scholarly journals miRNA-124-3p.1 Inhibits the Osteogenic and Odontogenic Differentiation of SCAPs via MACF1/smad7 Axis

2021 ◽  
Author(s):  
Na Li ◽  
Zehan Li ◽  
Ming Yan ◽  
Yanqiu Wang ◽  
Yongchun Gu ◽  
...  
Keyword(s):  
Protein Level ◽  
Nuclear Translocation ◽  
Inhibitory Effect ◽  
Luciferase Reporter ◽  
Alizarin Red ◽  
Differentiation Potential ◽  
Altered Expression ◽  
Odontogenic Differentiation ◽  
Micro Rnas ◽  
Apical Papilla

Abstract Background: Previous research has indicated that altered expression of micro-RNAs (miRNAs) is in connection with differentiation of stem cells from apical papillae (SCAPs). We investigated the mechanisms that miR-124-3p.1 inhibited osteogenic and odontogenic differentiation of SCAPs. Methods: SCAPs were isolated from dental apical papilla. MiR-124-3p.1 mimic and inhibitor were used for overexpression and knockdown assays. For overexpression and knockdown of microtubule actin cross‐linking factor 1 (MACF1), lentivirus infection and siRNA transfection were performed. Luciferase reporter assay was performed to determine the relationship between miR-124-3p.1 and MACF1. The osteogenic and odontogenic differentiation potential was analyzed by alkaline phosphatase activity analysis (ALP), alizarin red S (ARS) staining, quantitative real time reverse-transcription polymerase chain reaction (qRT-PCR), western blot and immunofluorescence (IF) staining. Results: We observed a time dependent decrease of miR‐124‐3p.1 level in mineralization induction of SCAPs. Further study found that miR‐124‐3p.1 exhibited an inhibitory effect on SCAPs osteo/odontogenic differentiation. Similarly, we found that the overexpression of miR‐124‐3p.1 dramatically inhibited MACF1 protein level in SCAPs and knockdown of miR‐124‐3p.1 significantly increased MACF1 protein level in SCAPs. Moreover, MACF1 was verified as the targeting of miR‐124‐3p.1. Meanwhile, the expression of MACF1 was related to smad7 nuclear translocation.Conclusion: Collectively, diverse data demonstrated that miR‐124‐3p.1 is a regulator of MACF1/smad7, playing plays an important role in osteogenic and odontogenic differentiation of SCAPs via MACF1/smad7 axis.

MiR-144-3p Inhibits BMSC Proliferation and Osteogenic Differentiation Via Targeting FZD4 in Steroid-Associated Osteonecrosis

2020 ◽  
Vol 25 (45) ◽  
pp. 4806-4812 ◽  
Author(s):  
Zhibo Sun ◽  
Fei Wu ◽  
Yue Yang ◽  
Feng Liu ◽  
Fengbo Mo ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Nuclear Translocation ◽  
Bone Diseases ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Alizarin Red ◽  
Over Expression ◽  
Negative Effect ◽  
Proliferation Ability

Background: MicroRNAs have recently been recognized to be engaged in the development of bone diseases. Objective: This study was performed to elucidate the effects of miR-144-3p on proliferation and osteogenesis of mesenchymal stem cells (MSCs) from the patients with steroid-associated osteonecrosis (ONFH) and its related mechanism. Method: The expression level of miR-144-3p in the MSCs from the proximal femur of the patients was examined by Real-time PCR. The cell proliferation ability was assayed by MTT. The differentiation ability of MSCs was assayed by Alizarin Red S (ARS) staining. The interaction between miR-144-3p and frizzled4 (FZD4) was investigated by Real-time PCR, western blot and luciferase reporter assay. Results: ONFH samples had the obviously high expression of miR-144-3p compared to the control. MiR-144-3p had a negative effect on the proliferation and osteogenesis of MSCs. Via targeting FZD4, miR-144-3p decreased β-catenin nuclear translocation, the transcription of RUNX2 and COL1A1. Over-expression of FZD4 partially reversed miR-144-3p-induced decrease in the proliferation and osteogenesis of MSCs. Conclusion: MiR-144-3p might play an important role in the development of ONFH and might be used as a novel class of therapeutic targets for this disease.

scholarly journals Wnt16 signaling promotes osteoblast differentiation of periosteal derived cells in vitro and in vivo

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e10374
Author(s):  
Ying Jin ◽  
Xiaoyan Sun ◽  
Fang Pei ◽  
Zhihe Zhao ◽  
Jeremy Mao
Keyword(s):  
Bone Formation ◽  
Mrna Expression ◽  
Nuclear Translocation ◽  
Luciferase Activity ◽  
Fracture Repair ◽  
Luciferase Reporter ◽  
Control Group ◽  
Type I ◽  
Alizarin Red ◽  
Osteogenic Induction

Background Periosteum plays critical roles in de novo bone formation and fracture repair. Wnt16 has been regarded as a key regulator in periosteum bone formation. However, the role of Wnt16 in periosteum derived cells (PDCs) osteogenic differentiation remains unclear. The study goal is to uncover whether and how Wnt16 acts on the osteogenesis of PDCs. Methods We detected the variation of Wnt16 mRNA expression in PDCs, which were isolated from mouse femur and identified by flow cytometry, cultured in osteogenic medium for 14 days, then knocked down and over-expressed Wnt16 in PDCs to analysis its effects in osteogenesis. Further, we seeded PDCs (Wnt16 over-expressed/vector) in β-tricalcium phosphate cubes, and transplanted this complex into a critical size calvarial defect. Lastly, we used immunofluorescence, Topflash and NFAT luciferase reporter assay to study the possible downstream signaling pathway of Wnt16. Results Wnt16 mRNA expression showed an increasing trend in PDCs under osteogenic induction for 14 days. Wnt16 shRNA reduced mRNA expression of Runx2, collage type I (Col-1) and osteocalcin (OCN) after 7 days of osteogenic induction, as well as alizarin red staining intensity after 21days. Wnt16 also increased the mRNA expression of Runx2 and OCN and the protein production of Runx2 and Col-1 after 2 days of osteogenic stimulation. In the orthotopic transplantation assay, more bone volume, trabecula number and less trabecula space were found in Wnt16 over-expressed group. Besides, in the newly formed tissue Brdu positive area was smaller and Col-1 was larger in Wnt16 over-expressed group compared to the control group. Finally, Wnt16 upregulated CTNNB1/β-catenin expression and its nuclear translocation in PDCs, also increased Topflash reporter luciferase activity. By contrast, Wnt16 failed to increase NFAT reporter luciferase activity. Conclusion Together, Wnt16 plays a positive role in regulating PDCs osteogenesis, and Wnt16 may have a potential use in improving bone regeneration.

scholarly journals PD-1 Suppresses the Osteogenic and Odontogenic Differentiation of Stem Cells From Dental Apical Papilla via Targeting SHP2/NF-κB axis

2021 ◽  
Author(s):  
Na Li ◽  
Zehan Li ◽  
Lin Fu ◽  
Ming Yan ◽  
Yanqiu Wang ◽  
...  
Keyword(s):  
Vital Role ◽  
Cell Counting ◽  
Alizarin Red ◽  
Odontogenic Differentiation ◽  
Root Dentin ◽  
Apical Papilla ◽  
Effect Of Pd ◽  
First Time ◽  
The Relationship

Abstract BackgroundStem cells from the apical papilla (SCAPs) are important for tooth root development and regeneration of root dentin. Here, we examined the expression of programmed cell death protein-1 (PD-1) in SCAPs and investigated the effect of PD-1 on odontogenic and osteogenic differentiation and the relationship between PD-1 and cell differentiation and SHP2/NF-κB signals.MethodsSCAPs were obtained culture in the related medium. The proliferation ability was evaluated by cell counting kit 8 and 5‐ethynyl‐20‐deoxyuridine (EdU) assay. Alkaline phosphatase (ALP) activity assay, ALP staining, western blot, real-time RT-PCR, Alizarin Red S staining, and immunofluorescence staining were performed to explore the osteo/odontogenic potential and the involvement of SHP2/NF-κB pathways. Besides, we transplanted SCAPs component into mouse calvaria defects to evaluate osteogenesis in vivo. ResultsWe found that human SCAPs expressed PD-1 for the first time. PD-1 knockdown enhanced the osteo/odontogenic differentiation of SCAPs by suppressing SHP2 pathway and activating NF-κB pathway. Overexpression of PD-1 inhibited the osteogenesis and odontogenesis of SCAPs via activation of SHP2 signal and inhibition of NF-κB pathway. ConclusionPD-1 activated SHP2 signal to block NF-κB signal and then played a vital role in osteo/odontogenic differentiation of SCAPs.

scholarly journals MicroRNA-346-5p Regulates Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells by Inhibiting Transmembrane Protein 9

2020 ◽  
Vol 2020 ◽  
pp. 1-7
Author(s):  
Yicai Zhang ◽  
Yi Sun ◽  
Jinlong Liu ◽  
Yu Han ◽  
Jinglong Yan
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Protein Level ◽  
Molecular Mechanisms ◽  
Transmembrane Protein ◽  
Luciferase Reporter ◽  
Alizarin Red ◽  
Protein Levels

The molecular mechanisms how bone marrow-derived mesenchymal stem cells (BMSCs) differentiate into osteoblast need to be investigated. MicroRNAs (miRNAs) contribute to the osteogenic differentiation of BMSCs. However, the effect of miR-346-5p on osteogenic differentiation of BMSCs is not clear. This study is aimed at elucidating the underlying mechanism by which miR-346-5p regulates osteogenic differentiation of human BMSCs. Results of alkaline phosphatase (ALP) and Alizarin Red S (ARS) staining indicated that upregulation of miR-346-5p suppressed osteogenic differentiation of BMSCs, whereas downregulation of miR-346-5p enhanced this process. The protein levels of the osteoblastic markers Osterix and Runt-related transcription factor 2 (Runx2) were decreased in cells treated with miR-346-5p mimic at day 7 and day 14 after being differentiated. By contrast, downregulation of miR-346-5p elevated the protein levels of Osterix and Runx2. Moreover, a dual-luciferase reporter assay revealed that Transmembrane Protein 9 (TMEM9) was a target of miR-346-5p. In addition, the Western Blot results demonstrated that the TMEM9 protein level was significantly reduced by the miR-346-5p mimic whereas downregulation of miR-346-5p improved the protein level of TMEM9. These results together demonstrated that miR-346-5p served a key role in BMSC osteogenic differentiation of through targeting TMEM9, which may provide a novel target for clinical treatments of bone injury.

scholarly journals miR-6807-5p Inhibited the Odontogenic Differentiation of Human Dental Pulp Stem Cells Through Directly Targeting METTL7A

2021 ◽  
Vol 9 ◽  
Author(s):  
Ning Wang ◽  
Xiao Han ◽  
Haoqing Yang ◽  
Dengsheng Xia ◽  
Zhipeng Fan
Keyword(s):  
Stem Cells ◽  
Dental Pulp ◽  
Binding Protein ◽  
Dental Pulp Stem Cells ◽  
Luciferase Reporter ◽  
Control Group ◽  
Alizarin Red ◽  
Odontogenic Differentiation ◽  
Human Dental Pulp ◽  
Tooth Tissue

Background: Tooth tissue regeneration mediated by mesenchymal stem cells (MSCs) has become the most ideal treatment. Although the known regulatory mechanism and some achievements have been discovered, directional differentiation cannot effectively induce regeneration of tooth tissue. In this study, we intended to explore the function and mechanism of miR-6807-5p and its target gene METTL7A in odontogenic differentiation.Methods: In this study, human dental pulp stem cells (DPSCs) were used. Alkaline phosphatase (ALP), Alizarin red staining (ARS), and calcium ion quantification were used to detect the odontogenic differentiation of miR-6807-5p and METTL7A. Real-time RT-PCR, western blot, dual-luciferase reporter assay, and pull-down assay with biotinylated miRNA were used to confirm that METTL7A was the downstream gene of miR-6807-5p. Protein mass spectrometry and co-immunoprecipitation (Co-IP) were used to detect that SNRNP200 was the co-binding protein of METTL7A.Results: After mineralized induction, the odontogenic differentiation was enhanced in the miR-6807-5p-knockdown group and weakened in the miR-6807-5p-overexpressed group compared with the control group. METTL7A was the downstream target of miR-6807-5p. After mineralized induction, the odontogenic differentiation was weakened in the METTL7A-knockdown group and enhanced in the METTL7A-overexpressed group compared with the control group. SNRNP200 was the co-binding protein of METTL7A. The knockdown of SNRNP200 inhibited the odontogenic differentiation of DPSCs.Conclusion: This study verified that miR-6807-5p inhibited the odontogenic differentiation of DPSCs. The binding site of miR-6807-5p was the 3′UTR region of METTL7A, which was silenced by miR-6807-5p. METTL7A promoted the odontogenic differentiation of DPSCs. SNRNP200, a co-binding protein of METTL7A, promoted the odontogenic differentiation of DPSCs.

scholarly journals Depletion of SNRNP200 inhibits the osteo−/dentinogenic differentiation and cell proliferation potential of stem cells from the apical papilla

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Xiaomin Su ◽  
Haoqing Yang ◽  
Ruitang Shi ◽  
Chen Zhang ◽  
Huina Liu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Cell Proliferation ◽  
S Phase ◽  
Alizarin Red ◽  
Differentiation Potential ◽  
Alp Activity ◽  
Proliferation And Differentiation ◽  
Apical Papilla ◽  
Dentinogenic Differentiation

Abstract Background Tissue regeneration mediated by mesenchymal stem cells (MSCs) is deemed a desirable way to repair teeth and craniomaxillofacial tissue defects. Nevertheless, the molecular mechanisms about cell proliferation and committed differentiation of MSCs remain obscure. Previous researches have proved that lysine demethylase 2A (KDM2A) performed significant function in the regulation of MSC proliferation and differentiation. SNRNP200, as a co-binding factor of KDM2A, its potential effect in regulating MSCs’ function is still unclear. Therefore, stem cells from the apical papilla (SCAPs) were used to investigate the function of SNRNP200 in this research. Methods The alkaline phosphatase (ALP) activity assay, Alizarin Red staining, and osteogenesis-related gene expressions were used to examine osteo−/dentinogenic differentiation potential. Carboxyfluorescein diacetate, succinimidyl ester (CFSE) and cell cycle analysis were applied to detect the cell proliferation. Western blot analysis was used to evaluate the expressions of cell cycle-related proteins. Results Depletion of SNRNP200 caused an obvious decrease of ALP activity, mineralization formation and the expressions of osteo−/dentinogenic genes including RUNX2, DSPP, DMP1 and BSP. Meanwhile, CFSE and cell cycle assays revealed that knock-down of SNRNP200 inhibited the cell proliferation and blocked cell cycle at the G2/M and S phase in SCAPs. In addition, it was found that depletion of SNRNP200 up-regulated p21 and p53, and down-regulated the CDK1, CyclinB, CyclinE and CDK2. Conclusions Depletion of SNRNP200 repressed osteo−/dentinogenic differentiation potentials and restrained cell proliferation through blocking cell cycle progression at the G2/M and S phase, further revealing that SNRNP200 has crucial effects on preserving the proliferation and differentiation potentials of dental tissue-derived MSCs.

scholarly journals miR-140-3p enhanced the osteo/odontogenic differentiation of DPSCs via inhibiting KMT5B under hypoxia condition

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Han Zheng ◽  
Ning Wang ◽  
Le Li ◽  
Lihua Ge ◽  
Haichao Jia ◽  
...  
Keyword(s):  
Stem Cells ◽  
Tissue Regeneration ◽  
Molecular Mechanisms ◽  
Bone Tissue Regeneration ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Alizarin Red ◽  
Odontogenic Differentiation ◽  
Gene Assay ◽  
Dentinogenic Differentiation

AbstractHuman dental pulp stem cells (DPSCs) have emerged as an important source of stem cells in the tissue engineering, and hypoxia will change various innate characteristics of DPSCs and then affect dental tissue regeneration. Nevertheless, little is known about the complicated molecular mechanisms. In this study, we aimed to investigate the influence and mechanism of miR-140-3p on DPSCs under hypoxia condition. Hypoxia was induced in DPSCs by Cobalt chloride (CoCl2) treatment. The osteo/dentinogenic differentiation capacity of DPSCs was assessed by alkaline phosphatase (ALP) activity, Alizarin Red S staining and main osteo/dentinogenic markers. A luciferase reporter gene assay was performed to verify the downstream target gene of miR-140-3p. This research exhibited that miR-140-3p promoted osteo/dentinogenic differentiation of DPSCs under normoxia environment. Furthermore, miR-140-3p rescued the CoCl2-induced decreased osteo/odontogenic differentiation potentials in DPSCs. Besides, we investigated that miR-140-3p directly targeted lysine methyltransferase 5B (KMT5B). Surprisingly, we found inhibition of KMT5B obviously enhanced osteo/dentinogenic differentiation of DPSCs both under normoxia and hypoxia conditions. In conclusion, our study revealed the role and mechanism of miR-140-3p for regulating osteo/dentinogenic differentiation of DPSCs under hypoxia, and discovered that miR-140-3p and KMT5B might be important targets for DPSC-mediated tooth or bone tissue regeneration.

scholarly journals MiR-27a-3p promotes the osteogenic differentiation by activating CRY2/ERK1/2 axis

2021 ◽  
Vol 27 (1) ◽  
Author(s):  
Li-Rong Ren ◽  
Ru-Bin Yao ◽  
Shi-Yong Wang ◽  
Xiang-Dong Gong ◽  
Ji-Tao Xu ◽  
...  
Keyword(s):  
Osteogenic Differentiation ◽  
Osteoblast Differentiation ◽  
Inhibitory Effect ◽  
Luciferase Reporter ◽  
Induction Medium ◽  
Luciferase Reporter Gene ◽  
Alizarin Red ◽  
Protein Levels ◽  
Phosphorylation Level ◽  
Gene Assay

Abstract Background Osteoporosis seriously disturbs the life of people. Meanwhile, inhibition or weakening of osteogenic differentiation is one of the important factors in the pathogenesis of osteoporosis. It was reported that miR-27a-3p reduced the symptoms of osteoporosis. However, the mechanism by which miR-27a-3p in osteogenic differentiation remains largely unknown. Methods To induce the osteogenic differentiation in MC3T3-E1 cells, cells were treated with osteogenic induction medium (OIM). RT-qPCR was used to evaluate the mRNA expression of miR-27a-3p and CRY2 in cells. The protein levels of CRY2, Runt-related transcription factor 2 (Runx2), osteopontin (OPN), osteocalcin (OCN) and the phosphorylation level of extracellular regulated protein kinases (ERK) 1/2 in MC3T3-E1 cells were evaluated by western blotting. Meanwhile, calcium nodules and ALP activity were tested by alizarin red staining and ALP kit, respectively. Luciferase reporter gene assay was used to analyze the correlation between CRY2 and miR-27a-3p. Results The expression of miR-27a-3p and the phosphorylation level of ERK1/2 were increased by OIM in MC3T3-E1 cells, while CRY2 expression was decreased. In addition, OIM-induced increase of calcified nodules, ALP content and osteogenesis-related protein expression was significantly reversed by downregulation of miR-27a-3p and overexpression of CRY2. In addition, miR-27a-3p directly targeted CRY2 and negatively regulated CRY2. Meanwhile, the inhibitory effect of miR-27a-3p inhibitor on osteogenic differentiation was reversed by knockdown of CRY2 or using honokiol (ERK1/2 signal activator). Furthermore, miR-27a-3p significantly inhibited the apoptosis of MC3T3-E1 cells treated by OIM. Taken together, miR-27a-3p/CRY2/ERK axis plays an important role in osteoblast differentiation. Conclusions MiR-27a-3p promoted osteoblast differentiation via mediation of CRY2/ERK1/2 axis. Thereby, miR-27a-3p might serve as a new target for the treatment of osteoporosis.

scholarly journals LINC00899 promotes osteogenic differentiation and alleviates osteoporosis via targeting miR-374a to regulate RUNX2

2020 ◽  
Author(s):  
Xiaoya Gao ◽  
Yun Xue ◽  
Kechun Yang
Keyword(s):  
Osteogenic Differentiation ◽  
Molecular Mechanisms ◽  
Rna Binding ◽  
Quantitative Polymerase Chain Reaction ◽  
Direct Interaction ◽  
Inhibitory Effect ◽  
Luciferase Reporter ◽  
Bone Mesenchymal Stem Cells ◽  
Alizarin Red ◽  
Osteogenic Induction

Abstract Objective: This study aims to illustrate the underlying molecular mechanisms of long noncoding RNAs (LncRNAs) LINC00899 in osteoporosis.Methods: Real Time-quantitative Polymerase Chain Reaction (RT-qPCR) was used to examine the levels of LINC00899, miR-374a and RUNX2 in clinical tissues or human bone mesenchymal stem cells (hBMSCs). The interaction between miR-374a and LINC00899 or RUNX2 was predicted by starBase and verified by luciferase reporter assay and RNA binding protein immunoprecipitation (RIP) assay. Alkaline phosphatase (ALP) activity and Alizarin Red S (ARS) staining were also used to evaluate the osteogenic ability of hBMSCs.Results: The expression levels of LINC00899 were gradually increased, but miR-374a expression was decreased with the prolongation of osteogenic induction. In addition, the expression of LINC00899 was lowly expressed in osteoporotic patients’ bone tissues and knockdown of LINC00899 decreased the expression of osteogenesis-related genes. Moreover, LINC00899 was confirmed to inhibit miR-374a expression by direct interaction. Finally, we demonstrated that RUNX2 was a target of miR-374a, and the silencing of miR-374a partially abolished the inhibitory effect of LINC00899 knockdown on the expression of RUNX2, OPN and OCN.Conclusions: We demonstrated that LINC00899 facilitated the osteogenic differentiation of hBMSCs and prevented osteoporosis by sponging miR-374a and enhancing RUNX2 expression, which might provide a useful therapeutic strategy for osteoporosis patients.

scholarly journals Circular RNA SIPA1L1 regulates osteoblastic differentiation of stem cells from apical papilla via miR-204-5p/ALPL pathway

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Yuzhi Li ◽  
Minxia Bian ◽  
Zhou Zhou ◽  
Xiao Wu ◽  
Xingyun Ge ◽  
...  
Keyword(s):  
Stem Cells ◽  
Osteogenic Differentiation ◽  
Molecular Mechanisms ◽  
Circular Rna ◽  
Osteoblastic Differentiation ◽  
Luciferase Reporter ◽  
Western Blot Assay ◽  
Alizarin Red ◽  
Complex Biological Process ◽  
Apical Papilla

Abstract Background Osteogenesis is a complex biological process which requires the coordination of multiple molecular mechanisms. This research aimed to explore the biological role and underlying regulatory mechanism of circSIPA1L1 during the osteogenic differentiation of stem cells from apical papilla (SCAPs). Methods EdU retention assay, flow cytometry assay, and CCK-8 assay were used to evaluate the proliferation capacity of SCAPs. Western blot assay, alkaline phosphatase (ALP), and alizarin red staining (ARS) were conducted to investigate the biological roles of circSIPA1L1 and miR-204-5p. Fluorescence in situ hybridization was applied for circSIPA1L1 localization. Dual-luciferase reporter assay was performed to prove the interaction of circSIPA1L1 and miR-204-5p. Results CircSIPA1L1 had no significant effect on the proliferative capacity of SCAPs. CircSIPA1L1 promotes osteogenic differentiation of SCAPs by serving as a miRNA sponge for miR-204-5p. Either knockdown of circSIPA1L1 or overexpression of miR-204-5p significantly suppresses osteogenic differentiation of SCAPs. Conclusions CircSIPA1L1 upregulates ALPL through targeting miR-204-5p and promotes the osteogenic differentiation of SCAPs.

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