MicroRNA-15b shuttled by bone marrow mesenchymal stem cell-derived extracellular vesicles binds to WWP1 and promotes osteogenic differentiation

Abstract Background Osteogenic differentiation is an essential process for bone regeneration involving bone marrow mesenchymal stem cells (BMSCs). BMSC-secreted extracellular vesicles (EVs) enriched with microRNAs (miRs) have vital roles to play in mediating osteogenic differentiation. Therefore, this study aimed to explore the effect of BMSC-derived EVs loaded with miR-15b on osteogenic differentiation. Methods Human BMSCs (hBMSCs) were cultured and treated with plasmids overexpressing or knocking down KLF2, WWP1, and miR-15b to define the role of derived EVs in osteogenic differentiation in vitro. The expression of osteogenic differentiation-related marker was measured by Western blot analysis. The interaction among miR-15b, WWP1, and ubiquitination of KLF2 was investigated by dual-luciferase reporter, immunoprecipitation, and GST pull-down assays. Moreover, EVs from hBMSCs transfected with miR-15b inhibitor (EV-miR-15b inhibitor) were injected into ovariectomized rats to verify the effect of miR-15b on bone loss in vivo. Results WWP1 was downregulated, and KLF2 was upregulated during osteogenic differentiation. After co-culture with EVs, miR-15b expression was elevated and WWP1 expression was reduced in hBMSCs. Upregulation of miR-15b or KLF2 or downregulation of WWP1 or NF-κB increased ALP activity and cell mineralization, as well as osteogenic differentiation-related marker expression in hBMSCs. Mechanistically, miR-15b targeted and inhibited WWP1, thus attenuating KLF2 degradation and inhibiting NF-κB activity. Co-culture of EVs increased the bone volume and trabecular number, but decreased bone loss in ovariectomized rats, which could be reversed after treatment with EV-miR-15b inhibitor. Conclusion Collectively, BMSC-derived EVs loaded with miR-15b promoted osteogenic differentiation by impairing WWP1-mediated KLF2 ubiquitination and inactivating the NF-κB signaling pathway. Graphical abstract

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Long non-coding RNA MIR22HG promotes osteogenic differentiation of bone marrow mesenchymal stem cells via PTEN/ AKT pathway

Cell Death and Disease ◽

10.1038/s41419-020-02813-2 ◽

2020 ◽

Vol 11 (7) ◽

Author(s):

Chanyuan Jin ◽

Lingfei Jia ◽

Zhihui Tang ◽

Yunfei Zheng

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Osteogenic Differentiation ◽

Mineral Density ◽

Tensin Homolog ◽

Human Bmscs ◽

Marrow Mesenchymal Stem Cells

Abstract Osteoporosis is a prevalent metabolic bone disease characterized by low bone mineral density and degenerative disorders of bone tissues. Previous studies showed the abnormal osteogenic differentiation of endogenous bone marrow mesenchymal stem cells (BMSCs) contributes to the development of osteoporosis. However, the underlying mechanisms by which BMSCs undergo osteogenic differentiation remain largely unexplored. Recently, long non-coding RNAs have been discovered to play important roles in regulating BMSC osteogenesis. In this study, we first showed MIR22HG, which has been demonstrated to be involved in the progression of several cancer types, played an important role in regulating BMSC osteogenesis. We found the expression of MIR22HG was significantly decreased in mouse BMSCs from the osteoporotic mice and it was upregulated during the osteogenic differentiation of human BMSCs. Overexpression of MIR22HG in human BMSCs enhanced osteogenic differentiation, whereas MIR22HG knockdown inhibited osteogenic differentiation both in vitro and in vivo. Mechanistically, MIR22HG promoted osteogenic differentiation by downregulating phosphatase and tensin homolog (PTEN) and therefore activating AKT signaling. Moreover, we found MIR22HG overexpression promoted osteoclastogenesis of RAW264.7 cells, which indicated that MIR22HG played a significant role in bone metabolism and could be a therapeutic target for osteoporosis and other bone-related diseases.

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ZEB1 Mediates Bone Marrow Mesenchymal Stem Cell Osteogenic Differentiation Partly via Wnt/β-Catenin Signaling

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.682728 ◽

2021 ◽

Vol 8 ◽

Author(s):

Cuidi Xu ◽

Hongli Shi ◽

Xin Jiang ◽

Yongqian Fan ◽

Donghui Huang ◽

...

Keyword(s):

Bone Marrow ◽

Transcription Factor ◽

Osteogenic Differentiation ◽

Zinc Finger ◽

Epithelial Mesenchymal Transition ◽

Mesenchymal Transition ◽

Marrow Mesenchymal Stem Cells ◽

Zeb1 Expression

Zinc finger E-box-binding homebox 1 (ZEB1) is a zinc-finger transcription factor best known for its role in promoting the epithelial-mesenchymal transition, which is also related to osteogenesis. Here, ZEB1 was investigated for its role in the commitment of bone marrow mesenchymal stem cells (BMSCs) to osteoblasts. In vitro, ZEB1 expression decreased following osteogenic differentiation. Furthermore, silencing of ZEB1 in BMSCs promoted osteogenic activity and mineralization. The increase in osteogenic differentiation induced by si-ZEB1 could be partly rescued by the inhibition of Wnt/β-catenin (si-β-catenin). In vivo, knockdown of ZEB1 in BMSCs inhibited the rapid bone loss of ovariectomized (OVX) mice. ZEB1 expression has also been negatively associated with bone mass and bone formation in postmenopausal women. In conclusion, ZEB1 is an essential transcription factor in BMSC differentiation and may serve as a potential anabolic strategy for treating and preventing postmenopausal osteoporosis (PMOP).

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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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(−)-Epigallocatechin-3-Gallate (EGCG) Enhances Osteogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells

Molecules ◽

10.3390/molecules23123221 ◽

2018 ◽

Vol 23 (12) ◽

pp. 3221 ◽

Cited By ~ 18

Author(s):

Sung-Yen Lin ◽

Lin Kang ◽

Chau-Zen Wang ◽

Han Huang ◽

Tsung-Lin Cheng ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Alkaline Phosphatase ◽

Mesenchymal Stem Cells ◽

Bone Loss ◽

Osteogenic Differentiation ◽

Mrna Expression ◽

Human Bmscs ◽

Marrow Mesenchymal Stem Cells ◽

Increase Mrna Expression

Osteoporosis is the second most-prevalent epidemiologic disease in the aging population worldwide. Cross-sectional and retrospective evidence indicates that tea consumption can mitigate bone loss and reduce risk of osteoporotic fractures. Tea polyphenols enhance osteoblastogenesis and suppress osteoclastogenesis in vitro. Previously, we showed that (−)-epigallocatechin-3-gallate (EGCG), one of the green tea polyphenols, increased osteogenic differentiation of murine bone marrow mesenchymal stem cells (BMSCs) by increasing the mRNA expression of osteogenesis-related genes, alkaline phosphatase activity and, eventually, mineralization. We also found that EGCG could mitigate bone loss and improve bone microarchitecture in ovariectomy-induced osteopenic rats, as well as enhancing bone defect healing partially via bone morphogenetic protein 2 (BMP2). The present study investigated the effects of EGCG in human BMSCs. We found that EGCG, at concentrations of both 1 and 10 µmol/L, can increase mRNA expression of BMP2, Runx2, alkaline phosphatase (ALP), osteonectin and osteocalcin 48 h after treatment. EGCG increased ALP activity both 7 and 14 days after treatment. Furthermore, EGCG can also enhance mineralization two weeks after treatment. EGCG without antioxidants also can enhance mineralization. In conclusion, EGCG can increase mRNA expression of BMP2 and subsequent osteogenic-related genes including Runx2, ALP, osteonectin and osteocalcin. EGCG further increased ALP activity and mineralization. Loss of antioxidant activity can still enhance mineralization of human BMSCs (hBMSCs).

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The osteogenic differentiation of dog bone marrow mesenchymal stem cells in a thermo-sensitive injectable chitosan/collagen/β-glycerophosphate hydrogel: in vitro and in vivo

Journal of Materials Science Materials in Medicine ◽

10.1007/s10856-011-4386-4 ◽

2011 ◽

Vol 22 (9) ◽

pp. 2111-2118 ◽

Cited By ~ 39

Author(s):

Bin Sun ◽

Wei Ma ◽

Fang Su ◽

Yi Wang ◽

Jiaqiang Liu ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Osteogenic Differentiation ◽

Dog Bone ◽

Marrow Mesenchymal Stem Cells

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Circular RNA AFF4 modulates osteogenic differentiation in BM-MSCs by activating SMAD1/5 pathway through miR-135a-5p/FNDC5/Irisin axis

Cell Death and Disease ◽

10.1038/s41419-021-03877-4 ◽

2021 ◽

Vol 12 (7) ◽

Author(s):

Chao Liu ◽

An-Song Liu ◽

Da Zhong ◽

Cheng-Gong Wang ◽

Mi Yu ◽

...

Keyword(s):

Bone Marrow ◽

Bone Formation ◽

Osteogenic Differentiation ◽

Molecular Mechanisms ◽

Expression Patterns ◽

Regulatory System ◽

Circular Rna ◽

Luciferase Reporter ◽

Integrin Αv

AbstractBone marrow-derived mesenchymal stem cells (BM-MSCs), the common progenitor cells of adipocytes and osteoblasts, have been recognized as the key mediator during bone formation. Herein, our study aim to investigate molecular mechanisms underlying circular RNA (circRNA) AFF4 (circ_AFF4)-regulated BM-MSCs osteogenesis. BM-MSCs were characterized by FACS, ARS, and ALP staining. Expression patterns of circ_AFF4, miR-135a-5p, FNDC5/Irisin, SMAD1/5, and osteogenesis markers, including ALP, BMP4, RUNX2, Spp1, and Colla1 were detected by qRT-PCR, western blot, or immunofluorescence staining, respectively. Interactions between circ_AFF4 and miR-135a-5p, FNDC5, and miR-135a-5p were analyzed using web tools including TargetScan, miRanda, and miRDB, and further confirmed by luciferase reporter assay and RNA pull-down. Complex formation between Irisin and Integrin αV was verified by Co-immunoprecipitation. To further verify the functional role of circ_AFF4 in vivo during bone formation, we conducted animal experiments harboring circ_AFF4 knockdown, and born samples were evaluated by immunohistochemistry, hematoxylin and eosin, and Masson staining. Circ_AFF4 was upregulated upon osteogenic differentiation induction in BM-MSCs, and miR-135a-5p expression declined as differentiation proceeds. Circ_AFF4 knockdown significantly inhibited osteogenesis potential in BM-MSCs. Circ_AFF4 stimulated FNDC5/Irisin expression through complementary binding to its downstream target molecule miR-135a-5p. Irisin formed an intermolecular complex with Integrin αV and activated the SMAD1/5 pathway during osteogenic differentiation. Our work revealed that circ_AFF4, acting as a sponge of miR-135a-5p, triggers the promotion of FNDC5/Irisin via activating the SMAD1/5 pathway to induce osteogenic differentiation in BM-MSCs. These findings gained a deeper insight into the circRNA-miRNA regulatory system in the bone marrow microenvironment and may improve our understanding of bone formation-related diseases at physiological and pathological levels.

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Exosome-transported circRNA_0001236 enhances chondrogenesis and suppress cartilage degradation via the miR-3677-3p/Sox9 axis

Stem Cell Research & Therapy ◽

10.1186/s13287-021-02431-5 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Guping Mao ◽

Yiyang Xu ◽

Dianbo Long ◽

Hong Sun ◽

Hongyi Li ◽

...

Keyword(s):

Bone Marrow ◽

Mouse Model ◽

Chondrogenic Differentiation ◽

Cartilage Degradation ◽

Human Bone ◽

Human Bone Marrow ◽

Luciferase Reporter ◽

Gene And Protein Expression

Abstract Objectives Aberrations in exosomal circular RNA (circRNA) expression have been identified in various human diseases. In this study, we investigated whether exosomal circRNAs could act as competing endogenous RNAs (ceRNAs) to regulate the pathological process of osteoarthritis (OA). This study aimed to elucidate the specific MSC-derived exosomal circRNAs responsible for MSC-mediated chondrogenic differentiation using human bone marrow-derived MSCs (hMSCs) and a destabilization of the medial meniscus (DMM) mouse model of OA. Methods Exosomal circRNA deep sequencing was performed to evaluate the expression of circRNAs in human bone marrow-derived MSCs (hMSCs) induced to undergo chondrogenesis from day 0 to day 21. The regulatory and functional roles of exosomal circRNA_0001236 were examined on day 21 after inducing chondrogenesis in hMSCs and were validated in vitro and in vivo. The downstream target of circRNA_0001236 was also explored in vitro and in vivo using bioinformatics analyses. A luciferase reporter assay was used to evaluate the interaction between circRNA_0001236 and miR-3677-3p as well as the target gene sex-determining region Y-box 9 (Sox9). The function and mechanism of exosomal circRNA_0001236 in OA were explored in the DMM mouse model. Results Upregulation of exosomal circRNA_0001236 enhanced the expression of Col2a1 and Sox9 but inhibited that of MMP13 in hMSCs induced to undergo chondrogenesis. Moreover, circRNA_0001236 acted as an miR-3677-3p sponge and functioned in human chondrocytes via targeting miR-3677-3p and Sox9. Intra-articular injection of exosomal circRNA_0001236 attenuated OA in the DMM mouse model. Conclusions Our results reveal an important role for a novel exosomal circRNA_0001236 in chondrogenic differentiation. Overexpression of exosomal circRNA_0001236 promoted cartilage-specific gene and protein expression through the miR-3677-3p/Sox9 axis. Thus, circRNA_0001236-overexpressing exosomes may alleviate cartilage degradation, suppressing OA progression and enhancing cartilage repair. Our findings provide a potentially effective therapeutic strategy for treating OA.

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Porcine Milk-Derived Small Extracellular Vesicles Promote Intestinal Immunoglobulin Production through pIgR

Animals ◽

10.3390/ani11061522 ◽

2021 ◽

Vol 11 (6) ◽

pp. 1522

Author(s):

Bin Zeng ◽

Hailong Wang ◽

Junyi Luo ◽

Meiying Xie ◽

Zhengjiang Zhao ◽

...

Keyword(s):

Small Intestine ◽

Extracellular Vesicles ◽

Immunoglobulin A ◽

Luciferase Reporter ◽

Epithelial Cell Line ◽

Intestinal Immunity ◽

In Vivo Experiments ◽

Porcine Milk

Secretory immunoglobulin A (SIgA) plays an important role in gut acquired immunity and mucosal homeostasis. Breast milk is the irreplaceable nutritional source for mammals after birth. Current studies have shown the potential functional role of milk-derived small extracellular vesicles (sEVs) and their RNAs cargo in intestinal health and immune regulation. However, there is a lack of studies to demonstrate how milk-derived sEVs affect intestinal immunity in recipient. In this study, through in vivo experiments, we found that porcine milk small extracellular vesicles (PM-sEVs) promoted intestinal SIgA levels, and increased the expression levels of polymeric immunoglobulin receptor (pIgR) both in mice and piglet. We examined the mechanism of how PM-sEVs increased the expression level of pIgR in vitro by using a porcine small intestine epithelial cell line (IPEC-J2). Through bioinformatics analysis, dual-luciferase reporter assays, and overexpression or knockdown of the corresponding non-coding RNAs, we identified circ-XPO4 in PM-sEVs as a crucial circRNA, which leads to the expression of pIgR via the suppression of miR-221-5p in intestinal cells. Importantly, we also observed that oral administration of PM-sEVs increased the level of circ-XPO4 and decreased the level of miR-221-5p in small intestine of piglets, indicating that circRNAs in milk-derived sEVs act as sponge for miRNAs in recipients. This study, for the first time, reveals that PM-sEVs have a capacity to stimulate intestinal SIgA production by delivering circRNAs to receptors and sponging the recipient’s original miRNAs, and also provides valuable data for insight into the role and mechanism of animal milk sEVs in intestinal immunity.

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Preclinical Evidence of Nanomedicine Formulation to Target Mycobacterium tuberculosis at Its Bone Marrow Niche

Pathogens ◽

10.3390/pathogens9050372 ◽

2020 ◽

Vol 9 (5) ◽

pp. 372 ◽

Cited By ~ 1

Author(s):

Jaishree Garhyan ◽

Surender Mohan ◽

Vinoth Rajendran ◽

Rakesh Bhatnagar

Keyword(s):

Bone Marrow ◽

Mycobacterium Tuberculosis ◽

In Vitro Release ◽

Bone Marrow Niche ◽

Mice Model ◽

Latently Infected ◽

Marrow Mesenchymal Stem Cells

One-third of the world’s population is estimated to be latently infected with Mycobacterium tuberculosis (Mtb). Recently, we found that dormant Mtb hides in bone marrow mesenchymal stem cells (BM-MSCs) post-chemotherapy in mice model and in clinical subjects. It is known that residual Mtb post-chemotherapy may be responsible for increased relapse rates. However, strategies for Mtb clearance post-chemotherapy are lacking. In this study, we engineered and formulated novel bone-homing PEGylated liposome nanoparticles (BTL-NPs) which actively targeted the bone microenvironment leading to Mtb clearance. Targeting of BM-resident Mtb was carried out through bone-homing liposomes tagged with alendronate (Ald). BTL characterization using TEM and DLS showed that the size of bone-homing isoniazid (INH) and rifampicin (RIF) BTLs were 100 ± 16.3 nm and 84 ± 18.4 nm, respectively, with the encapsulation efficiency of 69.5% ± 4.2% and 70.6% ± 4.7%. Further characterization of BTLs, displayed by sustained in vitro release patterns, increased in vivo tissue uptake and enhanced internalization of BTLs in RAW cells and CD271+BM-MSCs. The efficacy of isoniazid (INH)- and rifampicin (RIF)-loaded BTLs were shown using a mice model where the relapse rate of the tuberculosis was decreased significantly in targeted versus non-targeted groups. Our findings suggest that BTLs may play an important role in developing a clinical strategy for the clearance of dormant Mtb post-chemotherapy in BM cells.

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