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 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 ◽  
Osteogenic Induction ◽  
Rip Assay

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) 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. Results: 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. the expression levels of LINC00899 were gradually increased, but miR-374a expression was decreased with the prolongation of osteogenic induction. 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 osteogenic differentiation of hBMSCs and prevent osteoporosis by sponging miR-374a and enhancing RUNX2 expression, which might provide a useful therapeutic strategy for osteoporosis patients.

scholarly journals MiR-26b-5p Promotes Osteogenesis of Bone Mesenchymal Stem Cells via Suppressing FGF21

2021 ◽  
Author(s):  
Bin Wang ◽  
Zhenhui Li ◽  
Caiyuan Mai ◽  
Penglin Mou ◽  
Lei Pan
Keyword(s):  
Osteogenic Differentiation ◽  
Luciferase Reporter ◽  
Control Group ◽  
Osteogenic Differentiation Medium ◽  
Bone Mesenchymal Stem Cells ◽  
Alizarin Red ◽  
Osteogenic Induction ◽  
Direct Target ◽  
Inhibition Group ◽  
Fgf21 Expression

Abstract Introduction: It has been established that miR-26b-5p actively participate in the osteogenic differentiation of bone mesenchymal stem cells (BMSCs), which is of great value in osteoporosis treatment. Database showed that Fibroblast growth factor(FGF)21 is a potential binding site of miR-26b-5p. This study aimed to investigate the molecular osteogenic mechanisms of miR-26b-5p targeting FGF21 in postmenopausal osteoporosis (PMOP). Methods: 5ml of bone marrow was aspirated from the anterior superior iliac spine in 10 PMOP women during bone marrow puncture. BMSCs were used to establish an in vitro cell model, and BMSCs markers were analyzed by flow cytometry. miR-26b-5p and FGF21 were overexpressed for 48h, and then placed in an osteogenic induction medium for osteogenic induction culture, the expression of RNA was detect using RT-qPCR. Cells from miR-26b-5p group were collected on days 7, 14 and 21 of induction for ALP and alizarin red S staining. On day 7 of induction, RT-qPCR was used to measure Runx2, Osterix (Osx), and target gene FGF21 expression levels in each group. The dual-luciferase reporter gene system was used to verify that FGF21 was a direct target of miR-26b-5p. FGF21 was measured by western blotting in the miR-26b-5p overexpression group and in the miR-26b-5p inhibition group. Results: BMSCs were identified according with the antigenic characteristics. miR-26b-5p expression was significantly upregulated after the expression of miR-26b-5p mimics; however, FGF21 expression was downregulated after FGF21 mimics. After overexpression of miR-26b-5p, the alkaline phosphatase activity and nodules of alizarin red S in the culture medium gradually increased as the induction time increased. RT-qPCR showed that the expressions of master osteogenic factors Runx2 and Osx in the BMSC+ osteogenic differentiation medium group was significantly higher than in the BMSC group, the expressions of the factors in the BMSC+ miR-26b-5p overexpression group was significantly higher than in the control group. Target gene FGF21 expression was significantly lower in the BMSC+ osteogenic differentiation medium group than in the BMSC group, and was significantly lower in the BMSC+ miR-26b-5p overexpression group than in the control group. Luciferase reporter assays demonstrated that FGF21 was a direct target of miR-26b-5p. Finally, western blotting analysis showed that FGF21 expression was significantly downregulated in the miR-26b-5p overexpressed group and upregulated in the miR-26b-5p inhibition group. Conclusion: miR-26b-5p can regulate the osteogenic differentiation of BMSCs and participate in PMOP pathogenesis via suppressing FGF21. The present study provides the basis for further studies on PMOP.

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 MicroRNA-409-3p promotes osteoblastic differentiation via activation of Wnt/β-catenin signaling pathway by targeting SCAI

2020 ◽  
Author(s):  
Nan Chen ◽  
Hao Yang ◽  
Lijun Song ◽  
Hua Li ◽  
Yi Liu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Osteogenic Differentiation ◽  
Signaling Pathway ◽  
Quantitative Polymerase Chain Reaction ◽  
Osteoblastic Differentiation ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Complementary Sequence ◽  
Alizarin Red ◽  
Gene Assay

Osteogenic differentiation is an important process of new bone formation, miR-409-3p has been reported to be upregulated in osteogenic differentiation of human bone marrow mesenchymal stem cells (MSCs). To investigate the regulatory effect of miR-409-3p on osteogenic differentiation of MSCs and its molecular mechanism, the expression of miR-409-3p in osteoblast (HCO) and bone marrow-derived MSCs (MSC-A, MSC-B, MSC-U) were detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The binding of miR-409-3p to SCAI in MSC-B was investigated by dual-luciferase reporter gene assay. MSC-B were selected to transfect with miR-409-3p analog/complementary sequence (cs), miR-409-3p analog + SCAI and miR-409-3p cs + small interfering (si)-SCAI, as well as control, respectively. The alkaline phosphatase activity, alizarin red staining, and the expression of osteogenic markers in MSC-B during osteoblastic differentiation were tested by RT-qPCR and Western blotting, respectively. The Wnt/β-catenin pathway was inhibited by dickkopf-related protein 1 to get the roles of miR-409-3p during the osteoblastic differentiation of MSC-B when transfected with miR-409-3p analog. The expression of miR-409-3p in HCO was higher than that in these three MSCs, showing an increasing time-dependent trend on the 0 and 21th day of osteoblastic differentiation. MiR-409-3p directly regulated SCAI by targeting SCAI 3′UTR. Further, miR-409-3p suppressed SCAI expression, but SCAI upregulation suppressed the osteoblastic differentiation, as well as reduced the relative mRNA/protein expression of Wnt/β-catenin signaling pathway-related genes. Importantly, disruption of Wnt signaling also blocked miR-409-3p induced osteoblastic differentiation of MSCs. Therefore, miR-409-3p promotes osteoblastic differentiation through the activation of the Wnt/β-catenin pathway by downregulating SCAI expression.

miR-217 Suppresses Osteogenic Differentiation of Human Dental Pulp Stem Cells by Down-Regulating Sirtuin 1

2020 ◽  
Vol 10 (7) ◽  
pp. 978-986
Author(s):  
Haiquan Yue ◽  
Yidan Guo ◽  
Juan Song ◽  
Ruimin Liu
Keyword(s):  
Stem Cells ◽  
Osteogenic Differentiation ◽  
Dental Pulp ◽  
Dental Pulp Stem Cells ◽  
Sirtuin 1 ◽  
Luciferase Reporter ◽  
Induction Medium ◽  
Alizarin Red ◽  
Human Dental Pulp ◽  
Osteogenic Induction

The paper is committed to uncovering the effect of miR-217 on osteogenic differentiation of human dental pulp stem cells (hDPSCs) and its mechanism. hDPSCs were separated from human dental pulp tissues for measurement of stemness. The osteogenic differentiation of hDPSCs was induced in an osteogenic induction medium. The hDPSCs were transfected with miR-217 mimic, miR-217 inhibitor and/or sh-SIRT1 accordingly. The expressions of miR-217 and SIRT1 were detected in hDPSCs after cell transfection and osteogenic differentiation. Calcium nodules were showed by alizarin red staining. Moreover, the expressions of osteogenic differentiation-related genes were also assessed. The binding of miR-217 to SIRT1 was predicted on starBase and further determined by dual-luciferase reporter assay. Down-regulated miR-217 and up-regulated SIRT1 were found during osteogenic differentiation of hDPSCs. The osteogenic differentiation of hDPSCs was suppressed after transfection of miR-217 mimic or sh-SIRT1 while promoted by miR-217 inhibition. Taken together, miR-217 can suppress osteogenic differentiation of hDPSCs by negatively regulating SIRT1.

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 Hsa_circ_0066523 promotes the proliferation and osteogenic differentiation of bone mesenchymal stem cells by repressing PTEN

2021 ◽  
Vol 10 (8) ◽  
pp. 526-535
Author(s):  
Wei Xin ◽  
Shuai Yuan ◽  
Bo Wang ◽  
Qirong Qian ◽  
Yi Chen
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Akt Pathway ◽  
Bone Mesenchymal Stem Cells ◽  
Alizarin Red ◽  
Proliferation And Differentiation ◽  
The Impact

Aims Circular RNAs (circRNAs) are a novel type of non-coding RNA that plays major roles in the development of diverse diseases including osteonecrosis of the femoral head (ONFH). Here, we explored the impact of hsa_circ_0066523 derived from forkhead box P1 (FOXP1) (also called circFOXP1) on bone mesenchymal stem cells (BMSCs), which is important for ONFH development. Methods RNA or protein expression in BMSCs was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) or western blot, respectively. Cell Counting Kit 8 (CCK8) and 5-ethynyl-2’-deoxyuridine (EdU) were used to analyze cell proliferation. Alkaline phosphatase (ALP) activity, ALP staining, and Alizarin Red S staining were employed to evaluate the osteoblastic differentiation. Chromatin immunoprecipitation (ChIP), luciferase reporter, RNA pull down, and RNA immunoprecipitation (RIP) assays were combined for exploring molecular associations. Results Circ_0066523 was upregulated in osteogenic induction process of BMSCs. Silencing circ_0066523 restrained the proliferation and osteogenic differentiation of BMSCs. Mechanistically, circ_0066523 activated phosphatidylinositol-4,5-bisphosphate 3-kinase / AKT serine/threonine kinase 1 (PI3K/AKT) pathway via recruiting lysine demethylase 5B (KDM5B) to epigenetically repress the transcription of phosphatase and tensin homolog (PTEN). Functionally, AKT signalling pathway agonist or PTEN knockdown counteracted the effects of silenced circ_0066523 on BMSC proliferation and differentiation. Conclusion Circ_0066523 promotes the proliferation and differentiation of BMSCs by epigenetically repressing PTEN and therefore activating AKT pathway. This finding might open new avenues for the identification of therapeutic targets for osteoblast differentiation related diseases such as ONFH. Cite this article: Bone Joint Res 2021;10(8):526–535.

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.

scholarly journals Plastrum Testudinis Extracts Promote BMSC Proliferation and Osteogenic Differentiation by Regulating Let-7f-5p and the TNFR2/PI3K/AKT Signaling Pathway

2018 ◽  
Vol 47 (6) ◽  
pp. 2307-2318 ◽  
Author(s):  
Geng-Yang Shen ◽  
Hui Ren ◽  
Jin-Jing Huang ◽  
Zhi-Da Zhang ◽  
Wen-Hua Zhao ◽  
...  
Keyword(s):  
Protein Expression ◽  
Osteogenic Differentiation ◽  
Mrna Expression ◽  
Signaling Pathway ◽  
Quantitative Polymerase Chain Reaction ◽  
Akt Signaling ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Akt Signaling Pathway ◽  
Alizarin Red

Background/Aims: Plastrum testudinis extracts (PTE) show osteoprotective effects on glucocorticoid-induced osteoporosis in vivo and in vitro. However, the underlying molecular mechanism of PTE in promoting osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) is unclear. Methods: BMSC proliferation was investigated using the Cell Counting Kit-8 assay. BMSC differentiation and osteogenic mineralization were assayed using alkaline phosphatase and Alizarin red staining, respectively. The mRNA expression levels of Let-7f-5p, Tnfr2, Traf2, Pi3k, Akt, β-catenin, Gsk3β, Runx2, and Ocn were measured using real time quantitative polymerase chain reaction. Protein levels of TNFR2, TRAF2, p-PI3K, p-AKT, p-β-CATENIN, and p-GSK3β were analyzed by western blotting. The functional relationship of Let-7f-5p and Tnfr2 was determined by luciferase reporter assays. Results: The optimum concentration for PTE was 30 μg/ml. PTE significantly promoted BMSC osteogenic differentiation and mineralization after 7 and 14 days in culture, respectively. The combination of PTE and osteogenic induction exhibited significant synergy. PTE upregulated Let-7f-5p, β-catenin, Runx2, and Ocn mRNA expression, and downregulated Tnfr2, Traf2, Pi3k, Akt, and Gsk3β mRNA expression. PTE inhibited TNFR2, TRAF2, and p-β-CATENIN protein expression, and promoted p-PI3K, p-AKT, and p-GSK3β protein expression. In addition, Tnfr2 was a functional target of Let-7f-5p in 293T cells. Conclusions: Our results suggested that PTE may promote BMSC proliferation and osteogenic differentiation via a mechanism associated with the regulation of Let-7f-5p and the TNFR2/PI3K/AKT signaling pathway.

scholarly journals 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

2019 ◽  
Vol 10 (12) ◽  
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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