Osteogenic Differentiation Of hBMSCs Research Articles

SPA inhibits hBMSC osteogenic differentiation and M1 macrophage polarization by suppressing SETD2 in acute suppurative osteomyelitis

To clarify the impact of SETD2 on macrophage function in pediatric patients with acute suppurative osteomyelitis and to elucidate the precise underlying mechanism. To gain insights into the potential functions of SETD2, a comprehensive study was conducted utilizing a co-culture model of human bone mesenchymal stem cells (hBMSCs) and bone marrow-derived macrophages (THP-1). A range of techniques were employed, including quantitative polymerase chain reaction, western blotting, ELISA, alkaline phosphatase activity assays, alizarin red S staining, luciferase reporter gene assays, and chromatin immunoprecipitation, to unravel the intricate interactions and molecular mechanisms involving SETD2 in this system. It was observed that SETD2 expression was reduced in THP-1 cells stimulated by staphylococcal protein A (SPA). Furthermore, the downregulation of SETD2 resulted in elevated M1 macrophage polarization and glycolysis, effects that were mitigated by SPA stimulation. Notably, SPA-stimulated THP-1 cells exhibited an increase in HIF-1α expression, which exhibited an inverse correlation with SETD2 levels. Moreover, it was discovered that SETD2 functioned as a catalyst for H3K36me3 and bound to the HIF-1α gene, which, in turn, regulated HIF-1α expression. Furthermore, the suppression of HIF-1α abrogated the consequences of SETD2 downregulation on glycolysis and M1 macrophage polarization. Lastly, the study demonstrated that M1 macrophage polarization serves as a mediator for BMP4’s inhibitory effect on osteogenic differentiation of hBMSCs. This research has uncovered a previously unknown role of SETD2 in macrophages during osteomyelitis, revealing its significance in the pathogenesis of this condition. These findings suggest SETD2 as a novel target for the treatment of osteomyelitis.

A novel miR-466l-3p/FGF23 axis promotes osteogenic differentiation of human bone marrow mesenchymal stem cells

BackgroundMicroRNAs (miRNAs) regulate osteogenic differentiation processes and influence the development of osteoporosis (OP). This study aimed to investigate the potential role of miR-466 l-3p in OP. MethodsThe expression levels of miR-466 l-3p and fibroblast growth factor 23 (FGF23) were quantified in the trabeculae of the femoral neck of 40 individuals with or without OP using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The impact of miR-466 l-3p or FGF23 expression on cell proliferation and the expression levels of runt-related transcription factor 2 (RUNX2), type I collagen (Col1), osteocalcin (OCN), osterix (OSX) and dentin matrix protein 1 (DMP1) was quantified in human bone marrow mesenchymal stem cells (hBMSCs) overexpressing miR-466 l-3p. Furthermore, alkaline phosphatase (ALP) staining and alizarin red staining were performed to measure ALP activity and the levels of calcium deposition, respectively. In addition, bioinformatics analysis, luciferase reporter assays, and RNA pull-down assays were conducted to explore the molecular mechanisms underlying the effects of miR-466 l-3p and FGF23 in osteogenic differentiation of hBMSCs. ResultsThe expression levels of miR-466 l-3p were significantly lower in femoral neck trabeculae of patients with OP than in the control cohort, whereas FGF23 levels exhibited the opposite trend. Furthermore, miR-466 l-3p levels were upregulated and FGF23 levels were downregulated in hBMSCs during osteogenic differentiation. Moreover, the high miR-466 l-3p expression enhanced the mRNA expression of RUNX2, Col1, OCN, OSX and DMP1, as well as cell proliferation, ALP activity, and calcium deposition in hBMSCs. FGF23 was found to be a direct target of miR-466 l-3p. FGF23 overexpression downregulated the expression of osteoblast markers and inhibited the osteogenic differentiation induced by miR-466 l-3p overexpression. qRT-PCR and Western blot assays showed that miR-466 l-3p overexpression decreased the expression levels of mRNAs and proteins associated with the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway, whereas FGF23 upregulation exhibited the opposite trend. ConclusionIn conclusion, these findings suggest that miR-466 l-3p enhances the osteogenic differentiation of hBMSCs by suppressing FGF23 expression, ultimately preventing OP.

Long noncoding RNA KCNMA1-AS1 promotes osteogenic differentiation of human bone marrow mesenchymal stem cells by activating the SMAD9 signaling pathway

The human bone marrow mesenchymal stem cells (hBMSCs) undergo intense osteogenic differentiation, a crucial bone formation mechanism. Evidence from prior studies suggested an association between long noncoding RNAs (lncRNAs) and the osteogenic differentiation of hBMSCs. However, precise roles and molecular mechanisms are still largely unknown. In this work, we report for the first time that lncRNA KCNMA1 antisense RNA 1 (KCNMA1-AS1) plays a vital role in regulating hBMSCs’ osteogenic differentiation. Here, it was observed that the KCNMA1-AS1 expression levels were significantly upregulated during osteogenic differentiation. In addition, KCNMA1-AS1 overexpression enhanced in vitro osteogenic differentiation of hBMSCs and in vivo bone formation, whereas knockdown of KCNMA1-AS1 resulted in the opposite result. Additionally, the interaction between KCNMA1-AS1 and mothers against decapentaplegic homolog 9 (SMAD9) was confirmed by an RNA pull-down experiment, mass spectrometry, and RIP assay. This interaction regulated the activation of the SMAD9 signaling pathway. Moreover, rescue assays demonstrated that the inhibitor of the SMAD9 signaling pathway reversed the stimulative effects on osteogenic differentiation of hBMSCs by KCNMA1-AS1 overexpression. Altogether, our results stipulate that KCNMA1-AS1 promotes osteogenic differentiation of hBMSCs via activating the SMAD9 signaling pathway and can serve as a biomarker and therapeutic target in treating bone defects.

Notoginsenoside R1 promotes osteogenic differentiation of human bone marrow mesenchymal stem cells via ERα/GSK-3β/β-catenin signalling pathway.

Human bone marrow mesenchymal stem cells (hBMSCs) are attractive therapeutic agents for bone tissue regeneration owing to their osteogenic differentiation potential. Notoginsenoside R1 (NGR1) is a novel phytoestrogen with diverse pharmacological activities. Here, we probed whether NGR1 has an effect on the osteogenic differentiation of hBMSCs. EdU, CCK-8 and Transwell assays were used to measure proliferation and migration of hBMSCs after treatment with different doses of NGR1. hBMSCs were treated with osteogenic differentiation induction medium for osteogenesis. Alizarin red S (ARS) and alkaline phosphatase (ALP) staining were used to measure mineralized nodule formation and ALP activity in hBMSCs, respectively. ICI 182780, an antagonist of oestrogen receptor alpha (ERα) was used to inhibit ERα expression. The results showed that NGR1 enhanced hBMSC proliferation and migration. NGR1 increased ALP activity and mineralized nodule formation as well as promoting ALP, RUNX2 and OCN expression in hBMSCs. NGR1 enhanced ERα expression and promoted GSK-3β/β-catenin signal transduction in hBMSCs. ICI 182780 reversed NGR1-mediated activation of the GSK-3β/β-catenin signalling and promoted an effect on hBMSC behaviour. Thus NGR1 promotes proliferation, migration and osteogenic differentiation of hBMSCs via the ERα/GSK-3β/β-catenin signalling pathway.

Ubiquitination of ASCL1 mediates CD47 transcriptional activation of the AKT signaling pathway, and glycolysis promotes osteogenic differentiation of hBMSCs

Bones are extremely dynamic organs that continually develop and remodel. This process involves changes in numerous gene expressions. hBMSC cells can promote osteogenic differentiation. The purpose of this study was to elucidate the mechanism by which ASCL1 promotes osteogenic differentiation in hBMSC cells while decreasing glycolysis. hBMSCs were induced to differentiate into osteoblasts. The ASCL1 expression level during hBMSC osteogenic differentiation was measured by RT‒qPCR, Western blotting, and immunofluorescence. The differentiation level of osteoblasts was observed after staining with ALP and alizarin red. ChIP-qPCR were used to determine the relationship between ASCL1 and CD47, and the expression of glycolysis-related proteins was detected. Overexpression of ASCL1 was used to determine its impact on osteogenic differentiation. si-USP8 was used to verify the ubiquitination of ASCL1-mediated CD47/AKT pathway’s impact on hBMSC glycolysis and osteogenic differentiation. The results showed that the expression of ASCL1 was upregulated after the induction of osteogenic differentiation in hBMSCs. From a functional perspective, knocking down USP8 can promote the ubiquitination of ASCL1, while the osteogenic differentiation ability of hBMSCs was improved after the overexpression of ASCL1, indicating that ASCL1 can promote the osteogenic differentiation of hBMSCs. In addition, USP8 regulates the ubiquitination level of ASCL1 and mediates CD47 transcriptional regulation of the AKT pathway to increase the glycolysis level of hBMSCs and cell osteogenic differentiation. USP8 ubiquitination regulates the level of ASCL1. In addition, ubiquitination of ASCL1 mediates CD47 transcription to activate the AKT signaling pathway and increase hBMSC glycolysis to promote osteogenic differentiation.

TiO2 nanotubes promote osteogenic differentiation of human bone marrow stem cells via epigenetic regulation of RMRP/DLEU2/EZH2 pathway

TiO2 nanotubes (TNTs) significantly promote osteogenic differentiation and bone regeneration of cells. Nevertheless, the biological processes by which they promote osteogenesis are currently poorly understood. Long non-coding RNAs (lncRNAs) are essential for controlling osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Epigenetic chromatin modification is one of the pathways in which lncRNAs regulate osteogenic differentiation. Here, we reported that TNTs could upregulate lncRNA RMRP, and inhibition of lncRNA RMRP in human BMSCs (hBMSCs) grown on TNTs could decrease runt-related transcription factor 2 (RUNX2), alkaline phosphatase, osteopontin, and osteocalcin (OCN) expression. Furthermore, we discovered that inhibiting lncRNA RMRP elevated the expression of lncRNA DLEU2, and lncRNA DLEU2 knockdown promoted osteogenic differentiation in hBMSCs. RNA immunoprecipitation experiments showed that lncRNA DLEU2 could interact with EZH2 to induce H3K27 methylation in the promoter regions of RUNX2 and OCN, suppressing gene expression epigenetically. According to these results, lncRNA RMRP is upregulated by TNTs to promote osteogenic differentiation through DLEU2/EZH2-mediated epigenetic modifications.

Pmepa1 knockdown alleviates SpA-induced pyroptosis and osteogenic differentiation inhibition of hBMSCs via p38MAPK/NLRP3 axis

BackgroundOsteomyelitis is a refractory bone infectious disease, which usually results in progressive bone destruction and bone loss. The invasion of pathogens and subsequent inflammatory response could damage bone marrow mesenchymal stem cells (BMSCs) and inhibit osteogenic differentiation, and finally aggravate uncontrolled bone remodeling in osteomyelitis by affecting bone formation. Exploring the mechanisms of BMSCs injury and osteogenic differentiation inhibition may would help us to find potential therapeutic targets. MethodFirstly, staphylococcal protein A (SpA)-treated human bone marrow mesenchymal stem cells (hBMSCs) were used to construct cell models of osteomyelitis. Secondly, transcriptome sequencing was performed to screen differentially expressed genes and then verified the expression of target genes. Next, in vitro experiments were conducted to explore the functions and mechanisms of prostate transmembrane protein androgen induced 1 (Pmepa1) in SpA-treated hBMSCs. Finally, the rat model of osteomyelitis was established to provide an auxiliary validation of the in vitro experimental results. ResultsWe found that SpA treatment induced inflammatory injury and inhibited osteogenic differentiation in hBMSCs, then the transcriptome sequencing and further detection results showed that Pmepa1 was significantly upregulated in this process. Functionally, Pmepa1 knockdown alleviated inflammatory injury and promoted osteogenic differentiation in SpA-treated hBMSCs. Among them, it was demonstrated that Pmepa1 knockdown exerted cytoprotective effects by alleviating pyroptosis of SpA-infected hBMSCs. Furthermore, recovery experiments revealed that Pmepa1 knockdown reversed SpA-mediated adverse effects by downregulating the p38MAPK/NLRP3 axis. Finally, the detection results of rat femoral osteomyelitis showed that the expression of Pmepa1 was up-regulated, and the expression trends of other indicators including p38MAPK, NLRP3, and caspase-1 were also consistent with the in vitro model. ConclusionPmepa1 knockdown alleviates SpA-induced pyroptosis and inhibition of osteogenic differentiation in hBMSCs by downregulating p38MAPK/NLRP3 signaling axis. Modulating the expression of Pmepa1 may be a potential strategy to ameliorate osteomyelitis.

NEAT1/microRNA 339-5p/SPI1 Axis Feedback Loop Contributes to Osteogenic Differentiation in Acute Suppurative Osteomyelitis in Children.

Long non-coding RNA plays an important role in osteogenic differentiation. Nuclear enriched abundant transcript 1 (NEAT1) has been revealed to promote osteogenic differentiation in human bone marrow mesenchymal stem cells (hBMSCs), but the underlying regulatory mechanism remains unknown in acute suppurative osteomyelitis of children. Osteogenic medium (OM) was used to induce osteogenic differentiation. Quantitative real-time PCR and Western blotting were used to evaluate gene expression. The effects of NEAT1, microRNA 339-5p (miR-339-5p), and salmonella pathogenicity island 1 (SPI1) on osteogenic differentiation were assessed in vitro using alizarin red S staining assays and alkaline phosphatase activity. Interactions between NEAT1, miR-339-5p, and SPI1 were identified using immunoprecipitation, luciferase reporter assays, and chromatin immunoprecipitation. During osteogenic differentiation, expression of NEAT1 was up-regulated in hBMSCs, and miR-339-5p level was down during osteogenic differentiation. Knockdown of NEAT1 reduced the osteogenic differentiation of hBMSCs, and down-regulation of miR-339-5p may counteract the effect of NEAT1 silencing. SPI1 was a target of miR-339-5p by luciferase reporter assay and was also a transcription factor of NEAT1 by chromatin immunoprecipitation. A positive NEAT1-miR-339-5p-SPI1 feedback loop was found to be present during osteogenic differentiation in hBMSCs. It was the first study to reveal that the NEAT1-miR-339-5p-SPI1 feedback loop can promote osteogenic differentiation in hBMSCs and shed a new light on the role of NEAT1 during osteogenic differentiation.

Dose-dependent effects of zoledronic acid on the osteogenic differentiation of human bone marrow stem cells (hBMSCs)

Recent studies have shown that bisphosphonates can also impact osteoblasts besides osteoclasts. This study aimed to evaluate the effects of different concentrations of Zoledronic acid (ZA) during the osteogenic differentiation of human Bone Marrow Stem Cells (hBMSCs) in vitro. Thus, osteogenic differentiation of hBMSCs was conducted with different concentrations of Zoledronic Acid (ZA) (0, 0.1, 1.0, and 5.0 μM) for the first 3 days. Cell metabolism was quantified at 1-, 3-, 7-, and 14 days. At 7- and 14-days, the following analyses were performed: 1) mineralization nodule assay, 2) LIVE/DEAD™, 3) cell adhesion and spreading, 4) alkaline phosphatase (ALP) activity, and 5) qPCR analysis for RUNX-2), ALPL, and COL1 A1. Data were analyzed by ANOVA 2-way, followed by Tukey's post hoc test (p < 0.05). Cell metabolism (3-, 7-, and 14-days) (p < 0.001), mineralization (7-, 14-days) (p < 0.001), and ALP activity (14-days) (p < 0.001) were reduced in ZA 5.0 µM when compared to control (no ZA). Also, ZA 5.0 µM downregulated the expression of RUNX2 at 7- and 14-days (p < 0.001). It is possible to conclude that ZA (5.0 µM) can impair hBMSC differentiation into osteoblasts and interferes with its mineralization phase.

IRF-1-inhibited lncRNA XIST regulated the osteogenic differentiation via miR-450b/FBXW7 axis.

Osteoporosis influences life quality among elder people. Osteoblast dysfunction could cause the occurrence of osteoporosis. LncRNA XIST are involved in the progression of osteoporosis. However, the correlation between IRF-1 and XIST in osteogenic differentiation remains unclear. In the study, Clinical samples were collected for the analysis of XIST level. mRNA and protein levels were detected by RT-qPCR and western blot, respectively. H&E staining was performed to observe the histological changes in mice. Alizarin Red Staining was applied to assess the calcium deposits in hBMSCs. Meanwhile, the relation among XIST, miR-450b and FBXW7 was investigated by dual luciferase assay and ChIP. In vivo model was constructed to assess the impact of XIST in osteoporosis. XIST was found to be upregulated in osteoporosis, and XIST overexpression could inhibit the osteogenic differentiation in hBMSCs. IRF-1 could transcriptionally inhibit the expression of XIST, and XIST could inhibit osteogenic differentiation through binding with miR-450b in hBMSCs. In addition, miR-450b significantly promoted the osteogenic differentiation in hBMSCs via targeting FBXW7. Furthermore, XIST knockdown could inhibit the symptom of osteoporosis in vivo. IRF-1 promoted the osteogenic differentiation via mediation of lncRNA XIST/miR-450b/FBXW7 axis, and this finding might shed novel insights on exploring new ideas against osteoporosis.

Hsa_circ_0007334 Promotes the Osteogenic Differentiation and Proliferation of Human Bone Marrow Mesenchymal Stem Cells by Sponging miR-144-3p

This study aimed to identify the possible function and the molecular mechanism of hsa_circ_0007334 in human bone marrow mesenchymal stem cells (hBMSCs) osteogenic differentiation. The level of hsa_circ_0007334 was detected by means of quantitative real-time polymerase chain reaction (RT-qPCR). Alkaline phosphatase (ALP), RUNX2, osterix (OSX), and osteocalcin (OCN) were monitored to analyze the degree of osteogenic differentiation under routine culture or under the control of hsa_circ_0007334. The proliferation of hBMSCs was tested with a cell counting kit-8 (CCK-8) assay. The migration of hBMSCs was tested using the Transwell assay. Bioinformatics analysis was used to predict the possible targets of hsa_circ_0007334 or miR-144-3p. Dual-luciferase reporter assay system was used to analyze the combination between hsa_circ_0007334 and miR-144-3p. Hsa_circ_0007334 was upregulated in osteogenic differentiation of hBMSCs. Osteogenic differentiation increased by hsa_circ_0007334 in vitro was confirmed with levels of ALP and bone markers (RUNX2, OCN, OSX). hsa_circ_0007334 overexpression promoted osteogenic differentiation, proliferation, and migration of hBMSCs, and knockdown of hsa_circ_0007334 has the opposite effects. miR-144-3p was identified as the target of hsa_circ_0007334. The targeting genes of miR-144-3p are involved in osteogenic-differentia-tion-related biological processes (such as bone development, epithelial cell proliferation, and mesenchymal cell apoptotic prosess) and pathways (including FoxO and VEGF signaling pathway). Hsa_circ_0007334, therefore, presents itself as a promising biological for osteogenic differentiation.

Circular RNA CircSLC8A1 contributes to osteogenic differentiation in hBMSCs via CircSLC8A1/miR-144-3p/RUNX1 in periprosthetic osteolysis.

Circular RNAs (circRNAs) are often found in eukaryocyte and have a role in the pathogenesis of a variety of human disorders. Our related research has shown the differential expression of circRNAs in periprosthetic osteolysis (PPOL). However, the involvement of circRNAs in the exact process is yet unknown. CircSLC8A1 expression was evaluated in clinical samples and human bone marrow mesenchymal stem cells (hBMSCs) in this investigation using quantitative real-time PCR. In vitro and in vivo studies were conducted to explicate its functional role and pathway. We demonstrated CircSLC8A1 is involved in PPOL using gain- and loss-of-function methods. The association of CircSLC8A1 and miR-144-3p, along with miR-144-3p and RUNX1, was predicted using bioinformatics. RNA pull-down and luciferase assays confirmed it. The impact of CircSLC8A1 in the PPOL-mouse model was also investigated using adeno-associated virus. CircSLC8A1 was found to be downregulated in PPOL patients' periprosthetic tissues. Overexpression of CircSLC8A1 promoted osteogenic differentiation (OD) and inhibited apoptosis of hBMSCs in vitro. The osteogenic markers of RUNX1, osteopontin (OPN) and osteocalcin (OCN) were significantly upregulated in hBMSCs after miR-144-3p inhibitor was transferred. Mechanistic analysis demonstrated that CircSLC8A1 directly bound to miR-144-3p and participated in PPOL through the miR-144-3p/RUNX1 pathway in hBMSCs. Micro-CT and quantitative analysis showed that CircSLC8A1 markedly inhibited PPOL, and osteogenic markers (RUNX1, OPN and OCN) were significantly increased (P<0.05) in the mice model. Our findings prove that CircSLC8A1 exerted a regulatory role in promoting osteogenic differentiation in hBMSCs, and CircSLC8A1/miR-144-3p/RUNX1 pathway may provide a potential target for prevention of PPOL.

Identification and validation of autophagy-related genes during osteogenic differentiation of bone marrow mesenchymal stem cells.

Osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) is an essential stage in bone formation. Autophagy plays a pivotal role in the self-renewal potential and pluripotency of stem cells. This study aimed to explore the function of autophagy-related genes during osteogenic differentiation of BMSCs. The differentially expressed autophagy-related genes (ARGs) were obtained from the GEO and HADb databases. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed using R software. The PPI and hub gene mining networks were constructed using the STRING database and Cytoscape. Finally, the RT-qPCR was conducted to validate the expression level of ARGs in BMSCs. Thirty-seven differentially expressed ARGs were finally obtained, including 12 upregulated and 25 downregulated genes. GO and KEGG enrichment analysis showed that most of these genes were enriched in apoptosis and autophagy. The PPI network revealed strong interactions between differentially expressed ARGs. The expression level of differentially expressed ARGs tested by RT-qPCR showed 6 upregulated ARGs, including FOXO1, MAP1LC3C, CTSB, FOXO3, CALCOCO2, FKBP1A, and 4 downregulated ARGs, including MAPK8IP1, NRG1, VEGFA, and ITGA6 were consistent with the expression of high-throughput sequencing data. We identified 37 ARGs during osteogenic differentiation using bioinformatics analysis. FOXO1, MAP1LC3C, CTSB, FOXO3, CALCOCO2, FKBP1A, MAPK8IP1, NRG1, VEGFA, and ITGA6 may regulate osteogenic differentiation of hBMSCs by involving autophagy pathway. This study provides new insight into the osteogenic differentiation of hBMSCs and may be available in developing therapeutic strategies for maxillofacial bone defects.

Long Noncoding RNA P53 Upregulated Regulator of P53 Levels Promotes Osteogenic Differentiation in Osteoporosis Progression Through Sponging miR-135a-5p

This study aimed to explore the expression of long noncoding RNA p53 upregulated regulator of P53 levels (lncRNA PURPL) and microRNA (miR)-135a-5p in osteoporosis and their role in osteogenic differentiation. The relationship between lncRNA PURPL and miR-135a-5p was confirmed by Star-Base and luciferase reporter assay. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay was used to detect lncRNA PURPL and miR-135a-5p expression. RT-qPCR and western blot analysis were used to measure osteogenic markers expression. Alkaline phosphatase (ALP) activity was also determined. Results indicated that lncRNA PURPL binds to miR-135a-5p. lncRNA PURPL expression was decreased and miR-135a-5p expression was increased in patients with osteoporosis. In the process of osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs), the expression levels of osteoblast markers including RUNX family transcription factor 2 (Runx2), ALP and Osterix, and ALP activity were significantly increased. Besides, lncRNA PURPL was improved, while miR-135a-5p was down-regulated during the osteogenic differentiation of hBMSCs. Moreover, lncRNA PURPL-siRNA significantly decreased the expression of ALP, Runx2 and Osterix, and reduced ALP activity in hBMSCs subjected to osteogenic induction, while all of these effects were reversed by miR-135a-5p inhibitor. In conclusion, lncRNA PURPL/miR-135a-5p may be a new axis for osteoporosis treatment.

CircZNF367 suppresses osteogenic differentiation of human bone marrow mesenchymal stromal/stem cells via reducing HuR-mediated mRNA stability of LRP5.

Osteoporosis is a highly prevalent disease characterized by bone mass loss and structural deterioration. There are evidences that altered differentiation of human bone marrow mesenchymal stromal/stem cells (hBMSCs) is a major cause for osteoporosis. Recent studies suggest that circular RNAs (circRNAs) are dysregulated in osteoporosis patients and involved in the pathogenesis of osteoporosis. In the present study, we are aimed to analyze the circRNA expression profiles in osteoporosis patients and identify potential circRNAs that involved in the differentiation of hBMSCs during osteoporosis. Transcriptome RNA-sequencing was conducted to search for differentially expressed circRNAs. Transwell assay, ARS and ALP staining, and ectopic bone formation model were performed to evaluate osteogenic differentiation of hBMSCs. RNA pull-down assay, RNA immunoprecipitation, western blot, and in vitro binding assay were conducted to evaluate the interaction of circRNAs and RNA-binding protein HuR. We found that hsa_circ_0008842 (designated as circZNF367) was upregulated in osteoporosis patients and decreased in hBMSCs during osteogenic differentiation. CircZNF367 overexpression suppressed migration, invasion and osteogenic differentiation of hBMSCs in vitro and in vivo. In comparison, knockdown of circZNF367 promoted migration, invasion and osteogenic differentiation of hBMSCs. CircZNF367 could interact with the RNA-binding protein HuR, thus reduced the mRNA stability of LRP5. Furthermore, HuR overexpression or LRP5 restoration abrogated the effects of circZNF367 overexpression on osteogenic differentiation of hBMSCs. Our results indicated that circZNF367 played a role in osteogenic differentiation of hBMSCs via reducing HuR-mediated mRNA stability of LRP5.

MiR-29a-3p Inhibits Proliferation and Osteogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells via Targeting FOXO3 and Repressing Wnt/β-Catenin Signaling in Steroid-Associated Osteonecrosis.

Background and ObjectivesThis study was to investigate the role of microRNA-29a-3p (miR-29a-3p) in human bone marrow mesenchymal stem cells (hBMSCs), and its relationship with steroid-associated osteonecrosis.Methods and ResultsThe online tool GEO2R was used to screen out the differentially expressed genes (DEGs) in GSE123568 dataset. Quantitative real time-polymerase chain reaction (qRT-PCR) was performed to detect the expression of miR-29a-3p, forkhead box O3 (FOXO3), alkaline phosphatase (ALP), bone gamma-carboxyglutamate protein (OCN) and RUNX family transcription factor 2 (Runx2) in the hBMSCs isolated from the patients with steroid-associated osteonecrosis. CCK-8 assay was executed to measure cell viability; western blot assay was utilized to detect FOXO3, ALP, Runx2, OCN and β-catenin expression. Cell apoptosis and cell cycle were detected by flow cytometry. Immunofluorescence assay was used to detect the sub-cellular localization of β-catenin. Bioinformatics analysis and luciferase reporter gene assay were performed to confirm whether miR-29a-3p can combine with FOXO3 3’UTR. MiR-29a-3p was markedly up-regulated in the hBMSCs of patients with steroid-associated osteonecrosis, while FOXO3 mRNA was significantly down-regulated. Transfection of miR-29a-3p mimics significantly inhibited the hBMSCs’ proliferation, osteogenic differentiation markers’ expressions, including ALP, Runx2, OCN, and repressed the ALP activity, as well as promoted cell apoptosis and cell-cycle arrest. FOXO3 was identified as a target gene of miR-29a-3p, and miR-29a-3p can inhibit the expression of FOXO3 and β-catenin, and inhibition of miR-29a-3p promoted translocation of β-catenin to the nucleus.ConclusionsMiR-29a-3p can modulate FOXO3 expression and Wnt/β-catenin signaling to inhibit viability and osteogenic differentiation of hBMSCs, thereby promoting the development of steroid-associated osteonecrosis.

Circ_0062582 Promotes Osteogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells in vitro by Elevating SMAD5 Expression through Sponging miR-197-3p

Circular RNAs (circRNAs) play crucial roles in many human diseases. However, the functions of circRNAs in osteoporosis (OP) are barely reported. In this study, we aimed to explore the function of circ_0062582 in osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) in vitro. circ_0062582 and SMAD5 were downregulated and miR-197-3p was upregulated in OP patients and increased in osteoblast medium (OM)-induced hBMSCs in vitro. circ_0062582 knockdown inhibited the viability and osteogenic differentiation of hBMSCs. circ_0062582 directly targeted miR-197-3p, and miR-197-3p inhibition reversed the effects of circ_0062582 on hBMSC viability and osteogenic differentiation. SMAD5 was the target gene of miR-197-3p. SMAD5 overexpression promoted the viability and osteogenic differentiation of hBMSCs and attenuated miR-197-3p-mediated suppressive roles in hBMSC viability and osteogenic differentiation. In conclusion, circ_0062582 sponged miR-197-3p to elevate SMAD5 expression, thereby inducing hBMSC proliferation and osteogenic differentiation in vitro.

LncRNA RAD51-AS1 Regulates Human Bone Marrow Mesenchymal Stem Cells via Interaction with YBX1 to Ameliorate Osteoporosis.

Long noncoding RNA (lncRNA) is a new key regulatory molecule in the occurrence of osteoporosis, but its research is still in the primary stage. In order to study the role and mechanism of lncRNA in the occurrence of osteoporosis, we reannotated the GSE35956 datasets, compared and analyzed the differential expression profiles of lncRNAs between bone marrow mesenchymal stem cells (hBMSCs) from healthy and osteoporotic patients, and then screened a lncRNA RAD51-AS1 with low expression in hBMSCs from osteoporotic patients, and its role in the occurrence of osteoporosis has not been studied. We confirmed that the expression level of lncRNA RAD51-AS1 in hBMSCs from patients with osteoporosis was significantly lower than those from healthy donors. A nuclear cytoplasmic separation experiment and RNA fluorescence in situ hybridization showed that RAD51-AS1 was mainly located in the nucleus. RAD51-AS1 knockdown significantly inhibited the proliferation and osteogenic differentiation of hBMSCs and significantly increased their apoptosis, while RAD51-AS1 overexpression significantly promoted the proliferation, osteogenic differentiation, and ectopic bone formation of hBMSCs. Mechanistically, we found that RAD51-AS1 banded to YBX1 and then activated the TGF-β signal pathway by binding to Smad7 and Smurf2 mRNA to inhibit their translation and transcription up-regulated PCNA and SIVA1 by binding to their promoter regions. In conclusion, RAD51-AS1 promoted the proliferation and osteogenic differentiation of hBMSCs by binding YBX1, inhibiting the translation of Smad7 and Smurf2, and transcriptionally up-regulated PCNA and SIVA1.

Super-Enhancer-Associated Long Non-Coding RNA LINC01485 Promotes Osteogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells by Regulating MiR-619-5p/RUNX2 Axis

ObjectiveTo investigate the mechanisms of super-enhancer-associated LINC01485/miR-619-5p/RUNX2 signaling axis involvement in osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs).MethodsOsteogenic differentiation of hBMSCs was induced in vitro. The expression levels of LINC01485 and miR-619-5p during osteogenesis were measured using quantitative real-time polymerase chain reaction (qRT-PCR). Osteogenic differentiation was examined by qRT-PCR, western blot, alkaline phosphatase (ALP) staining, ALP activity measurement, and Alizarin Red S (ARS) staining assays. Thereafter, the effects of LINC01485 and miR-619-5p on osteogenic differentiation of hBMSCs were evaluated by performing loss- and gain-of-function experiments. Subsequently, a fluorescence in situ hybridization (FISH) assay was employed to determine the cellular localization of LINC01485. Bioinformatics analysis, RNA antisense purification (RAP) assay, and dual-luciferase reporter assays were conducted to analyze the interactions of LINC01485, miR-619-5p, and RUNX2. Rescue experiments were performed to further delineate the role of the competitive endogenous RNA (ceRNA) signaling axis consisting of LINC01485/miR-619-5p/RUNX2 in osteogenic differentiation of hBMSCs.ResultsThe expression of LINC01485 was up-regulated during osteogenic differentiation of hBMSCs. The overexpression of LINC01485 promoted osteogenic differentiation of hBMSCs by up-regulating the expression of osteogenesis-related genes [e.g., runt-related transcription factor 2 (RUNX2), osterix (OSX), collagen type 1 alpha 1 (COL1A1), osteocalcin (OCN), and osteopontin (OPN)], and increasing the activity of ALP. ALP staining and ARS staining were also found to be increased upon overexpression of LINC01485. The opposing results were obtained upon LINC01485 interference in hBMSCs. miR-619-5p was found to inhibit osteogenic differentiation. FISH assay displayed that LINC01485 was mainly localized in the cytoplasm. RAP assay results showed that LINC01485 bound to miR-619-5p, and dual-luciferase reporter assay verified that LINC01485 bound to miR-619-5p, while miR-619-5p and RUNX2 bound to each other. Rescue experiments illustrated that LINC01485 could promote osteogenesis by increasing RUNX2 expression by sponging miR-619-5p.ConclusionLINC01485 could influence RUNX2 expression by acting as a ceRNA of miR-619-5p, thereby promoting osteogenic differentiation of hBMSCs. The LINC01485/miR-619-5p/RUNX2 axis might comprise a novel target in the bone tissue engineering field.

Knockdown of has_circ_0010452 Promotes Proliferation and Osteogenic Differentiation via Up-Regulating miR-543 in Human Bone Marrow Mesenchymal Stem Cells

Objective: The aim of this study was to explore the role of has_circ_0010452 in the progression of osteoporosis (OP) targeting miR-543, as well as their functions in regulating proliferation and osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs). Methods: The expression levels of circ_0010452 and miR-543 in hBMSCs at different time points of osteogenic differentiation were determined by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). After transfection of circ_0010452 siRNA or miR-543 inhibitor in hBMSCs, the relative expression levels of osteogenic marker proteins, including oat spelt xylan (OSX), osteocalcin (OCN) and collagen I (Col-1), were determined by western blot. Cell proliferation of hBMSCs was valued by Cell Counting Kit 8 (CCK-8) assay. Dual-Luciferase reporter gene assay was performed to verify the relationship between circ_0010452 and miR-543. Subsequently, the regulatory effects of circ_0010452 and miR-543 on osteogenic differentiation and the capability of mineralization were evaluated by alkaline phosphatase (ALP) determination and alizarin red staining, respectively. Results: The expression of circ_0010452 decreased gradually and miR-543 increased in hBMSCs with the prolongation of osteogenic differentiation. circ_0010452 could bind to miR-543, which was negatively regulated by miR-543 in hBMSCs. Moreover, knockdown of circ_0010452 inhibited proliferation and osteogenic differentiation by upregulating miR-543, as well as upregulating expressions of OSX, OCN and Col-1. Furthermore, knockdown of circ_0010452 markedly promoted the capability of mineralization of hBMSCs, which was further reversed by transfection of miR-543 inhibitor. The knockdown of miR-543 partially reversed the inhibitory effect of circ_0010452 on the osteogenesis of hBMSCs. Conclusions: Silence of circ_0010452 promotes the development of OP via binding to miR-543 regulating proliferation and osteogenic differentiation of hBMSCs, thus promoting the progression of osteoporosis.