miR-206 inhibits osteogenic differentiation of bone marrow mesenchymal stem cells by targetting glutaminase

AbstractOsteoblast-mediated bone formation is a complex process involving various pathways and regulatory factors, including cytokines, growth factors, and hormones. Investigating the regulatory mechanisms behind osteoblast differentiation is important for bone regeneration therapy. miRNAs are known as important regulators, not only in a variety of cellular processes, but also in the pathogenesis of bone diseases. In the present study, we investigated the potential roles of miR-206 during osteoblast differentiation. We report that miR-206 expression was significantly down-regulated in human bone marrow mesenchymal stem cells (BMSCs) at days 7 and 14 during osteogenic induction. Furthermore, miR-206 overexpressing BMSCs showed attenuated alkaline phosphatase (ALP) activity, Alizarin Red staining, and osteocalcin secretion. The mRNA levels of osteogenic markers, Runx2 and Osteopontin (OPN), were significantly down-regulated in miR-206 overexpressing BMSCs. We observed that significantly increased glutamine uptake at days 7 and 14 during the osteogenic induction and inhibition of glutamine metabolism by knocking down glutaminase (GLS)-suppressed osteogenic differentiation of BMSCs. Here, we discover that miR-206 could directly bind to the 3′-UTR region of GLS mRNA, resulting in suppressed GLS expression and glutamine metabolism. Finally, restoration of GLS in miR-206 overexpressing BMSCs led to recovery of glutamine metabolism and osteogenic differentiation. In summary, these results reveal a new insight into the mechanisms of the miR-206-mediated osteogenesis through regulating glutamine metabolism. Our study may contribute to the development of therapeutic agents against bone diseases.

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Icariin Promotes the Osteogenesis of Bone Marrow Mesenchymal Stem Cells through Regulating Sclerostin and Activating the Wnt/β-Catenin Signaling Pathway

BioMed Research International ◽

10.1155/2021/6666836 ◽

2021 ◽

Vol 2021 ◽

pp. 1-10

Author(s):

Jianliang Gao ◽

Shouyu Xiang ◽

Xiao Wei ◽

Ram Ishwar Yadav ◽

Menghu Han ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Osteogenic Differentiation ◽

Signaling Pathway ◽

Fragility Fractures ◽

Therapeutic Efficiency ◽

Increased Risk ◽

Marrow Mesenchymal Stem Cells ◽

Osteogenic Induction

Osteoporosis (OP) is a metabolic disease characterized by decreased bone mass and increased risk of fragility fractures, which significantly reduces the quality of life. Stem cell-based therapies, especially using bone marrow mesenchymal stem cells (BMSCs), are a promising strategy for treating OP. Nevertheless, the survival and differentiation rates of the transplanted BMSCs are low, which limits their therapeutic efficiency. Icariin (ICA) is a traditional Chinese medicine formulation that is prescribed for tonifying the kidneys. It also promotes the proliferation and osteogenic differentiation of BMSCs, although the specific mechanism remains unclear. Based on our previous research, we hypothesized that ICA promotes bone formation via the sclerostin/Wnt/β-catenin signaling pathway. We isolated rat BMSCs and transfected them with sclerostin gene (SOST) overexpressing or knockdown constructs and assessed osteogenic induction in the presence or absence of ICA. Sclerostin significantly inhibited BMSC proliferation and osteogenic differentiation, whereas the presence of ICA not only increased the number of viable BMSCs but also enhanced ALP activity and formation of calcium nodules during osteogenic induction. In addition, the osteogenic genes including Runx2, β-catenin, and c-myc as well as antioxidant factors (Prdx1, Cata, and Nqo1) were downregulated by sclerostin and restored by ICA treatment. Mechanistically, ICA exerted these effects by activating the Wnt/β-catenin pathway. In conclusion, ICA can promote the proliferation and osteogenic differentiation of BMSCs in situ and therefore may enhance the therapeutic efficiency of BMSC transplantation in OP.

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Cajanolactone A from Cajanus cajan Promoted Osteoblast Differentiation in Human Bone Marrow Mesenchymal Stem Cells via Stimulating Wnt/LRP5/β-Catenin Signaling

Molecules ◽

10.3390/molecules24020271 ◽

2019 ◽

Vol 24 (2) ◽

pp. 271

Author(s):

Shan Liu ◽

Zhuo-Hui Luo ◽

Gui-Mei Ji ◽

Wei Guo ◽

Jia-Zhong Cai ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Signaling Pathway ◽

Cajanus Cajan ◽

Osteoblast Differentiation ◽

Human Bone ◽

Human Bone Marrow ◽

Mrna Levels ◽

Marrow Mesenchymal Stem Cells

Cajanolactone A (CLA) is a stilbenoid discovered by us from Cajanus cajan (L.) Millsp. In our study, CLA was found to promote osteoblast differentiation in human bone marrow mesenchymal stem cells (hBMSCs), as judged by increased cellular alkaline phosphatase activity and extracellular calcium deposits, and elevated protein expression of Runx2, collagen-1, bone morphogenetic protein-2, and osteopontin. Mechanistic studies revealed that hBMSCs undergoing osteoblast differentiation expressed upregulated mRNA levels of Wnt3a, Wnt10b, LRP5/6, Frizzled 4, β-catenin, Runx2, and Osterix from the early stage of differentiation, indicating the role of activated Wnt/β-catenin signaling pathway in osteoblast differentiation. Addition of CLA to the differentiation medium further increased the mRNA level of Wnt3a, Wnt10b, Frizzled 4, LRP5, and β-catenin, inferring that CLA worked by stimulating Wnt/LRP5/β-catenin signaling. Wnt inhibitor dickkopf-1 antagonized CLA-promoted osteoblastogenesis, indicating that CLA did not target the downstream of canonical Wnt signaling pathway. Treatment with CLA caused no changes in mRNA expression level, as well as protein secretion of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-B ligand (RANKL), indicating that CLA did not affect the OPG/RANKL axis. Our results showed that CLA, which promoted osteoblast differentiation in hBMSCs, through activating Wnt/LRP5/β-catenin signaling transduction, is a promising anti-osteoporotic drug candidate.

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Exosomes from bone marrow mesenchymal stem cells promoted osteogenic differentiation by delivering miR-196a that targeted Dkk1 to activate Wnt/β-catenin pathway

10.21203/rs.3.rs-25044/v1 ◽

2020 ◽

Author(s):

Zhi Peng ◽

Zhenkai Lou ◽

Zhongjie Li ◽

Shaobo Li ◽

Kaishun Yang ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Western Blot ◽

Osteogenic Differentiation ◽

Osteoblast Differentiation ◽

Negative Regulator ◽

Luciferase Assay ◽

Alizarin Red ◽

Marrow Mesenchymal Stem Cells

Abstract Background: Osteoporosis is the most common bone metabolic disease. Emerging evidence suggests that exosomes are secreted by diverse cells such as bone marrow mesenchymal stem cells (BMSCs), and play important role in cell-to-cell communication and tissue homeostasis. Recently, the discovery of exosomes has attracted attention in the field of bone remodeling. Methods: The exosomes were extracted from BMSCs and labeled by PKH-67, and then incubated with hFOB1.19 cells to investigate the miR-196a function on the osteoblast differentiation of hFOB1.19. The osteoblast differentiation was detected via alizarin red staining and the expression of osteoblast genes were detected by western blot. The cell apoptosis was detected by flow cytometer. The target relationship of miR-196a and Dickkopf-1 (Dkk1) were verified by luciferase assay and western blot. Results: Here we demonstrated that exosomes extracted from BMSCs (BMSC-exo) significantly promoted hFOB1.19 differentiation to osteoblasts. We found that BMSC-exo were enriched with miR-196a and delivered miR-196a to hFOB1.19 cells to inhibit its target Dkk1, which is a negative regulator of Wnt/β-catenin pathway. Conclusion: BMSC-exo activated Wnt/β-catenin pathway to promote osteogenic differentiation, while BMSC-exo failed to exert the effects when miR-196a was deprived. In conclusion, miR-196a delivered by exosomes from BMSCs plays an essential role in enhancing osteoblastic differentiation by targeting Dkk1 to activate Wnt/β-catenin pathway.

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Semaphorin3B Promotes Proliferation and Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells in a High-Glucose Microenvironment

Stem Cells International ◽

10.1155/2021/6637176 ◽

2021 ◽

Vol 2021 ◽

pp. 1-9

Author(s):

Quan Xing ◽

Jingyi Feng ◽

Xiaolei Zhang

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Osteogenic Differentiation ◽

Bone Metabolism ◽

High Glucose ◽

Bone Diseases ◽

Akt Pathway ◽

Bone Formation Markers ◽

Marrow Mesenchymal Stem Cells

Bone marrow mesenchymal stem cells (BMSCs) play an essential role in osteogenesis and bone metabolism and have already been recognized as one of the most popular seed cells for bone tissue engineering for bone diseases. However, high-glucose (HG) conditions in type 2 diabetes mellitus (T2DM) exert deleterious effects on BMSC proliferation and osteogenic differentiation. Semaphorin 3B (Sema3B) increases osteoblast differentiation in bone metabolism. Here, we determined the role of Sema3B in the proliferation and osteogenic differentiation of BMSCs in the HG microenvironment. The HG microenvironment decreased Sema3B expression in BMSCs. Moreover, HG inhibited BMSC proliferation. Furthermore, HG inhibited osteogenic differentiation in BMSCs by decreasing the expression of bone formation markers, alkaline phosphatase (ALP) activity, and mineralization. However, the administration of recombinant Sema3B reversed all of these effects. Moreover, our study found that Sema3B could activate the Akt pathway in BMSCs. Sema3B rescues defects in BMSC proliferation and osteogenic differentiation in the HG microenvironment by activating the Akt pathway. These effects were significantly reduced by treatment with an Akt inhibitor. Together, these findings demonstrate that Sema3B promotes the proliferation and osteogenic differentiation of BMSCs via the Akt pathway under HG conditions. Our study provides new insights into the potential ability of Sema3B to ameliorate BMSC proliferation and osteogenic differentiation in an HG microenvironment.

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lncRNA MALAT1 Inhibits Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells by Down-Regulating WNT5A

Journal of Biomaterials and Tissue Engineering ◽

10.1166/jbt.2019.2167 ◽

2019 ◽

Vol 9 (11) ◽

pp. 1520-1527

Author(s):

Xiaoliang Li ◽

Guofeng Xia ◽

Hongmei Xin ◽

Chunsheng Tao ◽

Weiwei Lai ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Osteogenic Differentiation ◽

Wnt Signaling Pathway ◽

Luciferase Reporter ◽

Protein Levels ◽

Lncrna Malat1 ◽

Marrow Mesenchymal Stem Cells ◽

Osteogenic Induction

ncRNA involves in osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). WNT5A participates in the growth and development of osteogenic differentiation. This study aims to investigate whether lncRNA MALAT1 regulates BMSCs osteogenesis through WNT5A. qRT-PCR was done to detect the lncRNA MALAT1 level and osteogenic markers in osteoporosis patients and control groups. The above markers and WNT5A protein levels were detected by Western blot. The degree of osteogenic differentiation was detected by ALP activity assay and ALP staining. The differentiation ability of BMSCs after lncRNA MALAT1 overexpression was detected by ARS staining. The binding site of lncRNA MALAT1 to WNT5A was determined by dual luciferase reporter assay. lncRNA MALAT1 expression was higher in patients with osteoporosis, and decreased significantly with increased osteogenic induction. Overexpression of lncRNA MALAT1 in BMSCS reduced WNT5A level, while interference with lncRNA MALAT1 increased WNT5A levels. In cells with overexpression of lncRNA MALAT1, transfection of si-WNT5A can significantly downregulate the RUNX2, OSX, ALP, OCN, OPN, and COL1A1, thereby inhibiting osteogenic differentiation, interfering with the regulation of WNT signaling pathway and regulating BMSCs osteogenic differentiation. lncRNA MALAT1 and WNT5A can regulate BMSCs osteogenesis, thus accelerating the progression of osteoporosis.

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miR-218 Regulates Glycogen Synthase Kinase-3β and Promotes Differentiation of Human Bone Marrow Mesenchymal Stem Cells into Osteoblasts

Journal of Biomaterials and Tissue Engineering ◽

10.1166/jbt.2020.2233 ◽

2020 ◽

Vol 10 (2) ◽

pp. 176-181

Author(s):

Long Ling ◽

Hailan Hu ◽

Ram Ishwar Yadav ◽

Jianliang Gao ◽

Xiao Wei ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Osteoblast Differentiation ◽

Glycogen Synthase ◽

Induction Medium ◽

Alizarin Red ◽

Osteogenic Induction Medium ◽

Marrow Mesenchymal Stem Cells ◽

Osteogenic Induction

miR-218 is associated with osteogenesis. Bioinformatics analysis showed a targeting relationship between miR-218 and GSK-3β 3′-UTR. Our study assessed whether miR-218 regulates GSK-3β expression and affects osteoblast differentiation of bone marrow mesenchymal stem cells (BMSCs). Osteogenic induction medium was used to induce BMSCs to differentiate into osteoblasts. miR-218, GSK-3β, β-catenin and RUNX2 level was detected during D10 and D20 differentiation. BMSCs cells were divided into antagomir-NC and antagomir-218, and induced to differentiate for 20 days followed by analysis of GSK-3β, β-catenin and RUNX2 level, osteogenesis and cell differentiation by the alizarin red staining. Compared with pre-differentiation,the expression of miR-218, β-catenin and RUNX2 was gradually increased and GSK-3β expression was decreased during the differentiation of BMSCs into osteoblasts. There was a targeted regulation relationship between miR-218 and GSK-3β. Compared with the antagomir-NC group, GSK-3β protein expression was increased in antagomir-218 transfection group, with decreased the expression of β-catenin and RUNX2 protein, reduced ALP activity as well as weakened staining degree of alizarin red. GSK-3β expression is decreased and miR-218 expression is increased during osteoblast differentiation of BMSCs. Inhibition of miR-218 expression can up-regulate GSK-3β expression and attenuate the ability of BMSCs to differentiate into osteoblasts.

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