SIRT6 Promotes Osteogenic Differentiation of Adipose-Derived Mesenchymal Stem Cells Through Antagonizing DNMT1

BackgroundAdipose-derived stem cells (ADSCs) are increasingly used in regenerative medicine because of their potential to differentiate into multiple cell types, including osteogenic lineages. Sirtuin protein 6 (SIRT6) is a nicotinamide adenine dinucleotide (NAD)-dependent deacetylase that plays important roles in cell differentiation. NOTCH signaling has also been reported to involve in osteogenic differentiation. However, the function of SIRT6 in osteogenic differentiation of ADSCs and its relation to the NOTCH signaling pathways are yet to be explored.MethodsThe in vitro study with human ADSCs (hADSCs) and in vivo experiments with nude mice have been performed. Alkaline phosphatase (ALP) assays and ALP staining were used to detect osteogenic activity. Alizarin Red staining was performed to detect calcium deposition induced by osteogenic differentiation of ADSCs. Western blot, RT-qPCR, luciferase reporter assay, and co-immunoprecipitation assay were applied to explore the relationship between of SIRT6, DNA methyltransferases (DNMTs) and NOTCHs.ResultsSIRT6 promoted ALP activity, enhanced mineralization and upregulated expression of osteogenic-related genes of hADSCs in vitro and in vivo. Further mechanistic studies showed that SIRT6 deacetylated DNMT1, leading to its unstability at protein level. The decreased expression of DNMT1 prevented the abnormal DNA methylation of NOTCH1 and NOTCH2, resulting in the upregulation of their transcription. SIRT6 overexpression partially suppressed the abnormal DNA methylation of NOTCH1 and NOTCH2 by antagonizing DNMT1, leading to an increased capacity of ADSCs for their osteogenic differentiation.ConclusionThis study demonstrates that SIRT6 physical interacts with the DNMT1 protein, deacetylating and destabilizing DNMT1 protein, leading to the activation of NOTCH1 and NOTCH2, Which in turn promotes the osteogenic differentiation of ADSCs.

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SIRT6 Promotes Osteogenic Differentiation of Adipose-Derived Mesenchymal Stem Cells Through Activating Notch Signaling via Antagonizing DNMT1

10.21203/rs.3.rs-49927/v1 ◽

2020 ◽

Author(s):

Bo Jia ◽

Jun Chen ◽

Qin Wang ◽

Xiang Sun ◽

JiuSong Han ◽

...

Keyword(s):

Dna Methylation ◽

Stem Cells ◽

Alkaline Phosphatase ◽

Mesenchymal Stem Cells ◽

Osteogenic Differentiation ◽

Dna Methyltransferases ◽

Luciferase Reporter ◽

Physical Interaction ◽

Alizarin Red ◽

Alp Activity

Abstract Background: Adipose-derived stem cells (ADSCs) are increasingly accepted as one of ideal seed cells for regenerative medicine for its potential to differentiate into multiple cell types, including osteogenic lineages. Sirtuin proteins 6 (SIRT6) is a nicotinamide adenine dinucleotide (NAD)-dependent deacetylase and plays important roles in a variety of biological processes, including cell differentiation.Methods: Alkaline phosphatase (ALP) activity, ALP staining, and Alizarin Red Staining was performed to explore the roles of SIRT6 in the osteogenic differentiation of ADSCs. Western blot , RT-qPCR,Luciferase reporter assay and Co-Immunoprecipitation assay were applied to confirm the relationship between of Sirt6, DNA methyltransferases (DNMTs) and NOTCHs.Results: SIRT6 leads to increased alkaline phosphatase (ALP) activity, enhanced mineralization and upregulated expression of osteogenic-related genes of human adipose-derived mesenchymal stem cells (hADSCs) in vitro and in vivo. Further mechanistic studies showed that SIRT6 regulated osteogenic differentiation of hADSCs depending on its deacetylase activity. SIRT6 selectively prevents abnormal DNA methylation of NOTCH1, NOTCH2 in hADSCs by antagonizing DNMT1. DNMT1 expression was suppressed in SIRT6 overexpression hADSCs, and knockdown partially rescued abnormal DNA methylation of NOTCH1 and NOTCH2, leading to the increased capable of osteogenic differentiation. Conculsions: SIRT6 promotes the osteogenic differentiation of hADSCs.The SIRT6 protein suppresses DNMT level via physical interaction with the DNMT1 protein, deacetylating and destabilizing DNMT1 protein,leading the activation of NOTCH1 and NOTCH2.

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SIRT6 Promotes Osteogenic Differentiation of Adipose-Derived Mesenchymal Stem Cells Through Activating Notch Signaling Via Antagonizing DNMT1

10.21203/rs.3.rs-99004/v1 ◽

2020 ◽

Author(s):

BO JIA ◽

Jun Chen ◽

Qin Wang ◽

Xiang Sun ◽

Jiusong Han ◽

...

Keyword(s):

Dna Methylation ◽

Stem Cells ◽

Alkaline Phosphatase ◽

Mesenchymal Stem Cells ◽

Osteogenic Differentiation ◽

Dna Methyltransferases ◽

Luciferase Reporter ◽

Physical Interaction ◽

Alizarin Red ◽

Alp Activity

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LncRNA LOC100506178 promotes osteogenic differentiation via regulating miR-214-5p-BMP2 axis in human bone marrow mesenchymal stem cells

PeerJ ◽

10.7717/peerj.8909 ◽

2020 ◽

Vol 8 ◽

pp. e8909

Author(s):

Lina Li ◽

Jie Fang ◽

Yi Liu ◽

Li Xiao

Keyword(s):

Bone Marrow ◽

Osteogenic Differentiation ◽

Human Bone ◽

Human Bone Marrow ◽

Bone Morphogenetic Protein 2 ◽

Luciferase Reporter ◽

Alizarin Red ◽

Dental Implantation

Osteogenic differentiation is an important role in dental implantation. Long no coding RNAs (lncRNAs) are a novel class of noncoding RNAs that have significant effects in a variety of diseases. However, the function and mechanisms of LOC100506178 in osteogenic differentiation and migration of bone morphogenetic protein 2 (BMP2)-induced osteogenic differentiation of human bone marrow mesenchymalstem cells (hBMSCs) remain largely unclear. BMP2 was used to induce osteogenic differentiation of hBMSCs. Quantitative real time PCR (qRT-PCR) was used to examine the expression of LOC100506178, miR-214-5p, Runt-related transcription factor 2 (RUNX2), Osterix (Osx), and Alkaline Phosphatase (ALP) in BMP2-induced osteogenic differentiation of hBMSCs. The function of LOC100506178 and miR-214-5p was explored in vitro using Alizarin Red S Staining, ALP activity, as well as in vivo ectopic bone formation. Luciferase reporter assay was performed to assess the association between LOC100506178 and miR-214-5p, as well as miR-214-5p and BMP2. The miR-214-5p sponging potential of LOC100506178 was evaluated by RNA immunoprecipitation. In the present study, the expression of LOC100506178 was found to be increased in BMP2-induced osteogenic differentiation of hBMSCs, accompanied with decreased miR-214-5p expression and increased RUNX2, Osx and ALP expression. LOC100506178 significantly induced, while miR-214-5p suppressed the BMP2-induced osteogenic differentiation of hBMSCs. Mechanistically, LOC100506178 was directly bound to miR-214-5p and miR-214-5p targeted the 3′-untranslated region of BMP2 to negatively regulate its expression. In conclusion, our data indicate a novel molecular pathway LOC100506178/miR-214-5p/BMP2 in relation to hBMSCs differentiation into osteoblasts, which may facilitate bone anabolism.

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Role of Fzd6 in Regulating the Osteogenic Differentiation of Adipose-derived Stem Cells in Osteoporosis

10.21203/rs.3.rs-328417/v1 ◽

2021 ◽

Author(s):

Tianli Wu ◽

Zhihao Yao ◽

Gang Tao ◽

Fangzhi Lou ◽

Hui Tang ◽

...

Keyword(s):

Stem Cells ◽

Osteogenic Differentiation ◽

Wnt Signaling Pathway ◽

Osteogenic Potential ◽

Adipose Derived Stem Cells ◽

Alizarin Red ◽

Differentiation Potential

Abstract Objective: Although it has been demonstrated that adipose-derived stem cells (ASCs) from osteoporosis mice (OP-ASCs) exhibit impaired osteogenic differentiation potential, the molecular mechanism has not yet been elucidated. We found that Fzd6 was decreased in OP-ASCs compared with ASCs. This study investigates the effects and underlying mechanisms of Fzd6 in the osteogenic potential of OP-ASCs. Methods: Fzd6 expression in ASCs and OP-ASCs was measured by PCR gene chip. Fzd6 overexpression and silencing lentiviruses were used to evaluate the role of Fzd6 in the osteogenic differentiation of OP-ASCs. Real-time PCR (qPCR) and western blotting (WB) was performed to detect the expression of Fzd6 and bone-related molecules, including runt-related transcription factor 2 (Runx2) and osteopontin (Opn). Alizarin red staining and Alkaline phosphatase (ALP) staining was performed following osteogenic induction. Microscopic CT (Micro-CT), hematoxylin and eosin staining (H&E) staining, and Masson staining were used to assess the role of Fzd6 in osteogenic differentiation of osteoporosis (OP) mice in vivo.Results: Expression of Fzd6 was decreased significantly in OP-ASCs. Fzd6 silencing down-regulated the osteogenic ability of OP-ASCs in vitro. Overexpression of Fzd6 rescued the impaired osteogenic capacity in OP-ASCs in vitro. We obtained similar results in vivo.Conclusions: Fzd6 plays an important role in regulating the osteogenic ability of OP-ASCs both in vivo and in vitro. Overexpression of Fzd6 associated with the Wnt signaling pathway promotes the osteogenic ability of OP-ASCs, which provides new insights for the prevention and treatment of OP.

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circAKT3 positively regulates osteogenic differentiation of human dental pulp stromal cells via miR-206/CX43 axis

Stem Cell Research & Therapy ◽

10.1186/s13287-020-02058-y ◽

2020 ◽

Vol 11 (1) ◽

Author(s):

Bo Zhang ◽

Sibei Huo ◽

Xiao Cen ◽

Xuefeng Pan ◽

Xinqi Huang ◽

...

Keyword(s):

Stem Cells ◽

Osteogenic Differentiation ◽

Stromal Cells ◽

Dental Pulp ◽

Luciferase Reporter ◽

Circular Rnas ◽

Alizarin Red ◽

Cx43 Expression ◽

Human Dental Pulp

Abstract Background Human dental pulp stromal cells (hDPSCs) are promising sources of mesenchymal stem cells (MSCs) for bone tissue regeneration. Circular RNAs (circRNAs) have been demonstrated to play critical roles in stem cell osteogenic differentiation. Herein, we aimed to investigate the role of circAKT3 during osteogenesis of hDPSCs and the underlying mechanisms of its function. Methods We performed circRNA sequencing to investigate the expression profiles of circular RNAs during osteogenesis of hDPSCs. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed to detect the expression pattern of circAKT3 and miR-206 in hDPSCs during osteogenesis. We knocked down circAKT3 and interfered the expression of miR-206 to verify their regulatory role in hDPSC osteogenesis. We detected hDPSCs mineralization by alkaline phosphatase (ALP) and Alizarin Red S (ARS) staining and used dual-luciferase reporter assay to validate the direct binding between circAKT3 and miR-206. To investigate in vivo mineralization, we performed subcutaneous transplantation in nude mice and used hematoxylin and eosin, Masson’s trichrome, and immunohistochemistry staining. Results Totally, 86 circRNAs were differentially expressed during hDPSC osteogenesis, in which 29 were downregulated while 57 were upregulated. circAKT3 was upregulated while miR-206 was downregulated during hDPSC osteogenesis. Knockdown of circAKT3 inhibited ALP/ARS staining and expression levels of osteogenic genes. circAKT3 directly interacted with miR-206, and the latter one suppressed osteogenesis of hDPSCs. Silencing miR-206 partially reversed the inhibitory effect of circAKT3 knockdown on osteogenesis. Connexin 43 (CX43), which positively regulates osteogenesis of stem cells, was predicted as a target of miR-206, and overexpression or knockdown of miR-206 could correspondingly decrease and increase the expression of CX43. In vivo study showed knockdown of circAKT3 suppressed the formation of mineralized nodules and expression of osteogenic proteins. Conclusion During osteogenesis of hDPSCs, circAKT3 could function as a positive regulator by directly sponging miR-206 and arresting the inhibitive effect of miR-206 on CX43 expression.

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186 The effects of different concentrations of MgSO4 in osteogenic differentiation

Reproduction Fertility and Development ◽

10.1071/rdv31n1ab186 ◽

2019 ◽

Vol 31 (1) ◽

pp. 217

Author(s):

L. R. Padoveze ◽

M. Rubessa ◽

C. E. Ambrosio ◽

M. B. Wheeler

Keyword(s):

Stem Cells ◽

Osteogenic Differentiation ◽

In Vitro Study ◽

Control Group ◽

Nodule Formation ◽

Osteogenic Medium ◽

Alizarin Red ◽

Physiological Processes

Tissue engineering offers a viable alternative to bone grafts in repairing large bone defects. Magnesium-based materials are biocompatible in vivo, and it is possible to determine the degradation period according to the necessities (Farraro et al. 2014 J. Biomech. 47, 1979-1986). Magnesium (Mg) is part of many physiological processes, and it promotes the osteogenesis of mesenchymal stem cells (Díaz-Tocados et al. 2017 Sci. Rep. 7, 7839.). Moreover, Mg up-regulates important genes associated with the osteogenic differentiation (Yoshizawa et al. 2014 Acta Biomater. 10, 2834-2842). The aim of this study was to evaluate the effect of different Mg concentrations in the osteogenic medium on the number of nodules of bone. Swine adipose stem cells (ASC) were previously isolated as described (Monaco et al. 2009 Open Tissue Eng. Regen. Med. J. 2, 20-33). In this in vitro study, ASC were cultured during 4 weeks in osteogenic medium with addition of 0.1, 0.2, 1, 2, 10, or 20mM MgSO4. The medium was changed twice a week. Alizarin Red and Von Kossa staining were performed to evaluate the formation of nodules by mineralization of extracellular matrix (ECM), evidenced by dark red nodules and calcium deposit. The experiment was replicated 3 times in triplicate. Data were analysed using the generalized linear model (GLM) procedure, and Bonferroni’s post hoc test was used to perform statistical multiple comparison (SPSS Inc./IBM Corp., Chicago, IL, USA). The results showed enhanced nodule formation with 2mM Mg in the osteogenic medium (35.6v. 15.3, respectively for 2mM and Control). This result confirms the ability of magnesium to act in bone formation. There was no statistical difference among the different groups when we evaluated the Von Kossa staining results, indicating that the quality of the new formations was comparable to that of the control group even in an elevated nodule formation. In conclusion, a higher concentration of magnesium can improve nodule formation into osteogenic differentiation in vitro; the 2mM concentration showed the best nodule formation compared with the other groups. These results showed the value of magnesium in bone physiology.

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CircRNA hsa_circ_0074834 promotes the osteogenesis-angiogenesis coupling process in bone mesenchymal stem cells (BMSCs) by acting as a ceRNA for miR-942-5p

Cell Death and Disease ◽

10.1038/s41419-019-2161-5 ◽

2019 ◽

Vol 10 (12) ◽

Cited By ~ 6

Author(s):

Zhengxiao Ouyang ◽

Tingting Tan ◽

Xianghong Zhang ◽

Jia Wan ◽

Yanling Zhou ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Osteogenic Differentiation ◽

Noncoding Rna ◽

Fracture Site ◽

Luciferase Reporter ◽

Circular Rnas ◽

Bone Mesenchymal Stem Cells ◽

Alizarin Red

AbstractBone tissue has a strong ability to repair itself. When treated properly, most fractures will heal well. However, some fractures are difficult to heal. When a fracture does not heal, it is called nonunion. Approximately, 5% of all fracture patients have difficulty healing. Because of the continuous movement of the fracture site, bone nonunion is usually accompanied by pain, which greatly reduces the quality of life of patients. Bone marrow mesenchymal stem cells (BMSCs) play an important role in the process of nonunion. Circular RNAs (circRNAs) are a unique kind of noncoding RNA and represent the latest research hotspot in the RNA field. At present, no studies have reported a role of circRNAs in the development of nonunion. After isolation of BMSCs from patients with nonunion, the expression of circRNAs in these cells was detected by using a circRNA microarray. Alkaline phosphatase and Alizarin red staining were used to detect the regulation of osteogenic differentiation of BMSCs by hsa_circ_0074834. The target gene of hsa_circ_0074834 was detected by RNA pull-down and double-luciferase reporter assay. The ability of hsa_circ_0074834 to regulate the osteogenesis of BMSCs in vivo was tested by heterotopic osteogenesis and single cortical bone defect experiments. The results showed that the expression of hsa_circ_0074834 in BMSCs from patients with nonunion was decreased. Hsa_circ_0074834 acts as a ceRNA to regulate the expression of ZEB1 and VEGF through microRNA-942-5p. Hsa_circ_0074834 can promote osteogenic differentiation of BMSCs and the repair of bone defects. These results suggest that circRNAs may be a key target for the treatment of nonunion.

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miR-129-5p Promotes Osteogenic Differentiation of BMSCs and Bone Regeneration via Repressing Dkk3

Stem Cells International ◽

10.1155/2021/7435605 ◽

2021 ◽

Vol 2021 ◽

pp. 1-18

Author(s):

Changming Zhao ◽

Yulin Gu ◽

Yan Wang ◽

Qiaozhen Qin ◽

Ting Wang ◽

...

Keyword(s):

Western Blot ◽

Bone Regeneration ◽

Osteogenic Differentiation ◽

Luciferase Reporter ◽

Luciferase Reporter Gene ◽

Alizarin Red ◽

Gene Assay

Objective. Accumulating evidence indicates that microRNAs (miRNAs) play crucial roles in osteogenic differentiation. However, the associated mechanisms remain elusive. This paper is aimed at exploring the role of miR-129-5p in regulating bone marrow mesenchymal stem cell (BMSC) differentiation and bone regeneration in vivo and in vitro. Methods. BMSCs were transduced by miR-129-5p mimic, miR-129-5p inhibitor, and negative control lentivirus. The ability of BMSC differentiation to osteoblast was tested by alkaline phosphatase (ALP) and alizarin red staining (ARS). The expression of osteogenic genes (Runx2, Bmp2, and OCN) was examined via quantitative RT-PCR and western blot. A mouse model of calvaria defect was investigated by Micro-CT, immunohistochemistry, and histological examination. The luciferase reporter gene assay was performed to confirm the binding between Dkk3 and miR-129-5p. For the transfection experiments, lipofectamine 3000 was used to transfect pcDNA-Dkk3 into BMSCs to overexpress Dkk3. Coimmunoprecipitation and immunofluorescent localization assay were included for exploring the role of Dkk3 and β-catenin. Results. miR-129-5p was induced in BMSCs and MSC cell line C3H10T1/2 cells under osteogenic medium. Overexpression of miR-129-5p significantly promoted osteogenic differentiation of BMSCs in vitro. Moreover, BMSCs transduced with miR-129-5p mimic exhibited better bone regeneration compared with BMSCs transduced with control counterpart in vivo. Luciferase and western blot data showed that Dickkopf3 (Dkk3) is a target gene of miR-129-5p and the expression of Dkk3 was inhibited in BMSCs transduced with miR-129-5p mimic but enhanced in BMSCs transduced with miR-129-5p inhibitor. In addition, Dkk3 interacted with β-catenin directly. Conclusions. miR-129-5p promotes osteogenic differentiation of BMSCs and bone regeneration, and miR-129-5p/Dkk3 axis may be new potential targets for the treatment of bone defect and bone loss.

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Expression Profile of New Marker Genes Involved in Differentiation of Canine Adipose-Derived Stem Cells into Osteoblasts

International Journal of Molecular Sciences ◽

10.3390/ijms22136663 ◽

2021 ◽

Vol 22 (13) ◽

pp. 6663

Author(s):

Maurycy Jankowski ◽

Mariusz Kaczmarek ◽

Grzegorz Wąsiatycz ◽

Claudia Dompe ◽

Paul Mozdziak ◽

...

Keyword(s):

Stem Cells ◽

In Vitro Culture ◽

Osteogenic Differentiation ◽

Molecular Mechanisms ◽

Marker Genes ◽

P Value ◽

Illumina Platform

Next-generation sequencing (RNAseq) analysis of gene expression changes during the long-term in vitro culture and osteogenic differentiation of ASCs remains to be important, as the analysis provides important clues toward employing stem cells as a therapeutic intervention. In this study, the cells were isolated from adipose tissue obtained during routine surgical procedures and subjected to 14-day in vitro culture and differentiation. The mRNA transcript levels were evaluated using the Illumina platform, resulting in the detection of 19,856 gene transcripts. The most differentially expressed genes (fold change >|2|, adjusted p value < 0.05), between day 1, day 14 and differentiated cell cultures were extracted and subjected to bioinformatical analysis based on the R programming language. The results of this study provide molecular insight into the processes that occur during long-term in vitro culture and osteogenic differentiation of ASCs, allowing the re-evaluation of the roles of some genes in MSC progression towards a range of lineages. The results improve the knowledge of the molecular mechanisms associated with long-term in vitro culture and differentiation of ASCs, as well as providing a point of reference for potential in vivo and clinical studies regarding these cells’ application in regenerative medicine.

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