPB7 knockdown-mediated osteogenic inhibition.ConclusionsOur findings suggest that HSPB7 plays a positive role in driving osteoblastic differentiation, and with the capability in maintaining the osteo-adipogenesis balance. It holds great promise as a potential therapeutic target in the treatment of bone metabolic diseases.

Changes in N6-methyladenosine RNA methylomes of human periodontal ligament cells in response to inflammatory conditions.

To investigate the changes in the m6A methylation modification profile of human periodontal ligament cells (hPDLCs) in response to inflammatory conditions. Periodontitis is an infectious disease of the periodontal support tissue that leads to the loss of alveolar bone. HPDLCs are primary cells that can repair periodontal tissue defects caused by periodontitis. However, the inflammatory conditions induce inflammatory damage and decrease ossification of hPDLCs. This inflammatory response depends on genetic and epigenetic mechanisms, including m6A methylation. HPDLCs were cultured with osteogenic induction medium (NC group), while TNF-α (10ng/mL) and IL-1β (5ng/mL) were added to simulate inflammatory conditions (Inflam group). Then RNA-seq and MeRIP-seq analyses were performed to identify m6A methylation modification in the transcriptome range of hPDLCs. The results showed that the osteogenic differentiation of hPDLCs was inhibited under inflammatory conditions. RNA-seq analysis also revealed that the decreased genes in response to inflammatory conditions were primarily annotated in processes associated with ossification. Compared with the NC group, differentially m6A-methylated genes were primarily enriched in histone modification processes. Among 145 histone modification genes, 25 genes have been reported to be involved in the regulation of osteogenic differentiation, and they include KAT6B, EP300, BMI1, and KDMs (KDM1A, KDM2A, KDM3A, KDM4B, and KDM5A). This study demonstrated that the m6A landscape of hPDLCs was changed in response to inflammation. M6A methylation differences among histone modification genes may act on the osteogenic differentiation of hPDLCs.

High glucose impairs osteogenic differentiation of embryonic stem cells via early diversion of beta-catenin from Forkhead box O to T cell factor interaction.

Diabetes, which is characterized by an increase in blood glucose concentration, is accompanied by low bone turnover, increased fracture risk, and the formation of embryonic skeletal malformations. Yet, there are few studies elucidating the underlying alterations in signaling pathways leading to these osteogenic defects. We hypothesized here that bone formation deficiencies in a high glucose environment result from altered activity of beta-catenin (CTNNB1), a key contributor to osteogenic differentiation, dysregulation of which has also been implicated in the development of diabetes. To test this hypothesis, we used a previously established embryonic stem cell (ESC) model of differentiation that mimics the diabetic environment of the developing embryo. We differentiated murine ESCs within osteogenic-inducing media containing either high (diabetic) or low (physiological) levels of D-glucose and performed time course analyses to study the influence of high glucose on early and late bone cell differentiation. Endpoint measures for osteogenic differentiation were reduced in a glucose-dependent manner and expression of precursor-specific markers altered at multiple time points. Furthermore, transcriptional activity of the lymphoid enhancer factor (LEF)/T cell factor (TCF) transcription factors during precursor formation stages was significantly elevated while levels of CTNNB1 complexed with Forkhead box O 3a (FOXO3a) declined. Modulation of AKT, a known upstream regulator of both LEF/TCF and FOXO3a, as well as CTNNB1 rescued some of the reductions in osteogenic output seen in the high glucose condition. Within our in vitro model, we found a clear involvement of LEF/TCF and FOXO3a signaling pathways in the regulation of osteogenic differentiation, which may account for the skeletal deficiencies found in newborns of diabetic mothers.

CUL4B Upregulates RUNX2 to Promote the Osteogenic Differentiation of Human Periodontal Ligament Stem Cells by Epigenetically Repressing the Expression of miR-320c and miR-372/373-3p.

Mesenchymal stem cells (MSCs) within the periodontal ligament (PDL), termed periodontal ligament stem cells (PDLSCs), have a self-renewing capability and a multidirectional differentiation potential. The molecular mechanisms that regulate multidirectional differentiation, such as the osteogenic differentiation of PDLSCs, remain to be elucidated. Cullin 4B (CUL4B), which assembles the CUL4B-RING ubiquitin ligase (CRL4B) complex, is involved in regulating a variety of developmental and physiological processes including the skeletal development and stemness of cancer stem cells. However, nothing is known about the possible role of CUL4B in the osteogenic differentiation of PDLSCs. Here, we found that knockdown of CUL4B decreased the proliferation, migration, stemness and osteogenic differentiation ability of PDLSCs. Mechanistically, we demonstrate that CUL4B cooperates with the PRC2 complex to repress the expression of miR-320c and miR-372/373-3p, which results in the upregulation of RUNX2, a master transcription factor (TF) that regulates osteogenic differentiation. In brief, the present study reveals the role of CUL4B as a new regulator of osteogenic differentiation in PDLSCs.

Regulation of osteogenic differentiation by the pro-inflammatory cytokines IL-1β and TNF-α: current conclusions and controversies.

Treatment of complex bone fracture diseases is still a complicated problem that is urged to be solved in orthopedics. In bone tissue engineering, the use of mesenchymal stromal/stem cells (MSCs) for tissue repair brings hope to the medical field of bone diseases. MSCs can differentiate into osteoblasts and promote bone regeneration. An increasing number of studies show that the inflammatory microenvironment affects the osteogenic differentiation of MSCs. It is shown that TNF-α and IL-1β play different roles in the osteogenic differentiation of MSCs via different signal pathways. The main factors that affect the role of TNF-α and IL-1β in osteogenic differentiation of MSCs include concentration and the source of stem cells (different species and different tissues). This review in-depth analyzes the roles of pro-inflammatory cytokines in the osteogenic differentiation of MSCs and reveals some current controversies to provide a reference of comprehensively understanding.

Bioinformatics-Guided Analysis Uncovers AOX1 as an Osteogenic Differentiation-Relevant Gene of Human Mesenchymal Stem Cells.

Background: The available therapeutic options of bone defects, fracture nonunion, and osteoporosis remain limited, which are closely related to the osteogenic differentiation of bone marrow–derived mesenchymal stem cells (BMSCs). Thus, there remains an urgent demand to develop a prediction method to infer osteogenic differentiation–related genes in BMSCs. Method: We performed differential expression analysis between hBMSCs and osteogenically induced samples. Association analysis, co-expression analysis, and PPI analysis are then carried out to identify potential osteogenesis-related regulators. GO enrichment analysis and GSEA are performed to identify significantly enriched pathways associated with AOX1. qRT-PCR and Western blotting were employed to investigate the expression of genes on osteogenic differentiation, and plasmid transfection was used to overexpress the gene AOX1 in hBMSCs. Result: We identified 25 upregulated genes and 17 downregulated genes. Association analysis and PPI network analysis among these differentially expressed genes show that AOX1 is a potential regulator of osteogenic differentiation. GO enrichment analysis and GSEA show that AOX1 is significantly associated with osteoblast-related pathways. The experiments revealed that AOX1 level was higher and increased gradually in differentiated BMSCs compared with undifferentiated BMSCs, and AOX1 overexpression significantly increased the expression of osteo-specific genes, thereby clearly indicating that AOX1 plays an important role in osteogenic differentiation. Moreover, our method has ability in discriminating genes with osteogenic differentiation properties and can facilitate the process of discovery of new osteogenic differentiation–related genes. Conclusion: These findings collectively demonstrate that AOX1 is an osteogenic differentiation-relevant gene and provide a novel method established with a good performance for osteogenic differentiation-relevant genes prediction.

MiR-138-5p targets RUNX2 to inhibit osteogenic differentiation of aortic valve interstitial cells via Wnt/\u03b2-catenin signaling pathway

BackgroundHuman aortic valve interstitial cells (hAVICs) are a key factor in the pathogenesis of calcific aortic valve disease (CAVD). This research examines the role and mechanism of microRNA miR-138-5p in osteogenic differentiation of hAVICs.MethodsRT-qPCR analysis was applied for detecting miR-138-5p and RUNX2 expression in valve tissues of CAVD patients and controls. On completion of induction of osteogenic differentiation of hAVICs, and after overexpression or interference of miR-138-5p expression, the condition of osteogenic differentiation and calcification of hAVICs was confirmed using alkaline phosphatase staining and alizarin red staining. Subsequently, western blot was utilized to detect the expression of osteogenesis-related proteins OPN and ALP, and Wnt/β-catenin signaling pathway-related proteins. Finally, the relationship between miR-138-5p and RUNX2 was validated by dual-luciferase reporter assay and Pearson’s correlation test.ResultsDown-regulation of miR-138-5p was found in CAVD patients and during osteogenic differentiation of hAVICs. Overexpression of miR-138-5p contribute to the inhibition of osteoblast differentiation and calcium deposition in hAVICs, and of ALP and OPN protein expression. RUNX2 was a target gene of miR-138-5p, and it was negatively correlated with miR-138-5p in CAVD. Additionally, overexpression of RUNX2 could reverse the inhibitory effect of miR-138-5p on osteogenic differentiation of hAVICs.ConclusionmiR-138-5p can act as a positive regulator of osteogenic differentiation in CAVD patients to involve in inhibiting valve calcification, which is achieved through RUNX2 and Wnt/β-catenin signaling pathway.

TSG Targeting KDM5A Affects Osteogenic Differentiation of Bone Mesenchymal Stem Cells Induced by Bone Morphogenetic Protein 2.

Objectives To investigate the effect of 2,3,5,4′-tetrahydroxystilbene-2-O-β-D-glucoside (TSG) on the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and its molecular mechanism. Methods After TSG treatment of rBMSCs, alkaline phosphatase (ALP) activity was compared between the drug treatment group and the control group. The effects of TSG on alkaline phosphatase positive cloning and mineralized nodule formation were also detected. Total mRNA and protein were extracted, and the effects of TSG on the expression levels of osteopontin (OPN), osteocalcin (OCN), Runt-related transcription factor 2 (Runx2), Osterix, and Col1a1 were detected by real-time fluorescence quantitative PCR. Western blotting was used to detect the inhibitory effect of TSG on KDM5A. BMSCs were transfected with small interfering RNA (siRNA) targeting KDM5A (si-KMD5A) and pcDNA3.1 KMD5A. Results TSG significantly increased the activity of ALP, the number of alkaline phosphatase clones, and calcified nodule formation. The OPN, OCN, Runx2, and Osterix expression levels were significantly increased among the osteoblasts after TSG treatment. A mechanistic study showed that the effect of TSG is realized by inhibiting KDM5A. Conclusions KDM5A signaling may be involved in the regulation of osteogenic differentiation of rBMSCs. TSG can promote osteogenic differentiation and maturation of rBMSCs at 0.1–50 μmol/L. The mechanism of action was realized by inhibiting the expression of KDM5A.

Genome-Wide CRISPR/Cas9-Based Screening for Deubiquitinase Subfamily Identifies Ubiquitin-Specific Protease 11 as a Novel Regulator of Osteogenic Differentiation

The osteoblast differentiation capacity of mesenchymal stem cells must be tightly regulated, as inadequate bone mineralization can lead to osteoporosis, and excess bone formation can cause the heterotopic ossification of soft tissues. The balanced protein level of Msh homeobox 1 (MSX1) is critical during normal osteogenesis. To understand the factors that prevent MSX1 protein degradation, the identification of deubiquitinating enzymes (DUBs) for MSX1 is essential. In this study, we performed loss-of-function-based screening for DUBs regulating MSX1 protein levels using the CRISPR/Cas9 system. We identified ubiquitin-specific protease 11 (USP11) as a protein regulator of MSX1 and further demonstrated that USP11 interacts and prevents MSX1 protein degradation by its deubiquitinating activity. Overexpression of USP11 enhanced the expression of several osteogenic transcriptional factors in human mesenchymal stem cells (hMSCs). Additionally, differentiation studies revealed reduced calcification and alkaline phosphatase activity in USP11-depleted cells, while overexpression of USP11 enhanced the differentiation potential of hMSCs. These results indicate the novel role of USP11 during osteogenic differentiation and suggest USP11 as a potential target for bone regeneration.