miR-143 Inhibits Osteogenic Differentiation of Umbilical Cord Mesenchymal Stem Cell by Targeting Zinc Fingers and Homeoboxes 3

2019 ◽  
Vol 9 (11) ◽  
pp. 1550-1556
Author(s):  
Haiying Chen ◽  
Lei Zhang ◽  
Lijun Tao
Keyword(s):  
Stem Cell ◽  
Mesenchymal Stem Cell ◽  
Western Blot ◽  
Osteogenic Differentiation ◽  
Umbilical Cord ◽  
Osteoblast Differentiation ◽  
Bioinformatics Analysis ◽  
Zinc Fingers ◽  
Alizarin Red ◽  
Msc Differentiation

The transcription regulating factor-zinc fingers and homeoboxes 3 (ZHX3) involve in osteoblast differentiation. miR-143 regulates osteoblast differentiation. Bioinformatics analysis revealed a relationship between miR-143 and the 3′ -UTR of ZHX3. This study assessed miR-143's role in UC-MSC differentiation. The UC-MSCs were induced to differentiate into osteoblasts. The expressions of miR-143, ZHX3, RUNX2, Osteopontin (OPN) and Osteocalcin (OC) genes were detected by qRTPCR on days 0, 20 and 30. The UC-MSCs cells were divided into agomir-NC group and miR-143 agomir group. After 30 days of differentiation, the expression of ZHX3, RUNX2, OPN and OC protein was detected by western blot. During differentiation of UC-MSCs into osteoblasts, miR-143 expression was decreased, ZHX3 expression, ALP activity and alizarin red staining was increased. There was a targeted regulation relationship between miR-143 and ZHX3. Transfection of agomir143 significantly downregulated ZHX3, RUNX2, OPN, OC genes and proteins, significantly reduced alizarin red staining, and decreased the ability of osteoblast differentiation. Decreased miR-143 expression plays a role in up-regulating ZHX3 expression and promoting osteoblast differentiation of UC-MSCs. Overexpression of miR-143 can inhibit UC-MSCs differentiation into osteoblasts by targeting ZHX3.

scholarly journals Melatonin promotes bone marrow mesenchymal stem cell osteogenic differentiation and prevents osteoporosis development through modulating circ_0003865 that sponges miR-3653-3p

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Xudong Wang ◽  
Taiqiu Chen ◽  
Zhihuai Deng ◽  
Wenjie Gao ◽  
Tongzhou Liang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Mesenchymal Stem Cell ◽  
Osteogenic Differentiation ◽  
Luciferase Reporter ◽  
Specific Gene ◽  
Biological Processes ◽  
Alizarin Red ◽  
Qrt Pcr ◽  
Dual Luciferase Reporter Assay

Abstract Background Little is known about the implications of circRNAs in the effects of melatonin (MEL) on bone marrow mesenchymal stem cell (BMSC) osteogenic differentiation and osteoporosis (OP) progression. The aim of our study was to investigate circRNAs in MEL-regulated BMSC differentiation and OP progression. Methods BMSC osteogenic differentiation was measured by qRT-PCR, western blot (WB), Alizarin Red, and alkaline phosphatase (ALP) staining. Differential circRNA and mRNA profiles of BMSCs treated by MEL were characterized by deep sequencing, followed by validation using RT-PCR, Sanger sequencing, and qRT-PCR. Silencing and overexpression of circ_0003865 were conducted for functional investigations. The sponged microRNAs and targeted mRNAs were predicted by bioinformatics and validated by qRT-PCR, RNA pull-down, and dual-luciferase reporter assay. The function of miR-3653-3p and circ_0003865/miR-3653-3p/growth arrest-specific gene 1 (GAS1) cascade was validated for the osteogenic differentiation of BMSCs by CCK-8, qRT-PCR, WB, Alizarin Red, and ALP staining. The effects of circ_0003865 on OP development were tested in murine OP model. Results MEL promoted osteogenic differentiation of BMSCs. RNA sequencing revealed significant alterations in circRNA and mRNA profiles associated with multiple biological processes and signaling pathways. Circ_0003865 expression in BMSCs was significantly decreased by MEL treatment. Silencing of circ_0003865 had no effect on proliferation while promoted osteogenic differentiation of BMSCs. Overexpression of circ_0003865 abrogated the promotion of BMSC osteogenic differentiation induced by MEL, but proliferation of BMSCs induced by MEL had no change whether circ_0003865 was overexpression or not. Furthermore, circ_0003865 sponged miR-3653-3p to promote GAS1 expression in BMSCs. BMSC osteogenic differentiation was enhanced by miR-3653-3p overexpression while BMSC proliferation was not affected. By contrast, miR-3653-3p silencing mitigated the promoted BMSC osteogenic differentiation caused by circ_0003865 silencing, but had no effect on proliferation. Finally, circ_0003865 silencing repressed OP development in mouse model. Conclusion MEL promotes BMSC osteogenic differentiation and inhibits OP pathogenesis by suppressing the expression of circ_0003865, which regulates GAS1 gene expression via sponging miR-3653-3p.

scholarly journals Bone Marrow Mesenchymal Stem Cell-Derived Exosomal miR-25 Regulates the Ubiquitination and Degradation of Runx2 by SMURF1 to Promote Fracture Healing in Mice

2020 ◽  
Vol 7 ◽  
Author(s):  
Yikun Jiang ◽  
Jun Zhang ◽  
Zhengwei Li ◽  
Guoliang Jia
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Mesenchymal Stem Cell ◽  
Fracture Healing ◽  
Osteogenic Differentiation ◽  
Bioinformatics Analysis ◽  
Luciferase Reporter ◽  
Gene Assay ◽  
And Migration ◽  
Proliferation And Migration

Recent evidence has demonstrated that mesenchymal stem cells (MSCs) can release a large number of functionally specific microRNA (miRNA) microvesicles that play a role in promoting osteogenic differentiation, but the specific mechanism is not yet clear. Under such context, this study aims to elucidate the mechanism of bone marrow mesenchymal stem cell-derived exosomes (BMSC-Exo) promoting fracture healing in mice. We isolated and identified the BMSC-Exo. Bioinformatics analysis predicted high expression of miRNA in exosomes and verified the transfer of miR-25 in exosomes by immunofluorescence. Targeting relationship between miR-25 and Smad ubiquitination regulatory factor-1 (SMURF1) was predicted and verified by dual-luciferase reporter gene assay. Immunoprecipitation and protein stability assays were used to detect Runt-related transcription factor 2 (Runx2) ubiquitination and the effect of SMURF1 on Runx2 ubiquitination, respectively. The effect of miR-25 in BMSC-Exo on fracture healing in mice was assessed using X-ray imaging. alkaline phosphatase, alizarin red staining, EdU, CCK-8, and Transwell were used to evaluate the effects of exosomes transferred miR-25 on osteogenic differentiation, proliferation, and migration of osteoblasts. Bioinformatics analysis predicted that miR-25 expression in exosomes increased significantly. Moreover, the targeted regulation of SMURF1 by miR-25 was verified. SMURF1 inhibited Runx2 protein expression by promoting ubiquitination degradation of Runx2. Notably, miR-25 secreted by BMSC-Exo can accelerate osteogenic differentiation, proliferation, and migration of osteoblasts through SMURF1/Runx2 axis. Our results demonstrate that miR-25 in BMSC-Exo regulates the ubiquitination degradation of Runx2 by SMURF1 to promote fracture healing in mice.

scholarly journals The Role and Mechanism of Exosomes from Umbilical Cord Mesenchymal Stem Cells in Inducing Osteogenesis and Preventing Osteoporosis

2021 ◽  
Vol 30 ◽  
pp. 096368972110574
Author(s):  
Ge Yahao ◽  
Wang Xinjia
Keyword(s):  
Stem Cell ◽  
Osteogenic Differentiation ◽  
Umbilical Cord ◽  
Cell Counting ◽  
Control Group ◽  
Human Umbilical Cord ◽  
Promoting Effect ◽  
Alizarin Red ◽  
Osteogenic Induction

Mesenchymal stem cell (MSC) exosomes promote tissue regeneration and repair, and thus might be used to treat many diseases; however, the influence of microenvironmental conditions on exosomes remains unclear. The present study aimed to analyze the effect of osteogenic induction on the functions of human umbilical cord MSC (HucMSC)-derived exosomes. Exosomes from standardized stem cell culture (Exo1) and osteogenic differentiation-exosomes (Exo2) were co-cultured with osteoblasts, separately. Cell counting kit-8 assays, alkaline phosphatase and alizarin red staining were used to observe the exosomes’ effects on osteoblast proliferation and differentiation. The levels of osteogenic differentiation-related proteins were analyzed using western blotting. Estrogen-deficient osteoporosis model mice were established, and treated with the two exosome preparations. Micro-computed tomography and hematoxylin and eosin staining were performed after 6 weeks. MicroRNAs in Exo1 and Exo2 were sequenced and analyzed using bioinformatic analyses. Compared with Exo1 group, Exo2 had a stronger osteogenic differentiation promoting effect, but a weaker proliferation promoting effect. In ovariectomy-induced osteoporosis mice, both Exo1 and Exo2 improved the tibial density and reversed osteoporosis in vivo. High-throughput microRNA sequencing identified 221 differentially expressed microRNAs in HucMSC-derived exosomes upon osteogenic induction as compared with the untreated control group. Importantly, we found that 41 of these microRNAs are potentially critical for MSC-secreted exosomes during osteogenic induction. Mechanistically, exosomal miRNAs derived from osteogenic induced-HucMSCs are involved in bone development and differentiation, such as osteoclast differentiation and the MAPK signaling pathway. The expression of hsa-mir-2110 and hsa-mir-328-3p gradually increased with prolonged osteogenic differentiation and regulated target genes associated with bone differentiation, suggesting that they are probably the most important osteogenesis regulatory microRNAs in exosomes. In conclusion, we examined the contribution of osteogenic induction to the function of exosomes secreted by HucMSCs following osteogenic differentiation in vitro and in vivo, and reveal the underlying molecular mechanisms of exosome action during osteoporosis.

scholarly journals Melatonin Promotes Bone Marrow Mesenchymal Stem Cell Osteogenic Differentiation and Prevents Osteoporosis Development Through Modulating circ_0003865 that Sponges miR-3653-3p

2020 ◽  
Author(s):  
Xudong Wang ◽  
Taiqiu Chen ◽  
Zhihuai Deng ◽  
Wenjie Gao ◽  
Tongzhou Liang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Mesenchymal Stem Cell ◽  
Osteogenic Differentiation ◽  
Luciferase Reporter ◽  
Specific Gene ◽  
Biological Processes ◽  
Alizarin Red ◽  
Qrt Pcr ◽  
Dual Luciferase Reporter Assay

Abstract Background: To investigate circRNAs in Melatonin (MEL)-regulated bone marrow mesenchymal stem cell (BMSC) differentiation and osteoporosis.Methods: BMSC osteogenic differentiation was measured by qRT-PCR, western blot (WB), Alizarin Red and alkaline phosphatase (ALP) staining. Differential circRNA and mRNA profiles of BMSCs treated by MEL were characterized by deep sequencing, followed by validation using RT-PCR, Sanger sequencing, and qRT-PCR. Silencing and overexpression of circ_0003865 were conducted for functional investigations. The sponged microRNAs and targeted mRNAs were predicted by bioinformatics and validated by qRT-PCR, RNA pull-down, and dual-luciferase reporter assay. The function of miR-3653-3p and circ_0003865/miR-3653-3p/growth arrest-specific gene 1 (GAS1) cascade were validated for the osteogenic differentiation of BMSCs by qRT-PCR, WB, Alizarin Red, and ALP staining. The effects of circ_0003865 on osteoporosis (OP) development was tested in murine osteoporosis model.Results: MEL promoted osteogenic differentiation of BMSCs. RNA sequencing revealed significant alterations in circRNA and mRNA profiles associated with multiple biological processes and signaling pathways. Circ_0003865 expression in BMSCs was significantly decreased by MEL treatment. Silencing of circ_0003865 promoted osteogenic differentiation of BMSCs. Overexpression of circ_0003865 abrogated the promotion of BMSCs osteogenic differentiation induced by MEL. Furthermore, circ_0003865 sponged miR-3653-3p to promote GAS1 expression in BMSCs. BMSC osteogenic differentiation was enhanced by miR-3653-3p overexpression. By contrast, miR-3653-3p silencing mitigated the promoted BMSC osteogenic differentiation caused by circ_0003865 silencing. Finally, circ_0003865 silencing repressed OP development in mouse model.Conclusion: MEL promotes BMSC osteogenic differentiation and inhibits osteoporosis pathogenesis by suppressing the expression of circ_0003865, which regulates GAS1 gene expression via sponging miR-3653-3p.

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

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.

scholarly journals Leonurine Promotes the Osteoblast Differentiation of Rat BMSCs by Activation of Autophagy via the PI3K/Akt/mTOR Pathway

2021 ◽  
Vol 9 ◽  
Author(s):  
Bingkun Zhao ◽  
Qian Peng ◽  
Enoch Hin Lok Poon ◽  
Fubo Chen ◽  
Rong Zhou ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Western Blot ◽  
Osteogenic Differentiation ◽  
Osteoblast Differentiation ◽  
Mtor Pathway ◽  
Control Group ◽  
Alizarin Red ◽  
Qrt Pcr

BackgroundLeonurine, a major bioactive component from Herba leonuri, has been shown to exhibit anti-inflammatory and antioxidant effects. The aim of this study was to investigate the effect of leonurine on bone marrow-derived mesenchymal stem cells (BMSCs) as a therapeutic approach for treating osteoporosis.Materials and MethodsRat bone marrow-derived mesenchymal stem cells (rBMSCs) were isolated from 4-weeks-old Sprague–Dawley rats. The cytocompatibility of leonurine on rBMSCs was tested via CCK-8 assays and flow cytometric analyses. The effects of leonurine on rBMSC osteogenic differentiation were analyzed via ALP staining, Alizarin red staining, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blot. Additionally, autophagy-related markers were examined via qRT-PCR and Western blot analyses of rBMSCs during osteogenic differentiation with leonurine and with or without 3-methyladenine (3-MA) as an autophagic inhibitor. Finally, the PI3K/Akt/mTOR signaling pathway was evaluated during rBMSC osteogenesis.ResultsLeonurine at 2–100 μM promoted the proliferation of rBMSCs. ALP and Alizarin red staining results showed that 10 μM leonurine promoted rBMSC osteoblastic differentiation, which was consistent with the qRT-PCR and Western blot results. Compared with those of the control group, the mRNA and protein levels of Atg5, Atg7, and LC3 were upregulated in the rBMSCs upon leonurine treatment. Furthermore, leonurine rescued rBMSC autophagy after inhibition by 3-MA. Additionally, the PI3K/AKT/mTOR pathway was activated in rBMSCs upon leonurine treatment.ConclusionLeonurine promotes the osteoblast differentiation of rBMSCs by activating autophagy, which depends on the PI3K/Akt/mTOR pathway. Our results suggest that leonurine may be a potential treatment for osteoporosis.

Impact of Zinc Fingers and Homeoboxes 3 on the Regulation of Mesenchymal Stem Cell Osteogenic Differentiation

2011 ◽  
Vol 20 (9) ◽  
pp. 1539-1547 ◽  
Author(s):  
Fumio Suehiro ◽  
Masahiro Nishimura ◽  
Takeshi Kawamoto ◽  
Masami Kanawa ◽  
Yuu Yoshizawa ◽  
...  
Keyword(s):  
Stem Cell ◽  
Mesenchymal Stem Cell ◽  
Osteogenic Differentiation ◽  
Zinc Fingers

The role and mechanism of exosomes from umbilical cord mesenchymal stem cells in inducing osteogenesis and preventing osteoporosis

2020 ◽  
Author(s):  
Yahao Ge ◽  
Xinjia Wang
Keyword(s):  
Stem Cell ◽  
Osteogenic Differentiation ◽  
Umbilical Cord ◽  
Osteoclast Differentiation ◽  
Cell Counting ◽  
Human Umbilical Cord ◽  
Dependent Manner ◽  
Promoting Effect ◽  
Alizarin Red ◽  
Gene And Protein Expression

Abstract BackgroundMesenchymal stem cell (MSC) exosomes promote tissue regeneration and repair, and thus might be used to treat many diseases; however, the influence of microenvironmental conditions on exosomes remains unclear. The present study aimed to analyze the effect of the osteogenic differentiation microenvironment on the functions of human umbilical cord MSCs (HucMSC)-derived exosomes. We explored the role and mechanism by which exosomes promote osteogenic differentiation and prevent osteoporosis, and propose a method to treat and prevent osteoporosis.MethodsHucMSCs were isolated from human umbilical cords, and after osteogenic differentiation and normal culture for 48 h, cell supernatants were collected to isolate exosomes. Exosomes from standardized stem cell culture (Exo1) and osteogenic differentiation-exosomes (Exo2) were co-cultured with osteoblasts, separately. Cell counting kit-8 assays, and alkaline phosphatase and alizarin red staining were used to observe the exosomes’ effects on osteoblast proliferation and differentiation. The levels of osteogenic differentiation-related proteins were analyzed using western blotting. Estrogen-deficient osteoporosis model mice were established, and treated with the two exosomes preparations. Micro-computed tomography and hematoxylin and eosin staining were performed after 6 weeks. MicroRNAs in Exo1 and Exo2 were sequenced and analyzed using bioinformatics.ResultsTreatment with Exo1 and Exo2 enhanced osteoblast osteogenic differentiation significantly, and osteogenic differentiation-related gene and protein expression increased significantly in a concentration-dependent manner. Compared with Exo1 group, Exo2 had a stronger osteogenic differentiation promoting effect, but a weaker proliferation promoting effect. Exo1 and Exo2 significantly improved the tibial density of osteoporosis model mice, with no significant differences between the groups. Sequencing and bioinformatics analysis showed that hsa-mir-328-3p and hsa-mir-2110 might be exosome osteogenesis regulatory microRNAs. Compared with Exo1, the target genes of Exo2-carried microRNAs are enriched in osteoclast differentiation and PPAR signaling. ConclusionsHucMSC-derived exosomes can promote osteogenic differentiation. Exosomes produced in an osteogenic differentiation microenvironment had a stronger osteogenic function, but a weaker proliferative effect. HucMSC-derived exosomes have a therapeutic effect on osteoporosis. The exosomes induced by osteoblasts carry microRNAs that regulate osteoblast differentiation, which might function via osteoblast, adipocyte, and osteoclast differentiation signaling pathways.

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