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Recent studies have revealed that long noncoding RNA HNF1A-antisense 1 (HNF1A-AS1) plays an important role in the development of several human malignancy entities. However, the expression and function of HNF1A-AS1 in the carcinogenesis and development of osteosarcoma remains unknown. In this study, we detected the HNF1A-AS1 levels in human osteosarcoma tissues and cell lines by quantitative real-time polymerase chain reaction (qRT-PCR), and investigated its role in osteosarcoma by using in vitro assays. Our study showed that HNF1A-AS1 expression was significantly up-regulated in human osteosarcoma tissues and cell lines compared with their normal counterparts, and its expression level was positively correlated with the distance metastasis (P = 0.009) and tumour stage (P = 0.019). Moreover, Kaplan-Meier curves with the log-rank test showed that higher expression of HNF1A-AS1 conferred a significantly poorer survival and multivariate Cox proportional hazards analysis revealed that HNF1A-AS1 was an independent risk factor of overall survival. In addition, the expression of HNF1A-AS1 in serum is correlated with patients' status and receiver operating characteristic (ROC) curve analysis demonstrated that HNF1A-AS1 could distinguish patients with osteosarcoma from healthy individuals (the area under curve 0.849, P < 0.001). Furthermore, in vitro knockdown of HNF1A-AS1 by siRNA significantly inhibited cell proliferation and G1 /S transition, and suppressed migration and invasion by reducing the epithelial-mesenchymal transition (EMT) program in osteosarcoma cells. Taken together, our data suggested that HNF1A-AS1 is a novel molecule involved in osteosarcoma progression, which may provide as a potential diagnostic, prognostic biomarker and therapeutic target.
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Neoplasias Óseas/genética , Carcinogénesis/genética , Regulación Neoplásica de la Expresión Génica , Osteosarcoma/genética , ARN Largo no Codificante/genética , Adulto , Área Bajo la Curva , Neoplasias Óseas/sangre , Neoplasias Óseas/diagnóstico , Neoplasias Óseas/mortalidad , Carcinogénesis/metabolismo , Carcinogénesis/patología , Línea Celular Tumoral , Proliferación Celular , Progresión de la Enfermedad , Transición Epitelial-Mesenquimal , Femenino , Puntos de Control de la Fase G1 del Ciclo Celular/genética , Humanos , Masculino , Persona de Mediana Edad , Osteosarcoma/sangre , Osteosarcoma/diagnóstico , Osteosarcoma/mortalidad , Pronóstico , Modelos de Riesgos Proporcionales , ARN Largo no Codificante/antagonistas & inhibidores , ARN Largo no Codificante/sangre , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Curva ROC , Factores de RiesgoRESUMEN
Osteogenesis and adipogenesis of bone marrow mesenchymal stem cells (BMSCs) are regarded as being of great importance in the regulation of bone remodeling. In this study, rat BMSCs were exposed to different levels of cyclic mechanical stress generated by liquid drops and cultured in general medium or adipogenic medium. Markers of osteogenic (Runx2 and Collagen I) and adipogenic (C/EBPα, PPARγ, and lipid droplets) differentiation were detected using Western blot and histological staining. The protein levels of members of the phosphatidylinositol 3-kinase (PI3K)/Akt/glycogen synthase kinase 3ß (GSK-3ß)/ß-catenin signaling pathway were also examined. Results showed that small-magnitude stress significantly upregulated Runx2 and Collagen I and downregulated PPARγ and C/EBPα expression in BMSCs cultured in adipogenic medium, while large-magnitude stress reversed the effect when compared with unloading groups. The PI3K/Akt signaling pathway could be strongly activated by mechanical stimulation; however, large-magnitude stress led to decreased activation of the signaling pathway when compared with small-magnitude stress. Activation of ß-catenin with LiCl led to increased expression of Runx2 and Collagen I and reduction of C/EBPα and PPARγ expression in BMSCs. Inhibition of PI3K/Akt signaling partially blocked the expression of ß-catenin. Taken together, our results indicate that mechanical stress-regulated osteogenesis and adipogenesis of rat BMSCs are mediated, at least in part, by the PI3K/Akt/GSK-3ß/ß-catenin signaling pathway.
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Adipogénesis , Diferenciación Celular , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Mecanotransducción Celular , Células Madre Mesenquimatosas/metabolismo , Osteogénesis , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Estrés Mecánico , beta Catenina/metabolismo , Animales , Células Madre Mesenquimatosas/citología , Ratas , Ratas Sprague-DawleyRESUMEN
BACKGROUND/AIMS: This study investigated the effect of mechanical stress on tendon-bone healing in a rabbit anterior cruciate ligament (ACL) reconstruction model as well as cell proliferation and matrix formation in co-culture of bone-marrow mesenchymal stem cells (BMSCs) and tendon cells (TCs). METHODS: The effect of continuous passive motion (CPM) therapy on tendon-bone healing in a rabbit ACL reconstruction model was evaluated by histological analysis, biomechanical testing and gene expressions at the tendon-bone interface. Furthermore, the effect of mechanical stretch on cell proliferation and matrix synthesis in BMSC/TC co-culture was also examined. RESULTS: Postoperative CPM therapy significantly enhanced tendon-bone healing, as evidenced by increased amount of fibrocartilage, elevated ultimate load to failure levels, and up-regulated gene expressions of Collagen I, alkaline phosphatase, osteopontin, Tenascin C and tenomodulin at the tendon-bone junction. In addition, BMSC/TC co-culture treated with mechanical stretch showed a higher rate of cell proliferation and enhanced expressions of Collagen I, Collagen III, alkaline phosphatase, osteopontin, Tenascin C and tenomodulin than that of controls. CONCLUSION: These results demonstrated that proliferation and differentiation of local precursor cells could be enhanced by mechanical stimulation, which results in enhanced regenerative potential of BMSCs and TCs in tendon-bone healing.
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Lesiones del Ligamento Cruzado Anterior/cirugía , Reconstrucción del Ligamento Cruzado Anterior/rehabilitación , Ligamento Cruzado Anterior/cirugía , Células Madre Mesenquimatosas/citología , Tenocitos/citología , Fosfatasa Alcalina/genética , Fosfatasa Alcalina/metabolismo , Animales , Lesiones del Ligamento Cruzado Anterior/fisiopatología , Biomarcadores/metabolismo , Fenómenos Biomecánicos , Proliferación Celular , Técnicas de Cocultivo , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Colágeno Tipo III/genética , Colágeno Tipo III/metabolismo , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Fémur/cirugía , Expresión Génica , Masculino , Células Madre Mesenquimatosas/metabolismo , Osteopontina/genética , Osteopontina/metabolismo , Presión , Conejos , Tenascina/genética , Tenascina/metabolismo , Tendones/citología , Tendones/metabolismo , Tendones/cirugía , Tenocitos/metabolismo , Tibia/cirugíaRESUMEN
Recent studies have shown that some members of the tripartite motif-containing protein (TRIM) family serve as important regulators of tumorigenesis. However, the biological role of TRIM14 in osteosarcoma remains to be established. In this study, we showed that TRIM14 is upregulated in human osteosarcoma specimens and cell lines, and correlated with osteosarcoma progression and shorter patient survival times. Functional studies demonstrated that overexpression of TRIM14 enhances osteosarcoma cell proliferation, clone formation, cell cycle procession, migration and invasion in vitro and promotes tumor growth in vivo, and conversely, its silencing has the opposite effects. Furthermore, TRIM14 overexpression induced activation of the AKT pathway. Inhibition of AKT expression reversed the TRIM14-mediated promotory effects on cell growth and mobility, in addition to TRIM14-induced epithelial-to-mesenchymal transition (EMT) and cyclin D1 upregulation. Our findings collectively suggest that TRIM14 functions as an oncogene by upregulating the AKT signaling pathway in osteosarcoma cells, supporting its potential utility as a therapeutic target for this disease.
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Neoplasias Óseas/genética , Neoplasias Óseas/metabolismo , Proteínas Portadoras/genética , Osteosarcoma/genética , Osteosarcoma/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Adulto , Animales , Neoplasias Óseas/mortalidad , Neoplasias Óseas/patología , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Xenoinjertos , Humanos , Péptidos y Proteínas de Señalización Intracelular , Masculino , Ratones , Persona de Mediana Edad , Clasificación del Tumor , Metástasis de la Neoplasia , Estadificación de Neoplasias , Osteosarcoma/mortalidad , Osteosarcoma/patología , Pronóstico , Proteínas de Motivos Tripartitos , Carga Tumoral , Adulto JovenRESUMEN
INTRODUCTION: MicroRNAs (miRNAs) has emerged as important factors in osteogenesis and chondrogenesis. This study aimed to determine whether miR-221 is involved in the regulation of osteoporosis and its underlying mechanism. METHODS: Total RNA was extracted from fresh femoral neck trabecular bone from women undergoing hip replacement due to either osteoporotic fracture (OP group, n = 12) or osteoarthritis in the absence of osteoporosis (Control group, n = 12). Gene expression was quantified using TaqMan quantitative RT-PCR assays and protein production was determined by western blot analysis. The role of miR-221 in osteoblast differentiation was identified by gain or loss function experiment. MiRNA targets were identified using bioinformatics and luciferase reporter assay. RESULTS: MiR-221 was down-regulated in the osteoporotic samples compared with non-osteoporotic controls, and decreased in a C2C12 cell model of osteogenic differentiation. Overexpression of miR-221 resulted in a decrease in the osteogenic potential, as indicated by the reduced expression levels of key osteoblast markers, including osteocalcin (OC), alkaline phosphatase (ALP) and collagen, type I, α 1 (COL1A1), whereas inhibition of miR-221 promoted the activity of OC, ALP and COL1A1. Then bioinformatic analysis identified potential target sites of the miR-221 located in the 3' untranslated regions of RUNX2. Western blot analysis demonstrated that miR-221 inhibited RUNX2 gene expression. Furthermore, dual-luciferase reporter assays confirmed that RUNX2 was a direct target of miR-221. Rescue experiments showed that overexpression of RUNX2 significantly attenuated the effect of miR-221 on osteoblast markers providing strong evidence that miR-221 mediated the osteoblast differentiation by targeting RUNX2. CONCLUSIONS: Taken together, these data implied that miR-221 played an important part in osteoporosis through regulating RUNX2 expression and osteoblast differentiation.
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It is widely accepted that physiological mechanical stimulation suppresses apoptosis and induces synthesis of extracellular matrix by osteoblasts; however, the effect of stress overloading on osteoblasts has not been fully illustrated. In the present study, we investigated the effect of cyclic compressive stress on rat osteoblasts apoptosis, using a novel liquid drop method to generate mechanical stress on osteoblast monolayers. After treatment with different levels of mechanical stress, apoptosis of osteoblasts and activations of mitogen-activated protein kinases (MAPKs) and PI3-kinase (PI3K)/Akt signaling pathways were investigated. Osteoblasts apoptosis was observed after treated with specific inhibitors prior to mechanical stimulation. Protein levels of Bax/Bcl-2/caspase-3 signaling were determined using western blot with or without inhibitors of PI3K/Akt and phosphorylation of c-jun N-terminal kinase (JNK) MAPK. Results showed that mechanical stimulation led to osteoblasts apoptosis in a dose-dependent manner and a remarkable activation of MAPKs and PI3K/Akt signaling pathways. Activation of PI3K/Akt protected against apoptosis, whereas JNK MAPK increased apoptosis via regulation of Bax/Bcl-2/caspase-3 activation. In summary, the PI3K/Akt and JNK MAPK signaling pathways played opposing roles in osteoblasts apoptosis, resulting in inhibition of apoptosis upon small-magnitude stress and increased apoptosis upon large-magnitude stress.
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Caspasa 3/metabolismo , Sistema de Señalización de MAP Quinasas , Osteoblastos/citología , Fosfatidilinositol 3-Quinasa/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Estrés Mecánico , Animales , Apoptosis , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Supervivencia Celular , Osteoblastos/metabolismo , Fosforilación , RatasRESUMEN
BACKGROUND/AIMS: This study investigated the effect of silencing TOB1 (Transducer of ERBB2, 1) expression in bone marrow-derived mesenchymal stem cells (MSCs) on MSC-facilitated tendon-bone healing in a rat supraspinatus repair model. METHODS: Rat MSCs were transduced with a recombinant lentivirus encoding short hairpin RNA (shRNA) against TOB1. MSC cell proliferation was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. The effect of MSCs with TOB1 deficiency on tendon-bone healing in a rat rotator cuff repair model was evaluated by biomechanical testing, histological analysis and collagen type I and II gene expression. An upstream regulator (miR-218) of TOB1 was determined in MSCs. RESULTS: We found that knockdown of TOB1 significantly increased the proliferative activity of rat MSCs in vitro. When MSCs with TOB1 deficiency were injected into injured rat supraspinatus tendon-bone junctions, the effect on tendon-bone healing was enhanced compared to treatment with control MSCs with normal TOB1 expression, as evidenced by elevated levels of ultimate load to failure and stiffness, increased amount of fibrocartilage and augmented expression of collagen type I and type II genes. In addition, we found that the TOB1 3' untranslated region is a direct target of miR-218. Similar to the effect of TOB1 deficiency, overexpression of miR-218 effectively promoted tendon-bone healing in rat. CONCLUSION: These results suggest that TOB1 may play a negative role in the effect of MSCs on tendon-bone healing, and imply that expression of TOB1 may be regulated by miR-218.
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Trasplante de Células Madre Mesenquimatosas , Proteínas Represoras/genética , Manguito de los Rotadores/patología , Traumatismos de los Tendones/terapia , Tendones/patología , Animales , Células de la Médula Ósea/citología , Proliferación Celular , Células Cultivadas , Colágeno Tipo I/metabolismo , Colágeno Tipo II/metabolismo , Modelos Animales de Enfermedad , Lentivirus/genética , Masculino , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , MicroARNs/antagonistas & inhibidores , MicroARNs/genética , MicroARNs/metabolismo , Oligonucleótidos Antisentido/metabolismo , Interferencia de ARN , Ratas , Ratas Sprague-Dawley , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/metabolismo , Traumatismos de los Tendones/patología , Cicatrización de HeridasRESUMEN
To compare the clinical efficacy and radiological outcome of treating 4-level cervical spondylotic myelopathy (CSM) with either anterior cervical discectomy and fusion (ACDF) or "skip" corpectomy and fusion, 48 patients with 4-level CSM who had undergone ACDF or SCF at our hospital were analyzed retrospectively between January 2008 and June 2011. Twenty-seven patients received ACDF (Group A) and 21 patients received SCF. Japanese Orthopaedic Association (JOA) score, Neck Disability Index (NDI) score, and Cobb's angles of the fused segments and C2-7 segments were compared in the two groups. The minimum patient follow-up was 2 years. No significant differences between the groups were found in demographic and baseline disease characteristics, duration of surgery, or follow-up time. Our study demonstrates that there was no significant difference in the clinical efficacy of ACDF and SCF, but ACDF involves less intraoperative blood loss, better cervical spine alignment, and fewer postoperative complications than SCF.
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Complicaciones Posoperatorias/diagnóstico por imagen , Enfermedades de la Médula Espinal/diagnóstico por imagen , Fusión Vertebral/efectos adversos , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Radiografía , Estudios Retrospectivos , Enfermedades de la Médula Espinal/etiologíaRESUMEN
Using the single-sided reflection response of an unknown layered medium, one-dimensional single-sided acoustic focusing is a process that constructs a normally incident plane waveform such that the sound field collapses to a point inside this medium at a specified time. A recursive method for finding the incident waveform of the discrete single-sided focusing in the Goupillaud layered model is presented. The method is formulated using the standard Z-transform and provides an intuitive way to understand how single-sided focusing works in an unknown medium. Two numerical scattering experiments, with different layered structures, are simulated to verify the proposed method. In addition, the responses to virtual sources in the examples are reconstructed.
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OBJECTIVE: To explore the functional effects of MAPK pathway in the pathogenesis of human osteosarcoma. METHODS: Gene microarray (Human Genome U133A, Affymetrix) was used to screen the differential expression of genes involved in MAPK pathway between osteosarcoma cell lines and 3 osteoblastic cell lines. KEGG metabolic pathway analysis was performed among significantly increased or decreased genes using the MATLAB software. Immunohistochemical technique was used to detect the expressions of ERK1/2, JNK and p38 proteins among 48 osteosarcoma and benign 24 osteoblastic tumor samples. RESULTS: Using an entrance limit of > or = 2.0, 18 differentially expressed MAPK pathway-related genes were selected (10 up-regulated, 8 down-regulated) to mapped to the MAPK pathway of KEGG which are all important node genes. The positive rates of ERK1/2, JNK and p38 proteins were 83.3% (40/48), 72.9% (35/48) and 85.4% (41/48) in osteosarcomas,and 12.5% (3/24), 8.3% (2/24) and 16.7% (4/24) in the control group, respectively. The positive rates and expression intensities were statistically different between the 2 groups (P<0.01). CONCLUSION: MAPK pathway plays an important role in the pathogenesis of osteosarcoma. ERK, JNK and p38 form an intercoordinating network and regulate the cell proliferation, differentiation, apoptosis, invasion and migration in osteosarcoma.