Differential long noncoding RNA/mRNA expression profiling and functional network analysis during osteogenic differentiation of human bone marrow mesenchymal stem cells

The purpose of this study was to investigate the differential expression and putative function of long noncoding RNAs (lncRNAs) during the osteogenic differentiation of human bone marrow mesenchymal stem cells (MSCs). The differential lncRNAs expression profiles of undifferentiated and differentiated cells during osteogenic differentiation were established by lncRNA microarray. Microarray data were validated using quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Bioinformatic analyses (gene ontology, pathway and co-expression network analysis) were applied for further study of these differentially expressed lncRNAs. A total of 1,206 differentially expressed lncRNAs were identified during the process of osteogenic differentiation. Among these lncRNAs, 687 were up-regulated and 519 were down-regulated more than two-fold. Bioinformatic analyses were applied for further study of these differentially expressed lncRNAs. Further analysis found 48 regulated enhancer-like lncRNA and 14 lincRNA. The dynamic expression trends H19 and uc022axw.1 were then observed using qRT-PCR. The results showed that the two up-regulated lncRNAs are likely to play important roles in osteogenic differentiation process. Taken together, our study first revealed the expression profiles of lncRNAs in osteogenic differentiation of human bone marrow MSCs. It provides an experimental basis for further research on lncRNAs functions during osteogenic differentiation of human bone marrow MSCs.

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.

BackgroundThe proliferation and osteogenic differentiation of human bone marrow mesenchymal stem cells (HBMScs) are modulated by a variety of microRNAs (miRNAs). SATB homeobox 2 (SATB2) is a critical transcription factor that contributes to maintain the balance of bone metabolism. However, it remains unclear how the regulatory relationship between miR-103 and SATB2 on HBMScs proliferation and osteogenic differentiation.MethodsHBMScs were obtained from Cyagen Biosciences and successful induced osteogenic differentiation. The proliferation abilities of HBMScs after treatment with agomiR-103 and antagomiR-103 were assessed using a cell counting Kit-8 (CCK-8) assay, and osteogenic differentiation was determined using alizarin red S staining and alkaline phosphatase (ALP) activity assay. The expression levels of miR-103, SATB2, and associated osteogenic differentiation biomarkers, including RUNX family transcription factor 2 (RUNX2), bone gamma-carboxyglutamate protein (BGLAP), and secreted phosphoprotein 1 (SPP1), were evaluated using real-time qPCR and Western blot. The regulatory sites of miR-103 on SATB2 were predicted using bioinformatics software and validated using a dual luciferase reporter assay. The underlying mechanism of miR-103 on SATB2-medicated HBMScs proliferation and osteogenic differentiation were confirmed by co-transfection of antagomiR-103 and SATB2 siRNA.ResultsThe expression of miR-103 in HBMScs after induction of osteogenic differentiation was reduced in a time-dependent way. Overexpression of miR-103 by transfection of agomiR-103 suppressed HBMScs proliferation and osteogenic differentiation, while silencing of miR-103 by antagomiR-103 abolished these inhibitory effects. Consistently, RUNX2, BGLAP and SPP1 mRNA and protein expression were decreased in agomiR-103 treated HBMScs compared with those in agomiR-NC group. Meanwhile, antagomiR-103 upregulated the mRNA and protein expression levels of RUNX2, BGLAP and SPP1 in HBMScs. Further studies revealed that SATB2 was a direct target gene of miR-103. BMSCs transfected with agomiR-103 exhibited significantly downregulated protein expression level of SATB2, whereas knockdown of miR-103 promoted it. Additionally, rescue assays confirmed that silencing of SATB2 partially reversed the effects of antagomiR-103 induced HBMScs proliferation and osteogenic differentiation.ConclusionsThe present results suggested that miR-103 negatively regulates SATB2 to serve an inhibitory role in the proliferation and osteogenic differentiation of HBMScs, which sheds light upon a potential therapeutic target for treating bone-related diseases.

We aimed to assess the regulatory effects of miR-28 on the osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMMSCs). HBMMSCs isolated, cultured and induced (at P3) to undergo osteogenic induction. The expressions of miRNAs were detected by gene microarray, and differentially expressed miRNAs in hBMMSCs compared with induced cells were obtained by significance analysis of microarrays. The microarray findings were confirmed by RT-PCR. TargetScan showed that signal transducer and activator of transcription 1 (STAT1) was the downstream target gene of miR-28. The relationship between miR-28 and STAT1 was validated using dual-luciferase reporter gene assay. HBMMSCs were transfected with miR-28 mimics and STAT1 siRNA, respectively. Samples were collected on day 10 after osteogenic differentiation, and the alkaline phosphatase (AKP) activity, Runt-related transcription factor 2 (RUNX2, a key regulator of osteogenic differentiation) and STAT1 expressions were determined using kits, PCR and Western blotting, respectively. Cell proliferation and migration were detected through CCK-8 and Transwell assays, respectively. During the osteogenic differentiation of hBMMSCs, the expression level of miR-28 increased. MiR-28 specifically bound the 3’-untranslated region (3’UTR) of STAT1 mRNA. It inhibited STAT1 expression in a targeted manner during osteogenic differentiation. Interference with STAT1 partially mimicked the regulatory effects of miR-28 overexpression on the osteogenic differentiation of hBMMSCs. Interference with STAT1 or overexpression of miR-28 did not affect proliferation or migration. MiR-28 has gradually increased expression during the osteogenic differentiation of hBMMSCs, which can directly bind STAT1 3’UTR and inhibit its expression, thereby up-regulating AKP and RUNX2, and promoting osteogenic differentiation.

Signal transducer and activator of transcription 3—A key molecular switch for human mesenchymal stem cell proliferation

The mechanisms underlying the osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) remain unclear. In the present study, we aimed to identify the key biological processes during osteogenic differentiation. To this end, we downloaded three microarray data sets from the Gene Expression Omnibus (GEO) database: GSE12266, GSE18043 and GSE37558. Differentially expressed genes (DEGs) were screened using the limma package, and enrichment analysis was performed. Protein‐protein interaction network (PPI) analysis and visualization analysis were performed with STRING and Cytoscape. A total of 240 DEGs were identified, including 147 up‐regulated genes and 93 down‐regulated genes. Functional enrichment and pathways of the present DEGs include extracellular matrix organization, ossification, cell division, spindle and microtubule. Functional enrichment analysis of 10 hub genes showed that these genes are mainly enriched in microtubule‐related biological changes, that is sister chromatid segregation, microtubule cytoskeleton organization involved in mitosis, and spindle microtubule. Moreover, immunofluorescence and Western blotting revealed dramatic quantitative and morphological changes in the microtubules during the osteogenic differentiation of human adipose‐derived stem cells. In summary, the present results provide novel insights into the microtubule‐ and cytoskeleton‐related biological process changes, identifying candidates for the further study of osteogenic differentiation of the mesenchymal stem cells.

BackgroundAs one of the most common chronic diseases in the world, osteoporosis occurs especially in postmenopausal women. Circular RNAs (circRNAs) are emerging as major drivers in human disease. The aim of the present study was to analyse circRNA expression profiles in osteoporosis and to explore the clinical significance and the regulatory molecular mechanism of hsa_circ_0006859 during osteoporosis.MethodsExosomes were isolated from clinically collected serum samples. A circRNA microarray was performed to screen differentially expressed circRNAs. Quantitative real-time PCR (qRT-PCR) and western blot were performed to analyse target gene mRNA expression and protein expression. Alizarin red staining (ARS) was performed to evaluate the mineralization ability of human bone marrow mesenchymal stem cells (hBMSCs). Oil Red O staining was performed to evaluate the lipid droplet formation ability of hBMSCs. Bioinformatics analysis and the luciferase reporter assay were performed to investigate the interaction between two genes.ResultsHsa_circ_0006859 was identified as one of the most upregulated circRNAs in the microarray analysis. Hsa_circ_0006859 in exosomes was upregulated in osteoporosis patients compared to healthy controls. Hsa_circ_0006859 differentiated osteopenia or osteoporosis patients from healthy controls with high sensitivity and specificity. Hsa_circ_0006859 suppressed osteoblastic differentiation and promoted adipogenic differentiation of hBMSCs. Hsa_circ_0006859 directly bound to miR-431-5p, and ROCK1 was identified as a novel target gene of miR-431-5p. Hsa_circ_0006859 is a competing endogenous RNA (ceRNA) of miR-431-5p that promotes ROCK1 expression. Hsa_circ_0006859 suppressed osteogenesis and promoted adipogenesis by sponging miR-431-5p to upregulate ROCK1.ConclusionsExosomal hsa_circ_0006859 is a potential biomarker for postmenopausal osteoporosis and controls the balance between osteogenesis and adipogenesis in hBMSCs by sponging miR-431-5p.

BackgroundHuman bone marrow mesenchymal stem cells (hBMSCs) are a major source of osteoblast precursor cells and are directly involved in osteoporosis (OP) progression. Bromodomain-containing protein 4 (BRD4) is an important regulator for osteogenic differentiation. Therefore, its role and mechanism in osteogenic differentiation process deserve further investigation.MethodshBMSCs osteogenic differentiation was evaluated by flow cytometry, alkaline phosphatase assay and alizarin red staining. Western blot was used to test osteogenic differentiation-related proteins, BRD4 protein, WNT family members-4 (WNT4)/NF-κB-related proteins, and glycolysis-related proteins. Metabolomics techniques were used to detect metabolite changes and metabolic pathways. BRD4 and WNT4 mRNA levels were determined using quantitative real-time PCR. Dual-luciferase reporter assay and chromatin immunoprecipitation assay were performed to detect BRD4 and WNT4 interaction. Glycolysis ability was assessed by testing glucose uptake, lactic acid production, and ATP levels.ResultsAfter successful induction of osteogenic differentiation, the expression of BRD4 was increased significantly. BRD4 knockdown inhibited hBMSCs osteogenic differentiation. Metabolomics analysis showed that BRD4 expression was related to glucose metabolism in osteogenic differentiation. Moreover, BRD4 could directly bind to the promoter of the WNT4 gene. Further experiments confirmed that recombinant WNT4 reversed the inhibition effect of BRD4 knockdown on glycolysis, and NF-κB inhibitors (Bardoxolone Methyl) overturned the suppressive effect of BRD4 knockdown on hBMSCs osteogenic differentiation.ConclusionBRD4 promoted hBMSCs osteogenic differentiation by inhibiting NF-κB pathway via enhancing WNT4 expression.

Objective: To investigate the role of autophagy mediated by mTOR signaling pathway in the inhibition of osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) induced by cadmium. Methods: HBMSCs were divided into 0, 2.5 or 5.0 μmol/L groups according to the exposure dose of cadmium chloride (CdCl2), and each group was treated for 1 day, 4 days and (or) 7 days. The ALP activity and mRNA and protein expression levels of osteogenesis markers (ALP, RUNX2 and OSTERIX), autophagy-related proteins (LC3 and Beclin-1) and mTOR signaling pathway related proteins (mTOR, p-mTOR and p-p70S6K) expression, alkaline phosphatase staining and alizarin red staining were detected. MHY 1485 was selected as the signaling pathway activator. The control group, CdCl2 group (5.0 μmol/L), MHY 1485 group and CdCl2+MHY 1485 combined treatment group were set. After 7 days of treatment, the expression levels of autophagy related proteins and mTOR signaling pathway related proteins of hBMSCs in each group were detected. Results: There was no significant difference in ALP activity between 0, 2.5 and 5.0 μmol/L groups on day 1 and 4 (P>0.05); On day 7, compared with the 0 μmol/L group, the ALP activity, expression of osteogenic markers (ALP, RUNX2, OSTERIX) and mTOR signaling pathway related proteins (mTOR, p-mTOR, p-p70S6K) expression decreased in the 2.5 and 5.0 μmol/L group (P<0.05). Compared with the 0 μmol/L group, the staining of the 2.5 and 5.0 μmol/L groups became lighter, and the formation of ALP and mineralized nodules was reduced. Compared with the CdCl2 group, the autophagy related protein expression in the CdCl2+MHY 1485 combined treatment group decreased, and the mTOR signaling pathway related protein expression increased. The difference was statistically significant (P<0.05). Conclusion: The inhibition of osteogenic differentiation of hBMSCs by cadmium may be related to autophagy mediated by mTOR signaling pathway.

Osteogenic differentiation (OD) of human bone marrow mesenchymal stem cells (hBMSCs) plays a vital role in bone formation, and its impairment contributes to osteoporosis, particularly estrogen-deficient osteoporosis (EDO). Baicalein (BN) and silent information regulator of transcription 1 (SIRT1) have been implicated in bone metabolism; however, the upstream regulatory mechanisms of SIRT1 and the potential synergistic effect of BN with the transcription factor growth factor independent 1 (GFI1) remain unclear. In this study, target genes were identified via SwissTargetPrediction and GeneCards databases, and protein-protein interaction (PPI) networks were constructed with Cytoscape software. The mRNA and protein expression levels were analyzed by RT-qPCR and Western blotting. The Alkaline phosphatase (ALP) Assay Kit was employed to analyze ALP activity in osteogenically differentiated hBMSCs. Alizarin red S (ARS) staining was utilized to assess cell calcification. Apoptosis was determined by flow cytometry. Kits were used to test changes in ferroptosis-related indicators such as malondialdehyde (MDA), glutathione (GSH), reactive oxygen species (ROS) and Fe2+ content. Cell viability was determined by the CCK-8 assay. The binding of GFI1 to the SIRT1 promoter was confirmed by dual-luciferase reporter and chromatin immunoprecipitation (ChIP) assays. A rat osteoporosis model was established for in vivo validation. Bioinformatics analysis identified SIRT1 as a common target of BN, ferroptosis, and EDO. SIRT1 expression was significantly decreased in osteoporosis patients' peripheral blood but upregulated duringOD, paralleling increased GFI1 expression. ChIP assays confirmed GFI1's direct binding to the SIRT1 promoter, activating its transcription. BN treatment enhanced SIRT1 protein level. BN and GFI1 synergistically regulated SIRT1 at protein and transcriptional levels, respectively, promoting OD and inhibiting apoptosis and ferroptosis in hBMSCs. In vivo, BN facilitated OD in osteoporosis rats. These findings revealed a novel dual-level regulatory mechanism of SIRT1 by BN and GFI1, providing new insights into osteoporostic pathogenesis and suggesting potential therapeutic targets.

To explore the effects of the shock wave on the capacity of mesenchymal stem cells(MSCs) to proliferate and differentiate into osteoblasts. MSCs were isolated from the bone marrow of healthy donors. The human bone marrow MSCs(BM-MSCs) were divided into 3 groups including blank control group,osteoinduced group and shock wave group. The MSCs in blank control group were cultured with common mediam; the MSCs in osteoinduced group were treated with osteogenic agents and cultured; the MSCs in shock wave group were cultured with common medium and stimulated by shock wave. The morphology of MSCs in each groups were observed by micoscopy; the CCK-8 was used to detect the proliferation ability of MSCs; the alkaline phosphatase staining and von Kossa staining were used to evaluale the differentiation potential of MSCs in each groups. The results of CCK-8 revealed the shock wave could promote cell proliferation as compared with blank control group. The results of alkaline phosphatase and Von Kossa staining showed that the shock wave displayed a stronger ability to promote the human BMMSC differentiation into osteoblasts cells in comparison with the osteoinduced group. The blank control group was weakly positively stainined. The shock wave treatment can promote proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells.

Objective(s):The application of stem cells holds great promises in cell transplants. Considering the lack of optimal in vitro model for hepatogenic differentiation, this study was designed to examine the effects of laminin matrix on the improvement of in vitro differentiation of human bone marrow mesenchymal stem cells (hBM-MSC) into the more functional hepatocyte-like cells.Materials and Methods:Characterization of the hBM-MSCs was performed by immunophenotyping and their differentiation into the mesenchymal-derived lineage. Then, cells were seeded on the laminin-coated or tissue culture polystyrene (TCPS). The differentiation was carried out during two steps. Afterward, the expression of hepatocyte markers such as AFP, ALB, CK-18, and CK-19 as well as the expression of C-MET, the secretion of urea, and the activity of CYP3A4 enzyme were determined. Moreover, the cytoplasmic glycogen storage was examined by periodic acid–Schiff (PAS) staining.Results:The results demonstrated that the culture of hBM-MSC on laminin considerably improved hepatogenic differentiation compared to TCP group. A significant elevated level of urea biosynthesis and CYP3A4 enzyme activity was observed in the media of the laminin-coated differentiated cells (P<0.05). Furthermore higher expressions of both AFP and ALB were determined in cells differentiated on laminin matrix. Glycogen accumulation was not detected in the undifferentiated hBM-MSCs, however, both differentiated cells in laminin and TCPS groups demonstrated the intracellular glycogen accumulation on day 21 of hepatogenic differentiation.Conclusion:Taken together, these findings may indicate that laminin matrix can improve terminal differentiation of hepatocyte-like cells from hBM-MSCs. Thus, laminin might be considered as a suitable coating in hepatic tissue engineering designs.

Ultrasound Irradiation Combined with Hepatocyte Growth Factor Accelerate the Hepatic Differentiation of Human Bone Marrow Mesenchymal Stem Cells

Human bone marrow mesenchymal stem cells (hBMSCs) are widely used cell source for clinical bone regeneration. Achieving the greatest therapeutic effect is dependent on the osteogenic differentiation potential of the stem cells to be implanted. However, there are still no practical methods to characterize such potential non-invasively or previously. Monitoring cellular morphology is a practical and non-invasive approach for evaluating osteogenic potential. Unfortunately, such image-based approaches had been historically qualitative and requiring experienced interpretation. By combining the non-invasive attributes of microscopy with the latest technology allowing higher throughput and quantitative imaging metrics, we studied the applicability of morphometric features to quantitatively predict cellular osteogenic potential. We applied computational machine learning, combining cell morphology features with their corresponding biochemical osteogenic assay results, to develop prediction model of osteogenic differentiation. Using a dataset of 9,990 images automatically acquired by BioStation CT during osteogenic differentiation culture of hBMSCs, 666 morphometric features were extracted as parameters. Two commonly used osteogenic markers, alkaline phosphatase (ALP) activity and calcium deposition were measured experimentally, and used as the true biological differentiation status to validate the prediction accuracy. Using time-course morphological features throughout differentiation culture, the prediction results highly correlated with the experimentally defined differentiation marker values (R>0.89 for both marker predictions). The clinical applicability of our morphology-based prediction was further examined with two scenarios: one using only historical cell images and the other using both historical images together with the patient's own cell images to predict a new patient's cellular potential. The prediction accuracy was found to be greatly enhanced by incorporation of patients' own cell features in the modeling, indicating the practical strategy for clinical usage. Consequently, our results provide strong evidence for the feasibility of using a quantitative time series of phase-contrast cellular morphology for non-invasive cell quality prediction in regenerative medicine.

MicroRNAs (miRNAs) regulate osteogenic differentiation and influence osteoporosis (OP). The aim of this study was to determine the potential role of miR-874-3p in OP. The expression levels of miR-874-3p and leptin (LEP) in the femoral neck trabeculae of 35 patients with or without OP were measured by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The effects of miR-874-3p or LEP on the cell proliferation and alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), and osterix (OSX) levels were observed by upregulating miR-874-3p in human bone marrow mesenchymal stem cells (hBMSCs). Additionally, calcium deposition levels were evaluated using alizarin red staining (ARS). Molecular mechanisms of miR-874-3p and LEP underlying the osteogenic differentiation of hBMSCs were also evaluated using bioinformatics analysis, luciferase reporter assays, and RNA pull-down assays. The miR-874-3p levels were significantly lower in the femoral neck trabeculae of patients with OP than those of the control group, while the opposite was observed regarding the levels of LEP. Expression levels of miR-874-3p in hBMSCs were upregulated during osteogenic differentiation, while those of LEP were downregulated. Moreover, miR-874-3p upregulation promoted ALP, RUNX2, OCN, and OSX mRNA expression, cell proliferation, and calcium deposition in hBMSCs. LEP was found to be a target gene of miR-874-3p. Overexpression of LEP inhibited the expression of osteoblast markers and reversed the effect of osteogenic differentiation induced by the upregulation of miR-874-3p. In conclusion, miR-874-3p promoted the proliferation and differentiation of hBMSCs by downregulating the expression of LEP, thus inhibiting OP. Abbreviations : miRNAs: microRNAs; OP: osteoporosis; hBMSCs: human Bone Marrow Mesenchymal stem cells; LEP: leptin; DEGs: differentially expressed genes