CircRNA hsa_circ_0006215 promotes osteogenic differentiation of BMSCs and enhances osteogenesis-angiogenesis coupling by competitively binding to miR-942-5p and regulating RUNX2 and VEGF.
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ABSTRACT:
SUBMITTER: Ji H
PROVIDER: S-EPMC8064180 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Bone 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.
| S-EPMC6895238 | biostudies-literature
Project description:Statistics show that the prognosis of cervical cancer (CC) is poor, and the death rate of CC in advanced stage has been rising in recent years. Increasing evidence has demonstrated that circular RNAs (circRNAs) serve as promising biomarkers in human cancers, including CC. The present study planned to find out the circRNA involved in CC and to explore its regulatory mechanism in CC. We discovered the new circRNA, circ-0033550, upregulated in CC. Its associated gene was AKT (also known as protein kinase B) serine/threonine kinase 1 (AKT1), so we renamed circ-0033550 as circ-AKT1. We confirmed the high expression of circ-AKT1 in CC samples and cell lines, as well as the circle structure of circ-AKT1. Functionally, gain- and loss-of-function experiments indicated that circ-AKT1 and AKT1 promoted CC cell proliferation and invasion. Moreover, circ-AKT1 and AKT1 were induced by transforming growth factor beta (TGF-β) and facilitated EMT (epithelial-mesenchymal transition) in CC. Mechanically, we illustrated that circ-AKT1 upregulated AKT1 by sponging miR-942-5p. Rescue assays confirmed the role of the circ-AKT1/miR-942-5p/AKT1 axis in CC progression. In vivo assays validated that circ-AKT1 promoted tumor growth in CC. Overall, circRNA-AKT1 sequestered miR-942-5p to upregulate AKT1 and promote CC progression, which may offer a new molecular target for the treatment improvement of CC.
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Project description:Human amniotic mesenchymal stem cells (hAMSCs) are multiple potent progenitor cells (MPCs) that can differentiate into different lineages (osteogenic, chondrogenic, and adipogenic cells) and have a favorable capacity for angiogenesis. Schnurri-3 (Shn3) is a large zinc finger protein related to Drosophila Shn, which is a critical mediator of postnatal bone formation. Bone morphogenetic protein 9 (BMP9), one of the most potent osteogenic BMPs, can strongly upregulate various osteogenesis- and angiogenesis-related factors in MSCs. It remains unclear how Shn3 is involved in BMP9-induced osteogenic differentiation coupled with angiogenesis in hAMSCs. In this investigation, we conducted a comprehensive study to identify the effect of Shn3 on BMP9-induced osteogenic differentiation and angiogenesis in hAMSCs and analyze the responsible signaling pathway. The results from in vitro and in vivo experimentation show that Shn3 notably inhibits BMP9-induced early and late osteogenic differentiation of hAMSCs, expression of osteogenesis-related factors, and subcutaneous ectopic bone formation from hAMSCs in nude mice. Shn3 also inhibited BMP9-induced angiogenic differentiation, expression of angiogenesis-related factors, and subcutaneous vascular invasion in mice. Mechanistically, we found that Shn3 prominently inhibited the expression of BMP9 and activation of the BMP/Smad and BMP/MAPK signaling pathways. In addition, we further found activity on runt-related transcription factor 2 (Runx2), vascular endothelial growth factor (VEGF), and the target genes shared by BMP and Shn3 signaling pathways. Silencing Shn3 could dramatically enhance the expression of Runx2, which directly regulates the downstream target VEGF to couple osteogenic differentiation with angiogenesis. To summarize, our findings suggested that Shn3 significantly inhibited the BMP9-induced osteogenic differentiation and angiogenesis in hAMSCs. The effect of Shn3 was primarily seen through inhibition of the BMP/Smad signaling pathway and depressed expression of Runx2, which directly regulates VEGF, which couples BMP9-induced osteogenic differentiation with angiogenesis.
| S-EPMC6989499 | biostudies-literature
Project description:Pain, physical dysfunction, and mental disorders caused by bone nonunion bring great burden to patients. Bone mesenchymal stem cells (BMSCs) isolated from bone nonunion patients with poor proliferation and osteogenic ability are compared with that from normal bone-healing patients. Long noncoding RNAs (lncRNAs) are a class of RNAs that are more than 200 nucleotides in length, lack an open-reading frame encoding a protein, and have little or no protein-coding function, and could regulate gene expression, which is involved in the regulation of important life activities, such as growth, development, aging, and death at epigenetic, transcriptional, and post-transcriptional levels. In this study, we intended to investigate the difference of lncRNA expression between patients with nonunion and normal fracture healing. Our result found that lncRNA ENST00000563492 was downregulated in bone nonunion tissues. LncRNA ENST00000563492 promotes osteogenic differentiation of BMSCs through upregulating the expression of CDH11. On the other hand, LncRNA ENST0000563492 could improve the osteogenesis-angiogenesis coupling process through enhancing the expression of VEGF during osteogenic differentiation of BMSCs. LncRNA ENST00000563492 functions as a ceRNA for miR-205-5p that was targeting CDH11 and VEGF. LncRNA ENST00000563492 could promote the osteogenesis of BMSCs in vivo. Our result indicated that lncRNA ENST00000563492 may be a new target for bone nonunion.
| S-EPMC7316863 | biostudies-literature
Project description:N-acetyltransferase 10 (NAT10) is the key enzyme for N4-acetylcytidine (ac4C) modification of mRNA, which participates in various cellular processes and is related to many diseases. Here, we explore the relationships among osteoblast differentiation, NAT10, and ac4C, and we found that NAT0 expression and the ac4C level of total RNA were decreased in the bone tissues of bilateral ovariectomized (OVX) mice and osteoporosis patients. Adenoviruses overexpressing NAT10 reversed bone loss, and Remodelin, an NAT10 inhibitor, enhanced the loss of bone mass in OVX mice. Moreover, bone marrow-derived mesenchymal stem cells (BMSCs) with low-level ac4C modification formed fewer calcium nodules in vitro with NAT10 silencing, whereas BMSCs with high-level ac4C modification formed more calcium nodules with NAT10 overexpression. Moreover, we demonstrated that the ac4C level of runt-related transcription factor 2 (RUNX2) mRNA was increased after BMSCs were cultured in osteogenic medium (OM) and decreased after NAT10 silencing. The RUNX2 mRNA half-life and protein expression decreased after silencing NAT10 in BMSCs. Therefore, NAT10-based ac4C modification promotes the osteogenic differentiation of BMSCs by regulating the RUNX2 ac4C level. Because abnormal levels of NAT10 are probably one of the mechanisms responsible for osteoporosis, NAT10 is a new potential therapeutic target for this disease.
| S-EPMC8413676 | biostudies-literature
Project description:Osteogenesis is tightly coupled with angiogenesis spatiotemporally. Previous studies have demonstrated that type H blood vessel formed by endothelial cells with high expression of CD31 and Emcn (CD31hi Emcnhi ECs) play a crucial role in bone regeneration. The mechanism of the molecular communication around CD31hi Emcnhi ECs and bone mesenchymal stem cells (BMSCs) in the osteogenic microenvironment is unclear. This study indicates that exosomes from bone mesenchymal stem cells with 7 days osteogenic differentiation (7D-BMSCs-exo) may promote CD31hi Emcnhi ECs angiogenesis, which was verified by tube formation assay, qRT-PCR, Western blot, immunofluorescence staining and µCT assays etc. in vitro and in vivo. Furthermore, by exosomal miRNA microarray and WGCNA assays, we identified downregulated miR-150-5p as the most relative hub gene coupling osteogenic differentiation and type H blood vessel angiogenesis. With bioinformatics assays, dual luciferase reporter experiments, qRT-PCR and Western blot assays, SOX2(SRY-Box Transcription Factor 2) was confirmed as a novel downstream target gene of miR-150-5p in exosomes, which might be a pivotal mechanism regulating CD31hi Emcnhi ECs formation. Additionally, JC-1 immunofluorescence staining, Western blot and seahorse assay results showed that the overexpression of SOX2 could shift metabolic reprogramming from oxidative phosphorylation (OXPHOS) to glycolysis to enhance the CD31hi Emcnhi ECs formation. The PI3k/Akt signaling pathway might play a key role in this process. In summary, BMSCs in osteogenic differentiation might secrete exosomes with low miR-150-5p expression to induce type H blood vessel formation by mediating SOX2 overexpression in ECs. These findings might reveal a molecular mechanism of osteogenesis coupled with type H blood vessel angiogenesis in the osteogenic microenvironment and provide a new therapeutic target or cell-free remedy for osteogenesis impaired diseases.
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Project description:Regulation of microRNAs (miRNA) has been extensively investigated in diseases; however, little is known about the roles of miRNAs in cleidocranial dysplasia (CCD). The aim of the present study was to investigate the potential involvement of miRNAs in CCD. In vitro site-directed mutagenesis was performed to construct three mutant Runx2 expression vectors, which were then transfected into LS8 cells and MC3T3-E1 cells, to determine the impact on amelogenesis and osteogenesis, respectively. miRCURY LNA miRNA microarray identify miR-185-5p as a miRNA target commonly induced by all three Runx2 mutants. Real-time quantitative PCR was applied to determine the expression of miR-185-5p and Dlx2 in samples. Dual-luciferase reporter assays were conducted to confirm Dlx2 as a legitimate target of miR-185-5p. The suppressive effect of miR-185-5p on amelogenesis and osteogenesis of miR-185-5p was evaluated by RT-PCR and western blot examination of Amelx, Enam, Klk4, and Mmp20 gene and protein expression, and by Alizarin Red stain. We found that mutant Runx2 suppressed amelogenesis and osteogenesis. miR-185-5p, induced by Runx2, suppressed amelogenesis and osteogenesis. Furthermore, we identified Dlx2 as direct target of miR-185-5p. Consistently, Dlx2 expression was inversely correlated with miR-185-5p levels. This study highlights the molecular etiology and significance of miR-185-5p in CCD, and suggests that targeting miR-185-5p may represent a new therapeutic strategy in prevention or intervention of CCD.
| S-EPMC5870583 | biostudies-literature
Project description:BackgroundStabilization and increased activity of hypoxia-inducible factor 1-α (HIF-1α) can directly increase cancellous bone formation and play an essential role in bone modeling and remodeling. However, whether an increased HIF-1α expression in adipose-derived stem cells (ADSCs) increases osteogenic capacity and promotes bone regeneration is not known.ResultsIn this study, ADSCs transfected with small interfering RNA and HIF-1α overexpression plasmid were established to investigate the proliferation, migration, adhesion, and osteogenic capacity of ADSCs and the angiogenic ability of human umbilical vein endothelial cells (HUVECs). Overexpression of HIF-1α could promote the biological functions of ADSCs, and the angiogenic ability of HUVECs. Western blotting showed that the protein levels of osteogenesis-related factors were increased when HIF-1α was overexpressed. Furthermore, the influence of upregulation of HIF-1α in ADSC sheets on osseointegration was evaluated using a Sprague-Dawley (SD) rats implant model, in which the bone mass and osteoid mineralization speed were evaluated by radiological and histological analysis. The overexpression of HIF-1α in ADSCs enhanced bone remodeling and osseointegration around titanium implants. However, transfecting the small interfering RNA (siRNA) of HIF-1α in ADSCs attenuated their osteogenic and angiogenic capacity. Finally, it was confirmed in vitro that HIF-1α promotes osteogenic differentiation and the biological functions in ADSCs via the VEGF/AKT/mTOR pathway.ConclusionsThis study demonstrates that HIF-1α has a critical ability to promote osteogenic differentiation in ADSCs by coupling osteogenesis and angiogenesis via the VEGF/AKT/mTOR signaling pathway, which in turn increases osteointegration and bone formation around titanium implants.
| S-EPMC10405507 | biostudies-literature
Project description:Type 1 diabetes mellitus is associated with a number of disorders of skeletal health, conditions that rely, in part, on dynamic bone formation. A mouse model of distraction osteogenesis was used to study the consequences of streptozotocin-induced diabetes and insulin treatment on bone formation and osteoblastogenesis. In diabetic mice compared with control mice, new bone formation was decreased, and adipogenesis was increased in and around, respectively, the distraction gaps. Although insulin treatment restored bone formation to levels observed in nondiabetic control mice, it failed to significantly decrease adipogenesis. Molecular events altered during de novo bone formation in untreated type 1 diabetes mellitus, yet restored with insulin treatment were examined so as to clarify specific osteogenic genes that may contribute to diabetic bone disease. RNA from distraction gaps was analyzed by gene microarray and quantitative RT-PCR for osteogenic genes of interest. Runt-related transcription factor 2 (RUNX2), and several RUNX2 target genes, including matrix metalloproteinase-9, Akp2, integrin binding sialoprotein, Dmp1, Col1a2, Phex, Vdr, osteocalcin, and osterix, were all significantly down-regulated in the insulin-deficient, hyperglycemic diabetic animals; however, insulin treatment of diabetic animals significantly restored their expression. Expression of bone morphogenic protein-2, transcriptional coactivator with PDZ-binding motif, and TWIST2, all important regulators of RUNX2, were not impacted by the diabetic condition, suggesting that the defect in osteogenesis resides at the level of RUNX2 expression and its activity. Together, these data demonstrate that insulin and/or glycemic status can regulate osteogenesis in vivo, and systemic insulin therapy can, in large part, rescue the diabetic bone phenotype at the tissue and molecular level.
| S-EPMC2276714 | biostudies-literature
Project description:BackgroundFractures heal through a process that involves angiogenesis and osteogenesis but may also lead to non-union or delayed healing. Bone marrow mesenchymal stem cells (BMSCs) have been reported to play a pivotal role in bone formation and vascular regeneration and the p75 neurotrophin receptor (p75NTR) as being an important regulator of osteogenesis. Herein, we aim to determine the potential mediation of BMSCs by p75NTR in bone healing.MethodsRat BMSCs were identified by flow cytometry (FCM) to detect cell cycle and surface markers. Then transfection of si/oe-p75NTR was performed in BMSCs, followed by Alizarin red staining to detect osteogenic differentiation of cells, immunofluorescence double staining was performed to detect the expression of p75NTR and sortilin, co-immunoprecipitation (CO-IP) was conducted to analyze the interaction between p75NTR and sortilin, and EdU staining and cell scratch assay to assess the proliferation and migration of human umbilical vein endothelial cells (HUVECs). The expression of HIF-1α, VEGF, and apoptosis-related proteins were also detected. In addition, a rat fracture healing model was constructed, and BMSCs-si-p75NTR were injected, following which the fracture condition was observed using micro-CT imaging, and the expression of platelet/endothelial cell adhesion molecule-1 (CD31) was assessed.ResultsThe results showed that BMSCs were successfully isolated, p75NTR inhibited apoptosis and the osteogenic differentiation of BMSCs, while si-p75NTR led to a decrease in sortilin expression in BMSCs, increased proliferation and migration in HUVECs, and upregulation of HIF-1α and VEGF expression. In addition, an interaction was observed between p75NTR and sortilin. The knockdown of p75NTR was found to reduce the severity of fracture in rats and increase the expression of CD31 and osteogenesis-related proteins.ConclusionSilencing p75NTR effectively modulates BMSCs to promote osteogenic differentiation and angiogenesis, offering a novel perspective for improving fracture healing.
| S-EPMC10953090 | biostudies-literature