To identify novel candidate genes associated with osteoporosis, RNA‑sequence analysis of human mesenchymal stem cells (hMSCs) from patients with osteoporosis (G3) and osteopenia (G2), and healthy controls (G1) was performed. Differentially expressed genes (DEGs) from among the three groups were identified. DEGs were separated into nine groups according to their gene expression patterns: UU (up and up), UF (up and flat), UD (up and down), FU (flat and up), FF (flat and flat), FD (flat and down), DU (down and up), DF (down and flat), and DD (down and down). Among the 42 DEGs between G3 and G1, eight candidate genes, namely stimulated by retinoic acid 6 (STRA6), melanophilin, neurotrophic receptor tyrosine kinase 2, cartilage oligomeric matrix protein, collagen type XI α 1 chain, integrin subunit β 2, monooxygenase DBH‑like 1 and selenoprotein P, were selected, as they demonstrated consistent gene expression patterns of UU, FU, FD, and DD. Among these eight genes, STRA6 was highly expressed in the osteoporosis group and based on additional data from quantitative polymerase chain reaction analysis, it was selected for further study. In order to investigate whether STRA6 served a functional role in osteoblast or adipocyte differentiation, the effects of STRA6 expression changes in pluripotent stem cell C3H10T1/2, preosteoblast MC3T3‑E1 and stromal ST2 cell lines were examined. Bone morphogenetic protein 2 enhanced STRA6 expression only at the early stage of osteoblast differe-ntiation, and overexpression of STRA6 temporally inhibited the expression of osteoblastogenesis markers, including runt related transcription factor 2, bone sialoprotein and osteocalcin. Furthermore, the knockdown of STRA6 slightly enhanced nodule formation at the late stage of osteoblast differentiation, and overexpression of STRA6 in ST2 cells enhanced adipocyte differentiation. Taken together, STRA6 expression could be associated with the pathogenesis of osteoporosis by promoting adipocyte differentiation over osteoblast differentiation in the hMSC population.

Biodegradable magnesium (Mg) materials are considered ideal as osteosynthesis implants. However, clinical application has proven complex. This is primarily associated with the issue of reducing the extent of implant degradation to a range acceptable for the human body, while simultaneously enhancing osteogenesis or osteoinduction. In the present study, a combination of Mg ions and low‑intensity pulsed ultrasound (LIPUS) treatment was applied in hFOB 1.19 human osteoblast cells as a potential strategy to resolve this issue. A total of 7,314 differentially expressed genes (DEGs) and 826 shared DEGs in hFOB1.19 osteoblast cells were identified by microarray analysis following treatment with Mg and/or LIPUS. Gene Ontology analysis demonstrated that among cells treated with a combination of Mg and LIPUS, DEGs were significantly enriched in various functional annotations, including 'wound healing', 'transforming growth factor beta receptor signaling pathway', 'transcription, DNA‑templated', 'receptor complex', 'nucleus', 'SMAD protein complex', 'DNA binding', 'metal ion binding' and 'GTPase activator activity'. Notably, the transforming growth factor (TGF)‑β, mitogen‑activated protein kinase (MAPK) and tumor necrosis factor (TNF) signaling pathways were preferentially overrepresented in the Mg and LIPUS combination group, which was subsequently confirmed by reverse transcription‑quantitative polymerase chain reaction. Furthermore, genes involved in osteoblast mineralization promotion, including bone morphogenetic protein 6, noggin, bone morphogenetic protein receptor (BMPR)1A, BMPR2 and SMAD 5/8, were significantly upregulated following combination treatment compared with the control group. Genes involved in the promotion of migration, including c‑Jun N‑terminal kinase, doublecortin, paxillin and Jun proto‑oncogene AP‑1 transcription factor subunit, were also upregulated in the combination treatment group compared with the control group. The DEG data were supported by morphological observations of the osteoblasts using alizarin red S staining and wound healing assays, which indicated that Mg and LIPUS combinative treatment had a synergistic effect on osteoblast mineralization and migration. Additionally, the combined treatment significantly upregulated metal transporter genes associated with Mg entry, including ATPase Na+/K+‑transporting subunit α1, cyclin and CBS domain divalent metal cation transport mediator 2, K+ voltage‑gated channel subfamily J member 14, transient receptor potential cation channel (TRP) subfamily M member 7 and TRP subfamily V member 2. In summary, the findings of the present study revealed that combined stimulation with Mg and LIPUS may exhibit a synergistic effect on human osteoblast bone formation through the TGF‑β, MAPK and TNF signaling pathways, while also facilitating Mg influx. The present study demonstrated the potential of combinative LIPUS and Mg treatment as a novel therapeutic strategy for enhancing the osteoinduction, biocompatibility and biosafety of biodegradable Mg implants.

MicroRNAs are identified as negative regulators in gene expression through silencing gene expression at the post-transcriptional and translational levels. Bone morphogenetic protein 9 (BMP9) is the most effective in inducing osteogenesis in the BMP family, the members of which were originally identified as osteoinductive cytokines. In the current study, the role of miR‑23b in the progression of BMP9‑induced C2C12 myoblasts was investigated. The results indicated that miR‑23b was significantly downregulated in C2C12 myoblasts induced by BMP9. Overexpression of miR‑23b significantly inhibited osteogenesis in the C2C12 myoblasts. In addition, it was observed that Runx2 was negatively regulated by miR‑23b at the post‑transcriptional level, via a specific target site within the 3'UTR of Runx2. Knockdown of Runx2 promoted miR‑23b‑induced inhibition of osteogenesis in C2C12 myoblasts. The expression of Runx2 was observed to be frequently upregulated in osteoblast cell lines and inversely correlated with miR‑23b expression. Thus, the results of the present study suggest that miR‑23b inhibits BMP9‑induced C2C12 myoblast osteogenesis via targeting of the Runx2 gene, acting as a suppressor. The current study contributes to the understanding of the functions of BMP9 in ossification.

Necrosis of the femoral head (NFH), a severe orthopedic disease in adults, involves the collapse of the femoral head. The pathophysiological mechanisms underlying NFH are yet to be fully investigated. The aim of the present study was to identify potentially important genes and signaling pathways involved in NFH and investigate their molecular mechanisms. Gene expression profiles of patients with NFH and healthy controls were compared using the Gene Expression Omnibus (GEO) database repository of the National Center of Biotechnology Information. GSE74089 from the GEO database included 4 patients with NFH and 4 healthy individuals. A total of 1,191 differentially expressed genes (DEGs) were identified between the patients with NFH and controls, including 743 upregulated and 448 downregulated DEGs. Then, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis revealed that upregulated DEGs were mainly involved in the phosphoinositide 3‑kinase/protein kinase B signaling pathway, focal adhesion and extracellular matrix‑receptor interactions. Additionally, protein‑protein interaction (PPI) analysis identified the most central DEGs as vascular endothelial growth factor A, Jun proto‑oncogene, cyclin D1, fibroblast growth factor 2, HECT domain and ankyrin repeat‑containing E3 ubiquitin protein ligase 1, protein kinase Cα, bone morphogenetic protein 2 and prostaglandin‑endoperoxide synthase 2. PPI analysis also identified guanine nucleotide‑binding protein, γ13 as the most commonly downregulated gene based on different centrality. The results of the present study may provide novel insight into the genes and associated pathways involved in NFH, and aid the identification of novel therapeutic targets and biomarkers in the treatment of NFH.

The present study investigated the expression of bone morphogenetic protein (BMP) 7 in a newborn rat model of bronchopulmonary dysplasia (BPD) and the biological effects of BMP7 on newborn rat lung fibroblast (LF) cells. For this purpose, a total of 196 newborn rats were randomly and equally assigned to a model group and a control group. Lung tissue was collected at days 3, 7, 14 and 21 for histological analysis. The location and expression of BMP7 was examined by immunohistochemical staining and reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) analysis. A total of 38 full‑term newborn rats on the day of birth were sacrificed and LF cells were isolated and treated with BMP7. The biological effects of BMP7 on LF cells were assessed by cell proliferation and cell cycle analysis. The findings demonstrated that abnormal alveolar development due to BPD was gradually intensified in the model group over time. Immunohistochemical staining revealed that the location of BMP7 in lung tissue was altered. Immunohistochemistry and RT‑qPCR assays demonstrated a gradual decrease in BMP7 expression in the model group induced by hyperoxia. MTT assays demonstrated that BMP7 inhibited LF cells and the inhibitory effect was dose‑dependent and time‑dependent. Flow cytometry revealed that the inhibitory effect of BMP7 in LF cells was causing cell cycle arrest at the G1 phase. The present study demonstrated that BMP7 may serve an important role in alveolar development in a BPD model. BMP7 may be involved in abnormal alveolar development through the regulation of LF proliferation.

The present study aimed to identify any association between bone morphogenetic protein‑7 (BMP‑7) and the expression of the transcriptional co‑repressor Ski‑related novel protein N (SnoN), in addition to alterations in tubulointerstitial fibrosis, during the development and progression of diabetic nephropathy (DN). Streptozotocin was injected into the tail veins of 20 healthy and specific pathogen‑free male Sprague‑Dawley rats. The rats were sacrificed to detect the appropriate biochemical indicators of renal pathological alterations following 24 weeks. Then, various doses of human recombinant BMP‑7 were added to high glucose‑cultured NRK‑52E cells. Immunohistochemistry, immunofluorescence staining and western blotting were used to determine the expression of SnoN, BMP‑7, Smad ubiquitin regulatory factor (Smurf)2, Arkadia, E‑cadherin, α‑smooth muscle actin and Collagen III. Reverse transcription‑quantitative polymerase chain reaction was used to detect SnoN mRNA expression. With the progression of DN, the expression of BMP‑7 in rat renal tissue was downregulated, whereas the expression of Smurf2 and Arkadia increased. Furthermore, the expression of SnoN mRNA increased however the expression of SnoN protein decreased, accompanied by renal tubular epithelial cell mesenchymal transition, extracellular matrix (ECM) deposition and severe renal function disorder. The exogenous recombinant human BMP‑7 alleviated high glucose‑induced phenotypic transformation and ECM synthesis of NRK‑52E in vitro and upregulated SnoN transcription and protein expression, however no effect was observed on the expression of Smurf2 and Arkadia. BMP‑7 may ameliorate DN and renal fibrosis via increasing the expression of SnoN mRNA and protein in renal tubular epithelial cells, rather than directly inhibiting the degradation of SnoN by E3 ubiquitin ligase.

Early‑onset preeclampsia (EOPE) is a serious threat to maternal and foetal health. The present study aimed to identify potential biomarkers and targets for the treatment of EOPE. Expression profiles of placenta from patients with EOPE and healthy controls (GSE103542, GSE74341 and GSE44711) were downloaded from the Gene Expression Omnibus database. Integrated analysis revealed 246 genes and 28 microRNAs (miRNAs) that were differentially expressed between patients with EOPE and healthy controls. Differentially expressed genes (DEGs) were primarily enriched in 'biological processes', such as 'cell adhesion', 'female pregnancy', 'extracellular matrix organization' and 'response to hypoxia'. Significant pathways associated with DEGs primarily included 'focal adhesion', 'ECM‑receptor interaction', 'PI3K‑Akt signaling' and 'ovarian steroidogenesis'. A Protein‑Protein Interaction network of DEGs was constructed using the Search Tool for the Retrieval of Interacting Genes/Proteins online database, and epidermal growth factor receptor, collagen α‑1(I) chain, secreted phosphoprotein 1, leptin (LEP), collagen α‑2(I) chain (COL1A2), plasminogen activator inhibitor 1 (SERPINE1), Thy‑1 membrane glycoprotein, bone morphogenetic protein 4, vascular cell adhesion protein 1 and matrix metallopeptidase 1 were identified as hub genes. The alterations of hsa‑miR‑937, hsa‑miR‑148b*, hsa‑miR‑3907, hsa‑miR‑367*, COL1A2, LEP and SERPINE1 in placenta were validated using our local samples. Our research showed that the expression of hsa‑miR‑937, hsa‑miR‑1486*, hsa‑miR‑3907, hsa‑miR‑367* and hub genes in the placenta were closely associated with the pathophysiology of EOPE. hsa‑miR‑937, hsa‑miR‑1486*, hsa‑miR‑3907, hsa‑miR‑367* and hub genes could serve as biomarkers for diagnosis and as potential targets for the treatment of EOPE.

Maternal diabetes has been reported to be a critical factor for congenital heart defects (CHD) in offspring. The present study aimed to screen the key genes that may be involved in CHD in offspring of diabetic mothers. The present study obtained the gene expression profile of GSE32078, including three embryonic heart tissue samples at embryonic day 13.5 (E13.5), three embryonic heart tissue samples at embryonic day 15.5 (E15.5) from diabetic mice and their respective controls from normal mice. The cut‑off criterion of P<0.08 was set to screen differentially expressed genes (DEGs). Their enrichment functions were predicted by Gene Ontology. The enriched pathways were forecasted by Kyoto Encyclopedia of Genes and Genomes and Reactome analysis. Protein‑protein interaction (PPI) networks for DEGs were constructed using Cytoscape. The present study identified 869 and 802 DEGs in E13.5 group and E15.5 group, respectively and 182 DEGs were shared by the two developmental stages. The pathway enrichment analysis results revealed that DEGs including intercellular adhesion molecule 1 (Icam1) and H2‑M9 were enriched in cell adhesion molecules; DEGs including bone morphogenetic protein receptor type 1A, transforming growth factor β receptor 1 and SMAD specific E3 ubiquitin protein ligase 1 were enriched in the tumor growth factor‑β signaling pathway. In addition, DEGs including Icam1, C1s and Fc fragment of IgG receptor IIb were enriched in Staphylococcus aureus infection. Furthermore, the shared DEGs including Icam1, nuclear receptor corepressor 1 (Ncor1) and AKT serine/threonine kinase 3 (Akt3) had high connectivity degrees in the PPI network. The shared DEGs including Icam1, Ncor1 and Akt3 may be important in the cardiogenesis of embryos. These genes may be involved in the development of CHD in the offspring of diabetic mothers.

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system (CNS). Repulsive guidance molecule a (RGMa) has been indicated to act as a bone morphogenetic protein (BMP) co‑receptor, enhancing BMP signalling activity. However, the role and downstream pathways of the BMP signalling pathway mediated by RGMa have yet to be fully elucidated. A recent study revealed that C‑C motif chemokine ligand 5 (CCL5) has a major role in the pathogenesis of MS via the recruitment of macrophages and T‑lymphocytes into the CNS. The present study aimed to evaluate whether RGMa regulates CCL5 via the BMP pathway in MS. The results demonstrated that RGMa regulated CCL5 expression in a BMP ligand‑dependent manner in experimental autoimmune encephalomyelitis (EAE) mice in vivo and in endothelial cells in vitro. First, specific inhibition of the expression of RGMa via RNA interference led to a significant reduction of the expression of RGMa and this was associated with a significant delay of EAE, an alleviated disease course and downregulation of CCL5 expression at both the protein and mRNA levels. Furthermore, exogenous noggin, an extracellular antagonist of BMP ligand, abolished the induction effect of RGMa on CCL5 in endothelial cells. Taken together, these results suggested that RGMa is an important regulator of MS and inflammatory mediators such as CCL5, and the present results should prove to be useful in terms of further elucidating the RGMa‑BMP receptor signalling pathway and the pathogenesis of RGMa on MS as far as the involvement of blood‑brain barrier permeability is concerned.

Stem cells from apical papilla (SCAP) possess clear osteo‑/odontogenic differentiation capabilities, and are regarded as the major cellular source for root dentin development. Bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor (VEGF) serve pivotal roles in the modulation of tooth development and dentin formation. However, the synergistic effects of BMP2 and VEGF on osteo‑/odontogenic differentiation of SCAP remain unclear. The current study aimed to investigate the proliferative and osteo‑/odontogenic differentiating capabilities of BMP2 and VEGF gene-co-transfected SCAP (SCAP-BMP2-VEGF) in vitro. The basic characteristics of the isolated SCAP were identified by the induction of multipotent differentiation and by flow cytometry. Lentiviral vector‑mediated gene transfection was conducted with SCAP in order to construct blank vector‑transfected SCAP (SCAP-green fluorescent protein), BMP2 gene-transfected SCAP (SCAP-BMP2), VEGF gene‑transfected SCAP (SCAP‑VEGF) and SCAP-BMP2-VEGF. The Cell Counting Kit 8 assay was used to analyze the proliferative capacities of the four groups of cells. The expression of osteo-/odontogenic genes and proteins in the cells were evaluated by reverse transcription-quantitative polymerase chain reaction and western blotting. The mineralized nodules formed by the four group cells were visualized by alkaline phosphatase (ALP) staining. Among the four groups of cells, SCAP‑VEGF was demonstrated to exhibit increased proliferation, and SCAP‑BMP2‑VEGF exhibited reduced proliferation during eight days observation. SCAP‑BMP2‑VEGF exhibited significantly increased expression levels of ALP, osteocalcin, dentin sialophosphoprotein, dentin matrix acidic phosphoprotein gene 1 and dentin sialoprotein than the other three groups at the majority of the time points. Furthermore, the SCAP‑BMP2‑VEGF group exhibited a significantly greater number of ALP‑positive mineralized nodules than the other groups following 16 days culture in vitro. In conclusion, lentiviral vector-mediated BMP2 and VEGF gene co-transfection significantly activated the osteo‑/odontogenic differentiation of human SCAP.

Increasing research has demonstrated that expression of brain and muscle ARNT‑like 1 (BMAL1) and other circadian clock genes can be regulated by drugs and toxicants. We previously demonstrated that icariin, extracted from Herba Epimedii, sromotes osteogenic differentiation. However, the mechanism underlying the association between icariin and BMAL1 in osteogenic differentiation of bone marrow‑derived mesenchymal stem cells (BMSCs) remains unclear. The present study was designed with an aim to clarify the association between icariin and BMAL1 in osteogenic differentiation of BMSCs. The Cell Counting Kit‑8 assay was used to evaluate cell proliferation. The expression of bone morphogenetic protein 2 (BMP2), RUNX family transcription factor 2 (RUNX2), alkaline phosphatase (ALP), osteocalcin (OC) and BMAL1 in BMSCs was evaluated by reverse transcription‑quantitative PCR and western blotting. ALP and Alizarin red S (ARS) staining were also performed. Icariin promoted BMSC proliferation, and upregulated expression of osteogenic genes and BMAL1. In addition, expression of the osteogenic genes BMP2, RUNX2, ALP and OC were upregulated by BMAL1 overexpression. Furthermore, we confirmed that BMAL1 deficiency suppressed osteogenic differentiation in BMSCs. Finally, ARS staining of BMAL1‑/‑ BMSCs revealed that BMAL1 was an essential intermediary in matrix mineralization during osteogenic differentiation. In conclusion, these results demonstrated that icariin promoted osteogenic differentiation through BMAL1‑BMP2 signaling in BMSCs. The present study thus described a novel target of icariin that has potential applications in the treatment of osteogenic disorders.

The occurrence of pelvic organ prolapse (POP) is closely associated with alterations in the extracellular matrix proteins of the supporting ligament. Bone marrow mesenchymal stem cells (BMSCs) have the potential to differentiate into a variety of cell types, including osteoblasts, chondroblasts and adipocytes. Therefore, BMSCs have the potential to improve the clinical outcomes of POP. Tenascin‑C is a large glycoprotein that is present in the ECM and is involved in morphogenetic movements, and tissue patterning and repair. The aim of the present study was to investigate the effect of mechanical stretching on tenascin‑C expression during the differentiation of BMSCs induced by pelvic ligament fibroblasts. BMSCs were isolated from 7‑day‑old Sprague Dawley rats. Fibroblasts were obtained from rat pelvic ligaments and, at the fourth passage, were subjected to 10% deformation with 1 Hz, periodic one‑way mechanical stretch stimulation, followed by co‑culture with BMSCs. The co‑culture with stretched fibroblasts increased tenascin‑C and transforming growth factor (TGF)‑β expression levels, compared with groups without mechanical stimulation. Neutralizing anti‑TGF‑β1 antibodies, and inhibitors of TGF‑β receptor, mitogen‑activated protein kinase (MAPK) kinase and MAPK, decreased tenascin‑C expression levels induced by TGF‑β and mechanical stretching. The results of the present study suggested that the regulation of tenascin‑C expression levels in BMSCs co‑cultured with mechanically stretched pelvic ligament fibroblasts is mediated via the soluble growth factor TGF‑β and the MAPK signaling pathway. In addition, these results indicated that in an indirect co‑culture system, pelvic ligament fibroblasts with mechanical stretch stimulation may promote the synthesis of tenascin‑C and BMSC differentiation into pelvic ligament fibroblasts.

The implications of epithelial‑mesenchymal transdifferentiation (EMT) have extended beyond the confines of renal fibrosis to renal tubulointerstitial fibrosis. It has been proposed that EMT may be one of the mechanisms involved in the pathogenesis of renal fibrosis. However, the underlying mechanisms remain unknown. Transforming growth factor (TGF)‑β1 is considered to be an important cytokine which regulates the transdifferentiation of tubular epithelial cells into myofibroblasts in renal tubulointerstitial fibrosis. In the present study, normal rat kidney tubular epithelial cells (NRK‑52E) were treated for 48 h with TGF‑β1 (5 ng/ml) and different concentrations of artesunate (ART; 0.01, 0.1 and 1 µg/ml). Western blotting, reverse transcription‑semi quantitative polymerase chain reaction analysis and immunofluorescence staining were used to evaluate the expression of bone morphogenetic protein (BMP)‑7, uterine sensitization‑associated gene (USAG)‑1, E‑cadherin, α‑smooth muscle actin (α‑SMA) and extracellular matrix collagen type I (Col I) mRNA. ART was able to attenuate renal injury in a unilateral ureteral obstruction model. However, its anti‑fibrotic effect remains to be elucidated. In the present study, it was observed that ART was able to ameliorate the TGF‑β1‑induced alterations in cellular morphology. In addition, ART inhibited the TGF‑β1‑induced USAG‑1 increase and the decrease in BMP‑7. Treatment with ART markedly attenuated the TGF‑β1‑induced upregulation of α‑SMA and downregulation of E‑cadherin. Additionally, ART was able to significantly attenuate the deposition of interstitial collagens, including Col I. The results of the present study further verified the therapeutic efficacy of ART in TGF‑β1‑induced renal interstitial fibrosis. These findings indicated that ART may hold the potential to prevent chronic kidney diseases via the suppression of USAG‑1 expression or by increasing BMP‑7 expression.

One key risk factor of aortic valve stenosis in clinical practice is bicuspid aortic valve (BAV). Increasing evidence indicates that numerous microRNAs (miRs/miRNAs) are involved in BAV calcification via their target genes. miR‑330‑3p was found to be involved in the deterioration of BAV calcification by miR profiling in human calcified BAV and tricuspid aortic valve (TAV) tissues in the present study and the underlying mechanism was investigated. RNA sequencing was performed on four BAV and four TAV tissues from patients with aortic stenosis before these leaflets were examined for the expression levels of miR‑330‑3p and CREB‑binding protein (CREBBP) by reverse transcription‑PCR. The alteration of functional factors associated with calcification was also assessed by Western blotting and immunohistochemistry in human aortic tissue samples. The putative target of miR‑330‑3p was detected by dual‑luciferase assay in 293 cells. Furthermore, the influence of miR‑330‑3p expression on osteogenic progression was explored in cultured porcine valve interstitial cells (VICs). Rescue experiments of CRBBP were performed to confirm the influence of the miR‑330‑3p‑CREBBP pathway in the calcification progress in porcine VICs. RNA sequencing indicated distinct expression of miR‑330‑3p in human BAV tissues compared with TAV, which was then confirmed by PCR. CREBBP expression levels in human BAV and TAV leaflets also demonstrated the opposite alterations. This negative correlation was then confirmed in cultured porcine VICs. Under an osteogenic environment, cellular calcification was promoted in miR‑330‑3p‑overexpressed porcine VICs expressing higher bone morphogenetic protein 2, Runt‑related transcription factor 2, matrix metalloproteinase (MMP)‑2, MMP‑9 and collagen I compared with controls. Rescue experiments further confirmed that miR‑330‑3p played its role via targeting CREBBP in porcine VICs. Collectively, miR‑330‑3p was upregulated in calcified BAV compared with TAV. The upregulation of miR‑330‑3p promotes the calcification progress partially via targeting CREBBP.

Due to the lack of potential organs, hepatocellular transplantation has been considered for treating end-stage liver disease. Induced pluripotent stem cells (iPSCs) are reverted from somatic cells and are able to differentiate into hepatocytes. The present study aimed to investigate the mechanisms underlying iPSC differentiation to hepatocytes. GSE66076 was downloaded from the Gene Expression Omnibus; this database includes data from 3 undifferentiated (T0), 3 definitive endoderm (T5), and 3 early hepatocyte (T24) samples across hepatic‑directed differentiation of iPSCs. Differentially expressed genes (DEGs) between T0 and T5 or T24 samples were identified using the linear models for microarray data package in Bioconductor, and enrichment analyses were performed. Using the weighted correlation network analysis package in R, clusters were identified for the merged DEGs. Cytoscape was used to construct protein‑protein interaction (PPI) networks for DEGs identified to belong to significant clusters. Using the ReactomeFI plugin in Cytoscape, functional interaction (FI) networks were constructed for the common genes. A total of 433 and 1,342 DEGs were identified in the T5 and T24 samples respectively, compared with the T0 samples. Blue and turquoise clusters were identified as significant gene clusters. In the PPI network for DEGs in the blue cluster, the key node fibroblast growth factor 2 (FGF2) could interact with bone morphogenetic protein 2 (BMP2). Cyclin‑dependent kinase 1 (CDK1) was demonstrated to have the highest degree (degree=71) in the PPI network for DEGs in the turquoise cluster. Enrichment analysis for the common genes, including hepatocyte nuclear factor 4α (HNF4A) and epidermal growth factor (EGF), in the FI network indicated that EGF and FGF2 were enriched in the Ras and Rap1 signaling pathways. The present results suggest that FGF2, BMP2, CDK1, HNF4A and EGF may participate in the differentiation of iPSCs into hepatocytes.

Ankylosing spondylitis (AS) is a chronic inflammatory systemic disease and is difficult to detect in the early stages. The present study aimed to investigate the role of microRNA (miR)‑204‑5p in osteogenic differentiation of AS fibroblasts. Bone morphogenetic protein 2 (BMP‑2) was used to induce osteogenic differentiation. Cells were divided into the following groups: AS group, AS + BMP‑2 group, AS + BMP‑2 + miR‑negative control group, AS + BMP‑2 + miR‑204‑5p mimics group and AS + BMP‑2 + miR‑204‑5p mimics + pcDNA‑Notch2 group. The expression levels of miR‑204‑5p, Notch2, runt‑related transcription factor 2 (RUNX2) and osteocalcin were detected via reverse transcription‑quantitative PCR analysis. The binding site between Notch2 and miR‑204‑5p was predicted using TargetScan software and verified via the dual‑luciferase reporter assay. Alkaline phosphatase (ALP) activity was assessed via the ALP assay, while the mineralized nodules area was determined via the Alizarin Red S staining assay. The results demonstrated that Notch2 is a target gene of miR‑204‑5p. Furthermore, treatment with BMP‑2 significantly decreased miR‑204‑5p expression, and significantly increased ALP activity, the mineralized nodules area and the expression levels of Notch2, RUNX2 and osteocalcin in ligament fibroblasts (all P<0.05). Conversely, transfection with miR‑204‑5p mimics significantly increased miR‑204‑5p expression, and significantly decreased ALP activity, the mineralized nodules area and the expression levels of Notch2, RUNX2 and osteocalcin in ligament fibroblasts (all P<0.05). Notably, transfection with pcDNA‑Notch2 significantly reversed the inhibitory effects induced by miR‑204‑5p mimics on the osteogenic differentiation of ligament fibroblasts (all P<0.05). Furthermore, miR‑204‑5p inhibited the osteogenic differentiation of ligament fibroblasts in patients with AS by targeting Notch2. Thus, miR‑204‑5p may negatively regulate Notch2 expression and may be a potential therapeutic target for AS. Collectively, the results of the present study provide a theoretical basis for the effective treatment of patients with AS.