Modulation of osteoblastogenesis by NRF2: NRF2 activation suppresses osteogenic differentiation and enhances mineralization in human bone marrow-derived mesenchymal stromal cells.

Mesenchymal stromal stem cells (MSCs) or skeletal stem cells (SSCs) play a major role in tissue repair due to their robust ability to differentiate into osteoblasts, chondrocytes, and adipocytes. Complex cell signaling cascades tightly regulate this differentiation. In osteogenic differentiation, Runt-related transcription factor 2 (RUNX2) and ALP activity are essential. Furthermore, during the latter stages of osteogenic differentiation, mineral formation mediated by the osteoblast occurs with the secretion of a collagenous extracellular matrix and calcium deposition. Activation of nuclear factor erythroid 2-related factor 2 (NRF2), an important transcription factor against oxidative stress, inhibits osteogenic differentiation and mineralization via modulation of RUNX2 function; however, the exact role of NRF2 in osteoblastogenesis remains unclear. Here, we demonstrate that NRF2 activation in human bone marrow-derived stromal cells (HBMSCs) suppressed osteogenic differentiation. NRF2 activation increased the expression of STRO-1 and KITLG (stem cell markers), indicating NRF2 protects HBMSCs stemness against osteogenic differentiation. In contrast, NRF2 activation enhanced mineralization, which is typically linked to osteogenic differentiation. We determined that these divergent results were due in part to the modulation of cellular calcium flux genes by NRF2 activation. The current findings demonstrate a dual role for NRF2 as a HBMSC maintenance factor as well as a central factor in mineralization, with implications therein for elucidation of bone formation and cellular Ca2+ kinetics, dystrophic calcification and, potentially, application in the modulation of bone formation.

SCUBE3 promotes osteogenic differentiation and mitophagy in human bone marrow mesenchymal stem cells through the BMP2/TGF-ß signaling pathway.

In clinical settings, addressing large bone defects remains a significant challenge for orthopedic surgeons. The use of genetically modified bone marrow mesenchymal stem cells (BMSCs) has emerged as a highly promising approach for these treatments. Signal peptide-CUB-EGF domain-containing protein 3 (SCUBE3) is a multifunctional secreted glycoprotein, the role of which remains unclear in human hBMSCs. This study used various experimental methods to elucidate the potential mechanism by which SCUBE3 influences osteogenic differentiation of hBMSCs in vitro. Additionally, the therapeutic efficacy of SCUBE3, in conjunction with porous GeLMA microspheres, was evaluated in vivo using a mouse bone defect model. Our findings indicate that SCUBE3 levels increase significantly during early osteogenic differentiation of hBMSCs, and that reducing SCUBE3 levels can hinder this differentiation. Overexpressing SCUBE3 elevated osteogenesis gene and protein levels and enhanced calcium deposition. Furthermore, treatment with recombinant human SCUBE3 (rhSCUBE3) protein boosted BMP2 and TGF-ß expression, activated mitophagy in hBMSCs, ameliorated oxidative stress, and restored osteogenic function through SMAD phosphorylation. In vivo, GELMA/OE treatment effectively accelerated bone healing in mice. In conclusion, SCUBE3 fosters osteogenic differentiation and mitophagy in hBMSCs by activating the BMP2/TGF-ß signaling pathway. When combined with engineered hydrogel cell therapy, it could offer valuable guidance for the clinical management of extensive bone defects.

Omaveloxolone ameliorates glucocorticoid-induced osteonecrosis of the femoral head by promoting osteogenesis and angiogenesis.

Steroid (glucocorticoid)-induced necrosis of the femoral head (SONFH) represents a prevalent, progressive, and challenging bone and joint disease characterized by diminished osteogenesis and angiogenesis. Omaveloxolone (OMA), a semi-synthetic oleanocarpane triterpenoid with antioxidant, anti-inflammatory, and osteogenic properties, emerges as a potential therapeutic agent for SONFH. This study investigates the therapeutic impact of OMA on SONFH and elucidates its underlying mechanism. The in vitro environment of SONFH cells was simulated by inducing human bone marrow mesenchymal stem cells (hBMSCs) and human umbilical vein endothelial cells (HUVECs) using dexamethasone (DEX).Various assays, including CCK-8, alizarin red staining, Western blot, qPCR, immunofluorescence, flow cytometry, and TUNNEL, were employed to assess cell viability, STING/NF-κB signaling pathway-related proteins, hBMSCs osteogenesis, HUVECs migration, angiogenesis, and apoptosis. The results demonstrate that OMA promotes DEX-induced osteogenesis, HUVECs migration, angiogenesis, and anti-apoptosis in hBMSCs by inhibiting the STING/NF-κB signaling pathway. This experimental evidence underscores the potential of OMA in regulating DEX-induced osteogenesis, HUVECs migration, angiogenesis, and anti-apoptosis in hBMSCs through the STING/NF-κB pathway, thereby offering a promising avenue for improving the progression of SONFH.

Notoginsenoside R1 promotes osteogenic differentiation of human bone marrow mesenchymal stem cells via ERα/GSK-3ß/ß-catenin signalling pathway.

Human bone marrow mesenchymal stem cells (hBMSCs) are attractive therapeutic agents for bone tissue regeneration owing to their osteogenic differentiation potential. Notoginsenoside R1 (NGR1) is a novel phytoestrogen with diverse pharmacological activities. Here, we probed whether NGR1 has an effect on the osteogenic differentiation of hBMSCs. EdU, CCK-8 and Transwell assays were used to measure proliferation and migration of hBMSCs after treatment with different doses of NGR1. hBMSCs were treated with osteogenic differentiation induction medium for osteogenesis. Alizarin red S (ARS) and alkaline phosphatase (ALP) staining were used to measure mineralized nodule formation and ALP activity in hBMSCs, respectively. ICI 182780, an antagonist of oestrogen receptor alpha (ERα) was used to inhibit ERα expression. The results showed that NGR1 enhanced hBMSC proliferation and migration. NGR1 increased ALP activity and mineralized nodule formation as well as promoting ALP, RUNX2 and OCN expression in hBMSCs. NGR1 enhanced ERα expression and promoted GSK-3ß/ß-catenin signal transduction in hBMSCs. ICI 182780 reversed NGR1-mediated activation of the GSK-3ß/ß-catenin signalling and promoted an effect on hBMSC behaviour. Thus NGR1 promotes proliferation, migration and osteogenic differentiation of hBMSCs via the ERα/GSK-3ß/ß-catenin signalling pathway.

MFG-E8 promotes osteogenic differentiation of human bone marrow mesenchymal stem cells through GSK3ß/ß-catenin signaling pathway.

Fracture nonunion and bone defects are challenging for orthopedic surgeons. Milk fat globule-epidermal growth factor 8 (MFG-E8), a glycoprotein possibly secreted by macrophages in a fracture hematoma, participates in bone development. However, the role of MFG-E8 in the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) is unclear. We investigated the osteogenic effect of MFG-E8 in vitro and in vivo. The CCK-8 assay was used to assess the effect of recombinant human MFG-E8 (rhMFG-E8) on the viability of hBMSCs. Osteogenesis was investigated using RT-PCR, Western blotting, and immunofluorescence. Alkaline phosphatase (ALP) and Alizarin red staining were used to evaluate ALP activity and mineralization, respectively. An enzyme-linked immunosorbent assay was conducted to evaluate the secretory MFG-E8 concentration. Knockdown and overexpression of MFG-E8 in hBMSCs were established via siRNA and lentivirus vector transfection, respectively. Exogenous rhMFG-E8 was used to verify the in vivo therapeutic effect in a tibia bone defect model based on radiographic analysis and histological evaluation. Endogenous and secretory MFG-E8 levels increased significantly during the early osteogenic differentiation of hBMSCs. Knockdown of MFG-E8 inhibited the osteogenic differentiation of hBMSCs. Overexpression of MFG-E8 and rhMFG-E8 protein increased the expression of osteogenesis-related genes and proteins and enhanced calcium deposition. The active ß-catenin to total ß-catenin ratio and the p-GSK3ß protein level were increased by MFG-E8. The MFG-E8-induced enhanced osteogenic differentiation of hBMSCs was partially attenuated by a GSK3ß/ß-catenin signaling inhibitor. Recombinant MFG-E8 accelerated bone healing in a rat tibial-defect model. In conclusion, MFG-E8 promotes the osteogenic differentiation of hBMSCs by regulating the GSK3ß/ß-catenin signaling pathway and so, is a potential therapeutic target.

MiR-26b-5p/TET3 regulates the osteogenic differentiation of human bone mesenchymal stem cells and bone reconstruction in female rats with calvarial defects.

BACKGROUND: MicroRNAs (miRNAs) play critical roles in the osteogenic differentiation of human bone mesenchymal stem cells (hBMSCs), but the mechanism by which miRNAs indirectly modulate osteogenesis remains unclear. Here, we explored the mechanism by which miRNAs indirectly modulate gene expression through histone demethylases to promote bone regeneration. METHODS AND RESULTS: Bioinformatics analysis was performed on hBMSCs after 7 days of osteogenic induction. The differentially expressed miRNAs were screened, and potential target mRNAs were identified. To determine the bioactivity and stemness of hBMSCs and their potential for bone repair, we performed wound healing, Cell Counting Kit-8 (CCK-8), real-time reverse transcription quantitative polymerase chain reaction (RT‒qPCR), alkaline phosphatase activity, alizarin red S (ARS) staining and radiological and histological analyses on SD rats with calvarial bone defects. Additionally, a dual-luciferase reporter assay was utilized to investigate the interaction between miR-26b-5p and ten-eleven translocation 3 (TET3) in human embryonic kidney 293T cells. The in vitro and in vivo results suggested that miR-26b-5p effectively promoted the migration, proliferation and osteogenic differentiation of hBMSCs, as well as the bone reconstruction of calvarial defects in SD rats. Mechanistically, miR-26b-5p bound to the 3' untranslated region of TET3 mRNA to mediate gene silencing. CONCLUSIONS: MiR-26b-5p downregulated the expression of TET3 to increase the osteogenic differentiation of hBMSCs and bone repair in rat calvarial defects. MiR-26b-5p/TET3 crosstalk might be useful in large-scale critical bone defects.

Circular RNA CircSLC8A1 contributes to osteogenic differentiation in hBMSCs via CircSLC8A1/miR-144-3p/RUNX1 in periprosthetic osteolysis.

Circular RNAs (circRNAs) are often found in eukaryocyte and have a role in the pathogenesis of a variety of human disorders. Our related research has shown the differential expression of circRNAs in periprosthetic osteolysis (PPOL). However, the involvement of circRNAs in the exact process is yet unknown. CircSLC8A1 expression was evaluated in clinical samples and human bone marrow mesenchymal stem cells (hBMSCs) in this investigation using quantitative real-time PCR. In vitro and in vivo studies were conducted to explicate its functional role and pathway. We demonstrated CircSLC8A1 is involved in PPOL using gain- and loss-of-function methods. The association of CircSLC8A1 and miR-144-3p, along with miR-144-3p and RUNX1, was predicted using bioinformatics. RNA pull-down and luciferase assays confirmed it. The impact of CircSLC8A1 in the PPOL-mouse model was also investigated using adeno-associated virus. CircSLC8A1 was found to be downregulated in PPOL patients' periprosthetic tissues. Overexpression of CircSLC8A1 promoted osteogenic differentiation (OD) and inhibited apoptosis of hBMSCs in vitro. The osteogenic markers of RUNX1, osteopontin (OPN) and osteocalcin (OCN) were significantly upregulated in hBMSCs after miR-144-3p inhibitor was transferred. Mechanistic analysis demonstrated that CircSLC8A1 directly bound to miR-144-3p and participated in PPOL through the miR-144-3p/RUNX1 pathway in hBMSCs. Micro-CT and quantitative analysis showed that CircSLC8A1 markedly inhibited PPOL, and osteogenic markers (RUNX1, OPN and OCN) were significantly increased (P<0.05) in the mice model. Our findings prove that CircSLC8A1 exerted a regulatory role in promoting osteogenic differentiation in hBMSCs, and CircSLC8A1/miR-144-3p/RUNX1 pathway may provide a potential target for prevention of PPOL.

Decreased CRISPLD2 expression impairs osteogenic differentiation of human mesenchymal stem cells during in vitro expansion.

Human mesenchymal stem cells (hMSCs) are the cornerstone of regenerative medicine; large quantities of hMSCs are required via in vitro expansion to meet therapeutic purposes. However, hMSCs quickly lose their osteogenic differentiation potential during in vitro expansion, which is a major roadblock to their clinical applications. In this study, we found that the osteogenic differentiation potential of human bone marrow stem cells (hBMSCs), dental pulp stem cells (hDPSCs), and adipose stem cells (hASCs) was severely impaired after in vitro expansion. To clarify the molecular mechanism underlying this in vitro expansion-related loss of osteogenic capacity in hMSCs, the transcriptome changes following in vitro expansion of these hMSCs were compared. Cysteine-rich secretory protein LCCL domain-containing 2 (CRISPLD2) was identified as the most downregulated gene shared by late passage hBMSCs, hDPSCs, and hASCs. Both the secreted and non-secreted CRISPLD2 proteins progressively declined in hMSCs during in vitro expansion when the cells gradually lost their osteogenic potential. We thus hypothesized that the expression of CRISPLD2 is critical for hMSCs to maintain their osteogenic differentiation potential during in vitro expansion. Our studies showed that the knockdown of CRISPLD2 in early passage hBMSCs inhibited the cells' osteogenic differentiation in a siRNA dose-dependent manner. Transcriptome analysis and immunoblotting indicated that the CRISPLD2 knockdown-induced osteogenesis suppression might be attributed to the downregulation of matrix metallopeptidase 1 (MMP1) and forkhead box Q1 (FOXQ1). Furthermore, adeno-associated virus (AAV)-mediated CRISPLD2 overexpression could somewhat rescue the impaired osteogenic differentiation of hBMSCs during in vitro expansion. These results revealed that the downregulation of CRISPLD2 contributes to the impaired osteogenic differentiation of hMSCs during in vitro expansion. Our findings shed light on understanding the loss of osteogenic differentiation in hMSCs and provide a potential therapeutic target gene for bone-related diseases.

Injectable platelet-rich fibrin positively regulates osteogenic differentiation of stem cells from implant hole via the ERK1/2 pathway.

Bone regeneration in dentistry is a dynamic approach for treating critical size bone defects that are unlikely to self-heal. Human bone marrow stem cell (hBMSCs) therapies are being tested clinically for various disorders and have remarkable clinical advancements in bone regeneration. Injectable platelet-rich fibrin (i-PRF), which is obtained from autologous blood centrifuged at 700 rpm (60 G) for 3 min can promote osteogenic differentiation of this cell, but the mechanism remains unclear. The objectives of this study were to explore the contents of i-PRF further and investigate its effect on the cell behavior of hBMSCs and the underlying molecular mechanisms. The results showed that i-PRF contained 41 cytokines, including macrophage colony-stimulating factor (M-CSF) and ß-nerve growth factor (ß-NGF), which had not been reported before. The Cell Counting Kit-8 and wound healing assay showed that 10% and 20% i-PRF improved the proliferation rate and the migration capacity of hBMSCs without toxicity to cells. Besides, the expression of osteogenic markers and the capacity to form mineralized nodules of hBMSCs were promoted by 20% i-PRF. Furthermore, i-PRF activated the ERK pathway, and the ERK inhibitor attenuated its effects. In summary, i-PRF promotes hBMSCs proliferation and migration and facilitates cell osteogenesis through the ERK pathway, which has promising potential in bone regeneration.

What is the context? Bone defects caused by trauma or tumor is a great challenge in clinical practice. However, there is the good news that the bone defect in the oral can self-regenerate, the bone remodeling may take several months to several years and shows apparent individual differences.Different strategies, surgical techniques, and materials have been employed to induce an optimal outcome in guided bone regeneration.Blood products have been widely used in dentistry due to their excellent biocompatibility, growth factor content, ease of collection, and ability to be produced by the human body.Limited data suggest that Injectable platelet-rich fibrin positively regulates osteogenic differentiation of stem cells, but further evidence is needed to quantify this effect.What is new? It is unclear how many growth factors i-PRF contains in previous studies, so we detected 41 kinds of growth factors, more than has been previously appreciated, and found that all growth factors were measured in the samples, and the difference was in the amount of expression.In our research, we explored the role of i-PRF in the osteogenesis of hBMSCs through the effects of different concentrations of i-PRF on the proliferation, migration, and osteogenic differentiation of hBMSCs.Currently, most current research focuses on observing phenomena, and we wondered by what mechanism the i-PRF regulates stem cell function. We found that i-PRF can regulate the molecular mechanism of the osteogenic differentiation of hBMSCs in vitro by activating the MAPK/ERK signaling pathway.What is the impact?I-PRF promotes hBMSCs proliferation and migration and facilitates cell osteogenesis through the ERK pathway. The favorable cytobiological effects of i-PRF on hBMSCs might be the basis for i-PRF applications in bone regenerative.

Calcium phytate reverses high glucose-inhibited osteogenesis of BMSCs via the MAPK/JNK pathway.

OBJECTIVES: Diabetes mellitus (DM) induces oxidative tissue impairment and suppresses bone formation. Some studies have shown that phytic acid has antioxidant and anti-diabetic properties. This study aimed to investigate the potential of calcium phytate (Ca-phytate) to reverse inhibited osteogenesis of human bone marrow mesenchymal stem cells (hBMSCs) in a high glucose (HG) environment and to determine the underlying mechanism. MATERIALS AND METHODS: hBMSCs were exposed to HG and palmitic acid to simulate DM in vitro. Osteogenic differentiation was measured using alkaline phosphatase staining and activity assay, alizarin red S staining, qRT-PCR, Western blot and immunofluorescence staining. A critical-size cranial defect model of type 2 diabetes mellitus (T2DM) rats was established to evaluate bone regeneration. A specific pathway inhibitor was used to explore whether the MAPK/JNK pathway was involved. RESULTS: Treatment with 34 µM Ca-phytate had the highest effect on osteogenic differentiation in HG. Ca-phytate improved cranial bone defect healing in T2DM rats. The long-term HG environment inhibited the activation of the MAPK/JNK signalling pathway, which was restored by Ca-phytate. Blocking the JNK pathway reduced the Ca-phytate-mediated osteogenic differentiation of hBMSCs. CONCLUSION: Ca-phytate induced bone regeneration in vivo and reversed HG-inhibited osteogenesis of hBMSCs in vitro via the MAPK/JNK signalling pathway.

DPA promotes hBMSCs osteogenic differentiation by miR-9-5p/ERK/ALP signaling pathway.

Docosahexaenoic acid (DHA) has been reported potentiate osteogenic differentiation, while Docosapentaenoic acid (DPA), another Omega-3 fatty acid, its contribution to the osteogenic differentiation of human bone-marrow-derived mesenchymal stromal cells (hBMSCs) is not entirely elucidated. The Alizarin Red S (ARS) staining and the expression of osteogenesis­associated genes were analyzed during osteogenic induction by DPA. Then, bioinformatics analysis and dual luciferase reporter assays were investigated to confirm the interactions between miR-9-5p and alkaline phosphatase (ALP). miR-9-5p mimics / inhibitor were transfected to human hBMSCs and the osteogenic assay above was also performed. Furthermore, DPA significantly promoted the phosphorylation of ERK via miR-9-5p. PD98059, a highly specific and potent ERK1/2 inhibitor, inhibited the activation of ALP and partially reversed the role of DPA during osteogenic differentiation. These data indicated that DPA promoted osteogenic differentiation of hBMSCs potentially through miR-9-5p/ERK/ALP signaling pathway, providing a potentially useful therapeutic strategy for patients to improve bone loss.

Deubiquitinating Enzyme USP7 Is Required for Self-Renewal and Multipotency of Human Bone Marrow-Derived Mesenchymal Stromal Cells.

Ubiquitin-specific protease 7 (USP7) is highly expressed in a variety of malignant tumors. However, the role of USP7 in regulating self-renewal and differentiation of human bone marrow derived mesenchymal stromal cells (hBMSCs) remains unknown. Herein, we report that USP7 regulates self-renewal of hBMSCs and is required during the early stages of osteogenic, adipogenic, and chondrogenic differentiation of hBMSCs. USP7, a deubiquitinating enzyme (DUB), was found to be downregulated during hBMSC differentiation. Furthermore, USP7 is an upstream regulator of the self-renewal regulating proteins SOX2 and NANOG in hBMSCs. Moreover, we observed that SOX2 and NANOG are poly-ubiquitinated and their expression is downregulated in USP7-deficient hBMSCs. Overall, this study showed that USP7 is required for maintaining self-renewal and multipotency in cultured hBMSCs. Targeting USP7 might be a novel strategy to preserve the self-renewal capacity of hBMSCs intended for stem cell therapy.

MiR-138-5p knockdown promotes osteogenic differentiation through FOXC1 up-regulation in human bone mesenchymal stem cells.

This study examined the hypothesis that the microRNA miR-138-5p reduces the osteodifferentiation of human bone mesenchymal stem cells (hBMSCs) by downregulating the expression of forkhead box C1 (FOXC1). For this, hBMSCs were separated from bone marrow and osteogenic induction medium was added to stimulate osteogenic differentiation. Flow cytometric analysis was applied to evaluate the expression of cell-surface antigens associated with hBMSCs, including CD29, CD44, CD90, CD45, and CD34. qRT-PCR assays and Western blot assays were used to measure the mRNA and protein expression of miR-138-5p, osteocalcin, runt-related transcription factor 2, bone sialoprotein, alkaline phosphatase (ALP), and FOXC1. ALP staining assays and Alizarin Red staining (ARS) assays were used to confirm osteogenic differentiation. We used a luciferase assay to test the interaction between miR-138-5p and FOXC1. We demonstrated that miR-138-5p is downregulated in osteogenic differentiated hBMSCs. Further, overexpression of miR-138-5p reduced the expression of markers for osteodifferentiation, ALP activity, and ARS activity. Furthermore, we showed that FOXC1 is a downstream target gene of miR-138-5p, and that knockdown of miR-138-5p improves the osteogenesis differentiation of hBMSCs by upregulating FOXC1. The results from this study indicate miR-138-5p as a new target for osteogenic differentiation of hBMSCs and the treatment of bone defects.

Circ_FBLN1 promotes the proliferation and osteogenic differentiation of human bone marrow-derived mesenchymal stem cells by regulating let-7i-5p/FZD4 axis and Wnt/ß-catenin pathway.

BACKGROUND: Recently, more and more circular RNAs (circRNAs) have been identified in osteogenesis. In this study, we aimed to explore the effect of circ_FBLN1 on the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs). METHODS: The protein levels of osteogenesis-related genes, let-7i-5p, frizzled class receptor 4 (FZD4), Ki67, Wnt6 and ß-catenin were measured by western blot assay. The levels of circ_FBLN1, FBLN1 mRNA and FZD4 mRNA were determined by quantitative real-time polymerase chain reaction (qRT-PCR) assay. The feature of circ_FBLN1 was investigated by RNase R and Actinomycin D assays. Cell proliferation ability was evaluated by colony formation assay and 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assay. The targeting relationship between let-7i-5p and circ_FBLN1 or FZD4 was verified by dual-luciferase reporter assay. RESULTS: Circ_FBLN1 level was enhanced during the osteogenic differentiation of hBMSCs. Silencing of circ_FBLN1 repressed cell proliferation and osteogenic differentiation in hBMSCs. For mechanism analysis, circ_FBLN1 was found to act as a sponge for let-7i-5p and FZD4 served as a direct target gene of let-7i-5p. Let-7i-5p was downregulated during the osteogenic differentiation of hBMSCs and let-7i-5p inhibition restored the effects of circ_FBLN1 knockdown on the proliferation and osteogenesis of hBMSCs. Moreover, let-7i-5p overexpression suppressed cell proliferation and osteogenesis in hBMSCs through targeting FZD4. In addition, circ_FBLN1 knockdown reduced the levels of Wnt6 and ß-catenin in hBMSCs, indicating the inactivation of Wnt/ß-catenin pathway. CONCLUSION: Knockdown of circ_FBLN1 inhibited the proliferation and osteogenesis of hBMSCs by regulating let-7i-5p/FZD4 axis and repressing Wnt/ß-catenin pathway.

LncRNA SNHG14 promotes osteogenic differentiation of human bone marrow-derived mesenchymal stem cells via regulating miR-185-5p/WISP2 axis.

Osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs) is vital for bone formation, and its dysfunction is linked to osteoporosis (OP). In this work, we explored the function of long non-coding RNA (lncRNA) small nucleolar RNA host gene 14 (SNHG14) in regulating osteogenic differentiation of hBMSCs. In the present study, the expression of SNHG14 in hBMSCs obtained from OP patients was measured by quantitative real-time polymerase chain reaction (qRT-PCR). SNHG14 was over-expressed or knocked down in hBMSCs, and the expression levels of OP-related genes (ALP, OCN, and OPN) in hBMSCs were detected by qRT-PCR and Western blot. StarBase database and miRanda database were used to predict the binding sites between SNHG14 and miR-185-5p, and between miR-185-5p and 3'UTR of WNT1 inducible signaling pathway protein 2 (WISP2), respectively. Luciferase reporter gene assay was used to validate the binding relationship between SNHG14 and miR-185-5p, and miR-185-5p and 3'UTR of WISP2, respectively. Here, we report that SNHG14 was significantly down-regulated in hBMSCs obtained from patients with OP. Overexpression of SNHG14 promoted osteogenic differentiation, while knockdown of SNHG14 worked oppositely. Mechanistically, miR-185-5p was demonstrated to be a target of SNHG14, and could reverse the function of SNHG14. Additionally, WISP2 was identified as a target gene of miR-185-5p in hBMSCs and could be indirectly regulated by SNHG14. Taken together, down-regulation of SNHG14 in hBMSCs accelerated the progression of OP via regulating miR-185-5p/WISP2 axis.

Bioinformatics analysis of the biological changes involved in the osteogenic differentiation of human mesenchymal stem cells.

The mechanisms underlying the osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) remain unclear. In the present study, we aimed to identify the key biological processes during osteogenic differentiation. To this end, we downloaded three microarray data sets from the Gene Expression Omnibus (GEO) database: GSE12266, GSE18043 and GSE37558. Differentially expressed genes (DEGs) were screened using the limma package, and enrichment analysis was performed. Protein-protein interaction network (PPI) analysis and visualization analysis were performed with STRING and Cytoscape. A total of 240 DEGs were identified, including 147 up-regulated genes and 93 down-regulated genes. Functional enrichment and pathways of the present DEGs include extracellular matrix organization, ossification, cell division, spindle and microtubule. Functional enrichment analysis of 10 hub genes showed that these genes are mainly enriched in microtubule-related biological changes, that is sister chromatid segregation, microtubule cytoskeleton organization involved in mitosis, and spindle microtubule. Moreover, immunofluorescence and Western blotting revealed dramatic quantitative and morphological changes in the microtubules during the osteogenic differentiation of human adipose-derived stem cells. In summary, the present results provide novel insights into the microtubule- and cytoskeleton-related biological process changes, identifying candidates for the further study of osteogenic differentiation of the mesenchymal stem cells.

Circular RNA circ_0011269 sponges miR-122 to regulate RUNX2 expression and promotes osteoporosis progression.

Circular RNAs (circRNAs) are a novel class of noncoding RNAs that are widely expressed in human disease. However, circRNAs expression profile and potential mechanism in osteoporosis pathogenesis remain to be further studied. In the present study, a total of 69 circRNAs were identified to be abnormally expressed in osteoporosis patient samples by microarray and bioinformatics analyses. We found that circ_0011269 was notably downregulated in osteoporosis (fold change, 3.94). By means of miRanda algorithm, we constructed the interaction network of circ_0011269-miRNAs in osteoporosis based on target binding and miR-122 was enrolled in the network. Dual-luciferase reporter assay verified the target relationship of miR-122 and circ_0011269/RUNX2. The expression of circ_0011269 and RUNX2 were gradually increased during osteogenic differentiation while miR-122 exhibited a decreased expression. Moreover, overexpression of circ_0011269 could promote RUNX2 expression and inhibit osteoporosis. In summary, this study found that circ_0011269 sponges miR-122 to regulate RUNX2 expression and promotes osteoporosis progression.

Neural precursor cell-expressed, developmentally down-regulated 4 (NEDD4) regulates hydrogen peroxide-induced cell proliferation and death through inhibition of Hippo signaling.

E3 ubiquitin ligases are involved in the regulation of oxidative stress-induced cell death. In this study, we investigated the role of neural precursor cell-expressed, developmentally down-regulated protein 4 (NEDD4) in regulation of hydrogen peroxide (H2O2)-induced cell proliferation and apoptosis in human bone marrow-derived stem cells (hBMSCs). Cell proliferation was increased in low doses of H2O2 (10-4 to 10-2 µM), whereas sublethal concentrations of H2O2 (>200 µM) induced apoptosis. A chromatin immunoprecipitation assay identified that recruitment of NF-κB onto the promoter region of NEDD4 mediated H2O2-induced NEDD4 expression. The increase of NEDD4 expression by H2O2 induced translocation of yes-associated protein (YAP) into the nucleus by decreasing the stability of large tumor suppressor kinase (LATS). Thus, the phosphorylation of serine 127 residue of YAP by LATS upstream kinase is decreased and thereby increased the transcriptional activity of YAP. The mRNA expression levels of catalase and manganese superoxide dismutase, which are well-known targets of YAP, were increased by H2O2 treatment but down-regulated by NEDD4 silencing using a specific small interfering RNA targeting NEDD4 (siNEDD4). H2O2-induced scavenging capacity of reactive oxygen species was also decreased by siNEDD4 in hBMSCs. Finally, hBMSC differentiation into osteoblast was decreased by siNEDD4 but reverted by reintroduction of the S127A mutant construction of YAP. Taken together, these results indicate that NEDD4 regulates H2O2-induced alteration of cell status through regulation of the Hippo signaling pathway.-Jeon, S.-A., Kim, D. W., Cho, J.-Y. Neural precursor cell-expressed, developmentally down-regulated 4 (NEDD4) regulates hydrogen peroxide-induced cell proliferation and death through inhibition of Hippo signaling.

circRNA_0016624 could sponge miR-98 to regulate BMP2 expression in postmenopausal osteoporosis.

BACKGROUND: Circular RNAs (circRNAs) are emerging as important regulators in human disease. The expression profile and mechanism of circRNAs in postmenopausal osteoporosis remains largely unknown. Bone morphogenetic protein 2 (BMP2) is known to play important role in inducing osteogenic differentiation. MiR-98 was reported to regulate osteogenic differentiation of human bone mesenchymal stromal cells by targeting BMP2. AIM: We aimed to analyze circRNA expression profiles in osteoporosis and explore the molecular mechanism of circRNA_0016624 and interaction between circRNA_0016624, miR-98 and BMP2 during osteogenic differentiation. METHODS: RNA-seq and bioinformatics analysis was performed in postmenopausal osteoporosis patients to screen for differentially expressed circRNAs. MiRanda and TargetScan were used to detect miR-98 binding sites of circRNA_0016624 and the target relationship was confirmed by dual luciferase assay. Expression level of circRNA_0016624, miR-98 and BMP2 were measured by qRT-PCR or Western blot. ARS staining was used to observe the level of osteogenic differentiation after transfection. RESULTS: There were 387 circRNAs were differentially expressed in osteoporosis (|fold change| > 2 and P-value < 0.01). circRNA_0016624 and BMP2 were down-regulated in osteoporosis. CircRNA_0016624 could sponge miR-98 and regulate miR-98 expression. Overexpression of circRNA_0016624 promoted the expression of BMP2 and prevented osteoporosis. CONCLUSION: circRNA_0016624 could sponge miR-98 and enhance BMP2 expression, thus circRNA_0016624 prevents osteoporosis and may provide a novel therapeutic strategy.

Long noncoding RNA MALAT1 promotes osterix expression to regulate osteogenic differentiation by targeting miRNA-143 in human bone marrow-derived mesenchymal stem cells.

Osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs) is essential for the human bone formation, and emerging evidence shows that long non-coding RNAs (lncRNAs) play important roles in hBMSC osteogenic differentiation. MALAT1 is often regarded as a tumor-related lncRNA, but its function in mesenchymal stem cell differentiation remains to be defined. In this study, we aimed to investigate whether MALAT1 regulates Osterix (Osx) expression by sponging miR-143 to promote hBMSC osteogenic differentiation. Firstly, we found that the expression of MALAT1 was much lower in hBMSCs from osteoporosis patients and miR-143 was contrarily higher. In addition, MALAT1 expression increased, and miR-143 decreased when hBMSCs were treated with osteogenic induction. Then, we used short hairpin RNAs to knockdown MALAT1, and the results showed that hBMSC osteogenic differentiation decreased significantly, indicating that MALAT1 is a positive regulator of osteogenic differentiation in hBMSCs. Furthermore, by luciferase assays, we found that MALAT1 could directly bind to miR-143 and negatively regulate its expression. Similarly, miR-143 could directly bind to the target site on the Osx 3'-UTR and then inhibit Osx expression. Knockdown of MALAT1 decreased Osx expression, and co-transfection of miR-143 inhibitor could rescue Osx mRNA expression. While Osx expression was increased in MALAT1-overexpressing hBMSCs, it was reversed by the miR-143 mimics. Moreover, Osx silencing decreased ALP, OCN, and OPN mRNA expression induced by the miR-143 inhibitor. Altogether, our findings suggest that MALAT1 acts to regulate Osx expression through targeting miR-143; thus, it is considered as a positive regulator in hBMSC osteogenic differentiation.