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1.
Gene ; 726: 144145, 2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-31743769

RESUMO

Long non-coding RNA SNHG12 (lncSNHG12) plays important roles in the onset and progression of various cancers. However, the role of lncSNHG12 in osteosarcoma (OS) remains unclear. Therefore, the aim of the present study was to determine the function of lncSNHG12 in OS. A bioinformatics website was used to predict the downstream targets of lncSNHG12. In addition, qRT-PCR was employed to assess lncSNHG12 expression in OS cells. Cell migration and proliferation in vitro were verified using the transwell migration, clone formation, and CCK8 assays. Tumor metastasis and xenograft formation were monitored in nude mice with or without downregulation of lncSNHG12. The results show that lncSNHG12 was upregulated in OS cell lines. Downregulation lncSNHG12 suppressed the metastasis and proliferation both in vitro and in vivo. Also, lncSNHG12 downregulation suppressed the expression of insulin growth factor 1 receptor (IGF1R) expression through sponging miR-195-5p, which was verified with the luciferase reporter assay and rescue experiments. These findings suggest that downregulation of lncSNHG12 may suppress aggressive OS phenotypes. Moreover, lncSNHG12 silencing inhibited OS metastasis and growth by targeting the miR-195-5p/IGF1R axis, which represents a candidate marker and target for OS treatment and management.


Assuntos
Proliferação de Células/genética , Regulação para Baixo/genética , MicroRNAs/genética , Metástase Neoplásica/genética , Osteossarcoma/genética , RNA Longo não Codificante/genética , Receptor IGF Tipo 1/genética , Animais , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular/genética , Progressão da Doença , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Osteoblastos/patologia , Osteossarcoma/patologia , Prognóstico , Regulação para Cima/genética
2.
Toxicol Lett ; 321: 122-130, 2020 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-31874197

RESUMO

Our previous studies confirmed that prenatal caffeine exposure (PCE) could induce susceptibility to osteoarthritis in adult offspring rats due to poor chondrocyte differentiation, but its mechanism remains to be further investigated. This study aimed to explore whether subchondral bone dysplasia mediates susceptibility to osteoarthritis in adult offspring rats induced by PCE. Pregnant Wistar rats were treated with caffeine (120 mg/kg.d) or saline from gestational day (GD) 9 to 20. The female offspring were euthanized to collect femurs at GD20, postnatal week (PW) 6, and PW28 (non-ovariectomy and ovariectomy groups) to detect osteoarthritis-like phenotype, subchondral bone mass, ossification center development, and other evidence. The results showed that PCE increased the Mankin score of pathological articular cartilage, but decreased articular cartilage thickness and subchondral bone mass, which were more obvious after ovariectomy. Meanwhile, the correlation analysis results demonstrated that the Mankin score of articular cartilage was significantly negatively correlated with subchondral bone mass, and the thickness of articular cartilage was significantly positively correlated with subchondral bone mass. Further, the length and area of the primary and secondary ossification centers, the number of osteoblasts, and the related genes' expression of osteogenic differentiation (e.g., Runx2, BSP, ALP, and OCN) were all significantly decreased in the PCE group before and after birth. Taken together, PCE induced susceptibility to osteoarthritis in adult female offspring, which was likely related to the subchondral bone dysplasia and reduction of subchondral bone mass production due to developmental disorder of primary and secondary ossification centers caused by osteoblast differentiation disability before and after birth.


Assuntos
Doenças do Desenvolvimento Ósseo/induzido quimicamente , Cafeína/toxicidade , Cartilagem Articular/efeitos dos fármacos , Estimulantes do Sistema Nervoso Central/toxicidade , Osteoartrite/induzido quimicamente , Osteogênese/efeitos dos fármacos , Efeitos Tardios da Exposição Pré-Natal , Fatores Etários , Animais , Doenças do Desenvolvimento Ósseo/genética , Doenças do Desenvolvimento Ósseo/metabolismo , Doenças do Desenvolvimento Ósseo/patologia , Cartilagem Articular/patologia , Diferenciação Celular/efeitos dos fármacos , Feminino , Fêmur/efeitos dos fármacos , Fêmur/metabolismo , Fêmur/patologia , Idade Gestacional , Osteoartrite/genética , Osteoartrite/metabolismo , Osteoartrite/patologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteoblastos/patologia , Osteogênese/genética , Ovariectomia , Gravidez , Ratos Wistar , Fatores Sexuais , Tíbia/efeitos dos fármacos , Tíbia/metabolismo , Tíbia/patologia
3.
Clin Nucl Med ; 45(1): 32-37, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31693615

RESUMO

PURPOSE: The objective of this study was to evaluate the accuracy of using CT attenuation and SUVs to differentiate enostoses from untreated and treated osteoblastic metastases on the attenuation-correction CT component of F-FDG PET/CTs. METHODS: We retrospectively reviewed F-FDG PET/CT studies of 117 patients (169 lesions), of which 65 had imaging of enostoses, and 52 had imaging showing the transition of lesions from untreated to treated osteoblastic metastases. We measured the mean CT attenuations and the SUVmax and SUVmean of each lesion. Receiver operating characteristic curve analyses were used to evaluate the accuracy of each metric in distinguishing enostoses from untreated and treated osteoblastic metastases. RESULTS: For differentiating enostoses from untreated osteoblastic metastases, mean CT attenuation achieved an area under the receiver operating characteristic curve (AUC) of 90.8%, with an optimized threshold of 795 HU. SUVmax achieved an AUC of 94.9%, with an optimized threshold of 2.2. For differentiating enostoses from treated osteoblastic metastases, the AUCs for every metric decreased, with mean CT attenuation being the best at 82.7%. A joint predictive model combining both CT attenuation and SUV increased the AUC to 88.3%, and performance was significantly better than SUVmax or SUVmean alone (P = 0.029 and P = 0.049, respectively). CONCLUSIONS: CT attenuation and SUV can reliably distinguish between enostoses and metastases on F-FDG PET/CT. However, the accuracy of these metrics decreases when used to differentiate enostoses from treated metastases. A joint prediction model combining CT attenuation with SUV can improve accuracy.


Assuntos
Neoplasias Ósseas/diagnóstico por imagem , Neoplasias Ósseas/secundário , Fluordesoxiglucose F18/metabolismo , Processamento de Imagem Assistida por Computador , Tomografia Computadorizada com Tomografia por Emissão de Pósitrons , Idoso , Transporte Biológico , Neoplasias Ósseas/patologia , Neoplasias Ósseas/terapia , Diagnóstico Diferencial , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Osteoblastos/patologia , Curva ROC , Estudos Retrospectivos
4.
Life Sci ; 239: 116980, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31704449

RESUMO

AIMS: 15-lipoxygenase-1 (15-LOX-1) plays a vital role in aggravating the inflammatory response in various pathological processes, including osteoarthritis (OA). Abnormal osteoblast phenotypes including elevated runt-related transcription factor 2 (RUNX2), collagen type 1 alpha 1 (COL1), and osteocalcin (OCN) lead to osteosclerosis of the subchondral bone, which eventually causes OA. However, the pathogenesis of OA is poorly defined, and it is unclear if 15-LOX-1 induces osteoblast abnormal phenotypes in OA. Therefore, this study aimed to determine the roles of 15-LOX-1 on the abnormal phenotypes present in osteoblasts of the subchondral bone in OA. MAIN METHODS: The expression levels of 15-LOX-1 were measured by Immunohistochemistry, qRT-PCR and western blotting from the OA subchondral bone osteoblasts. To further investigate the roles of 15-LOX-1 in abnormal phenotypes of osteoblasts and its mechanisms in OA, 15-LOX-1 siRNA or overexpressing lv-15-lox-1 were transfected into osteoblasts, respectively. The effects of 15-LOX-1 on abnormal phenotypes of osteoblasts in OA were assessed by qRT-PCR, and western blotting. We also examined the role of 15-LOX-1-inhibited autophagy in OA osteoblasts by qRT-PCR, and western blotting, transmission electron microscopy. KEY FINDINGS: The expression levels of 15-LOX-1 along with osteoblast phenotype markers such as RUNX2, COL1, and OCN were significantly increased in OA subchondral bone. Furthermore, 15-LOX-1 inhibited autophagy significantly upregulated the expression levels of RUNX2, COL1 and OCN through activated mTORC1. Similarly, treatment with autophagy inhibitors alleviated osteoblast abnormal phenotypes of osteoblasts in OA. SIGNIFICANCE: In conclusion, our results suggested that the expression of 15-LOX-1 on osteoblasts from the subchondral bone increased in OA. 15-LOX-1 inhibited autophagy by activated mTORC1, which in turn upregulated the markers of abnormal osteoblast phenotypes RUNX2, COL1, and OCN.


Assuntos
Araquidonato 15-Lipoxigenase/metabolismo , Lipoxigenase/metabolismo , Osteoartrite/metabolismo , Osteoblastos/metabolismo , Araquidonato 15-Lipoxigenase/sangue , Osso e Ossos/metabolismo , Células Cultivadas , Colágeno Tipo I/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Humanos , Lipoxigenase/sangue , Osteoblastos/patologia , Osteocalcina/metabolismo , Fenótipo
5.
Int J Nanomedicine ; 14: 7309-7322, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31571855

RESUMO

Introduction: The only treatment for aseptic loosening is the replacement of the prosthesis through revision surgery. A preventive approach, achieved through anti-inflammatory drugs released from the device, has shown to be a viable strategy; however, the performance of these devices is not yet satisfactory thus further improvements are necessary. Methods: We used titanium nanoparticles as a model for implant surfaces and developed a coating containing dexamethasone (DEX) using layer-by-layer deposition. Results: The amount of deposited drug depended on the number of layers and the release was sustained for months. The efficiency of the released DEX in reducing inflammation markers (tumor necrosis factor alpha and IL-6) produced by human monocytes and macrophages was similar to the pure drug at the same concentration without negative impacts on the viability and morphology of these cells. Conclusion: These coatings were not inferior to medical grade titanium (the standard material used in uncemented devices) regarding their ability to sustain osteoblasts and fibroblasts growth.


Assuntos
Anti-Inflamatórios/farmacologia , Cimentos para Ossos/farmacologia , Materiais Revestidos Biocompatíveis/farmacologia , Liberação Controlada de Fármacos , Nanopartículas/química , Falha de Prótese , Linhagem Celular , Forma Celular/efeitos dos fármacos , Dexametasona/farmacologia , Fibroblastos/efeitos dos fármacos , Humanos , Interleucina-6/metabolismo , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Monócitos/efeitos dos fármacos , Nanopartículas/ultraestrutura , Osteoblastos/efeitos dos fármacos , Osteoblastos/patologia , Tamanho da Partícula , Termogravimetria
6.
J Bone Joint Surg Am ; 101(19): 1741-1749, 2019 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-31577679

RESUMO

BACKGROUND: Local, intrawound use of antibiotic powder, such as vancomycin and tobramycin, in spinal fusion surgery has become an increasingly common prophylactic measure in an attempt to reduce rates of postsurgical infection. However, the effects of localized antibiotic delivery on fusion remain unclear. The objective of this study was to examine the in vivo effects of intraoperative local delivery of 2 antibiotics commonly used in bone-grafting surgery on spinal fusion outcomes in a rat model. METHODS: Single-level (L4-L5), bilateral posterolateral intertransverse process lumbar fusion surgery was performed on 60 female Lewis rats (6 to 8 weeks of age) using syngeneic iliac crest allograft mixed with clinical bone-graft substitute and varying concentrations of antibiotics (n = 12 each): (1) control without any antibiotics, (2) low-dose vancomycin (14.3 mg/kg), (3) high-dose vancomycin (71.5 mg/kg), (4) low-dose tobramycin (28.6 mg/kg), and (5) high-dose tobramycin (143 mg/kg). Eight weeks postoperatively, fusion was evaluated via micro-computed tomography (µCT), manual palpation, and histological analysis, with blinding to treatment group. In the µCT analysis, fusion-mass volumes were measured for each rat. Each spine specimen (L4-L5) was rated (manual palpation score) on a scale of 2 to 0 (2 = fused, 1 = partially fused, and 0 = non-fused). RESULTS: The mean fusion-mass volume on µCT (mm) was as follows: control, 29.3 ± 6.2; low-dose vancomycin, 26.3 ± 8.9; high-dose vancomycin, 18.8 ± 7.9; low-dose tobramycin, 32.7 ± 9.0; and high-dose tobramycin, 43.8 ± 11.9 (control versus high-dose vancomycin, p < 0.05; and control versus high-dose tobramycin, p < 0.05). The mean manual palpation score for each group was as follows: control, 1.46 ± 0.58; low-dose vancomycin, 0.86 ± 0.87; high-dose vancomycin, 0.68 ± 0.62; low-dose tobramycin, 1.25 ± 0.71; and high-dose tobramycin, 1.32 ± 0.72 (control versus high-dose vancomycin, p < 0.05). The histological analyses demonstrated a similar trend with regard to spinal fusion volume. CONCLUSIONS: Intraoperative local application of vancomycin, particularly at a supraphysiological dosage, may have detrimental effects on fusion-mass formation. No inhibitory effect of tobramycin on fusion-mass formation was observed. CLINICAL RELEVANCE: When spine surgeons decide to use intraoperative intrawound antibiotics in spinal fusion surgery, they should weigh the reduction in surgical site infection against a possible inhibitory effect on fusion.


Assuntos
Antibacterianos/administração & dosagem , Vértebras Lombares/cirurgia , Fusão Vertebral/métodos , Tobramicina/administração & dosagem , Vancomicina/administração & dosagem , Animais , Relação Dose-Resposta a Droga , Humanos , Vértebras Lombares/diagnóstico por imagem , Osteoblastos/patologia , Palpação/métodos , Pós , Ratos Endogâmicos Lew , Infecção da Ferida Cirúrgica/patologia , Infecção da Ferida Cirúrgica/prevenção & controle , Microtomografia por Raio-X
7.
Anticancer Res ; 39(10): 5663-5668, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31570464

RESUMO

BACKGROUND/AIM: Osteosarcoma of the upper extremities is rare, and characteristics in this location have not been described before. We aimed to analyze the characteristics and survival rate of osteosarcoma of the upper extremities. MATERIALS AND METHODS: A retrospective cohort study was performed by querying the National Cancer Database. Statistical analysis was performed using a multivariate logistic regression model and Kaplan-Meier log-rank tests for survival. RESULTS: A total of 991 patients were diagnosed with osteosarcoma of the upper extremities. Most tumors were osteogenic and osteoblastic (66.8%), larger than 8 cm (47.9%), high grade (64.3%), lymph node-negative (7.9%), and without metastasis to lungs (39.0%). Osteosarcomas of the hand and wrist were less likely to be high-grade when compared to osteosarcomas of the forearm, arm, and shoulder. CONCLUSION: The results of this study help us to approach patients promptly and avoid total amputation, increasing functionality and prognosis of the disease.


Assuntos
Osteossarcoma/patologia , Extremidade Superior/patologia , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Osteoblastos/patologia , Prognóstico , Estudos Retrospectivos , Taxa de Sobrevida , Adulto Jovem
8.
Mater Sci Eng C Mater Biol Appl ; 105: 110096, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31546344

RESUMO

The objective of this study is to understand the effect of sustained release of vitamin C from ß-tricalcium phosphate (ß-TCP) scaffold on proliferation, viability and differentiation of human fetal osteoblast cells (hFOB). The influence of pH, drug concentration, and presence of polymer on the sustained release of vitamin C from polycaprolactone (PCL) coated ß-TCP scaffolds are studied. Prolonged and sustained release of vitamin C, over 60 days is observed in PCL coated ß-TCP scaffolds compared to uncoated scaffolds. Presence of PCL helps to minimize the burst release of vitamin C from ß-TCP scaffolds in the initial 24 h of release. To evaluate the osteogenic potential of vitamin C incorporated ß-TCP scaffolds, osteoblast cells are cultured and cell morphology, proliferation, viability, and differentiation are assessed. Morphological characterization shows layer like osteoblast cell attachment in the presence of vitamin C compared to the control. MTT cell viability assay shows 2 folds increase in osteoblast cell density in the presence of vitamin C after 3,7 and 11 days of culture. Furthermore, increased ALP activity at 11 days of culture indicates the possible role of vitamin C on osteoblast differentiation. Additionally, a preliminary study shows vitamin C loaded scaffolds suppress osteosarcoma (MG-63) cell proliferation to 4 folds after 3 days compared to control. These results show a sustained release of vitamin C from PCL coated ß-TCP scaffolds improve proliferation, viability, and differentiation of osteoblasts cell as well as mitigate osteosarcoma cell proliferation, suggesting its potential application as synthetic bone graft substitutes in tissue engineering application.


Assuntos
Ácido Ascórbico , Neoplasias Ósseas , Fosfatos de Cálcio , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Osteoblastos/metabolismo , Osteossarcoma , Ácido Ascórbico/química , Ácido Ascórbico/farmacocinética , Ácido Ascórbico/farmacologia , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Fosfatos de Cálcio/química , Fosfatos de Cálcio/farmacocinética , Fosfatos de Cálcio/farmacologia , Linhagem Celular Tumoral , Preparações de Ação Retardada/química , Preparações de Ação Retardada/farmacocinética , Preparações de Ação Retardada/farmacologia , Humanos , Osteoblastos/patologia , Osteossarcoma/tratamento farmacológico , Osteossarcoma/metabolismo , Osteossarcoma/patologia
9.
Int J Mol Sci ; 20(18)2019 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-31505764

RESUMO

Wnt signaling plays a major role in bone metabolism. Advances in our understanding of secreted regulators of Wnt have yielded several therapeutic targets to stimulate osteoanabolism-the most promising of which is the Wnt inhibitor sclerostin. Sclerostin antibody recently gained approval for clinical use to treat osteoporosis, but the biology surrounding sclerostin antagonism is still incompletely understood. Numerous factors regulate the efficacy of sclerostin inhibition on bone formation, a process known as self-regulation. In previous communications we reported that the basic helix-loop-helix transcription factor Twist1-a gene know to regulate skeletal development-is highly upregulated among the osteocyte cell population in mice treated with sclerostin antibody. In this communication, we tested the hypothesis that preventing Twist1 upregulation by deletion of Twist1 from late-stage osteoblasts and osteocytes would increase the efficacy of sclerostin antibody treatment, since Twist1 is known to restrain osteoblast activity in many models. Twist1-floxed loss-of-function mice were crossed to the Dmp1-Cre driver to delete Twist1 in Dmp1-expressing cells. Conditional Twist1 deletion was associated with a mild but significant increase in bone mass, as assessed by dual energy x-ray absorptiometry (DXA) and microCT (µCT) for many endpoints in both male and female mice. Biomechanical properties of the femur were not affected by conditional mutation of Twist1. Sclerostin antibody improved all bone properties significantly, regardless of Twist1 status, sex, or endpoint examined. No interactions were detected when Twist1 status and antibody treatment were examined together, suggesting that Twist1 upregulation in the osteocyte population is not an endogenous mechanism that restrains the osteoanabolic effect of sclerostin antibody treatment. In summary, Twist1 inhibition in the late-stage osteoblast/osteocyte increases bone mass but does not affect the anabolic response to sclerostin neutralization.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Anticorpos Neutralizantes/farmacologia , Densidade Óssea , Proteínas da Matriz Extracelular/biossíntese , Fêmur/metabolismo , Osteogênese , Proteína 1 Relacionada a Twist/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas da Matriz Extracelular/genética , Feminino , Fêmur/patologia , Deleção de Genes , Masculino , Camundongos , Camundongos Transgênicos , Osteoblastos/metabolismo , Osteoblastos/patologia , Osteócitos/metabolismo , Osteócitos/patologia , Proteína 1 Relacionada a Twist/metabolismo , Microtomografia por Raio-X
10.
Pharm Biol ; 57(1): 586-594, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31492082

RESUMO

Context: Evidence suggests that microRNA (miRNA) regulate gene expression and bone tissue homoeostasis of osteoporosis. MiR-152 has found to be abnormally expressed in osteoporosis, but its role in osteoblast differentiation has not been elucidated. Objective: To understand the potential mechanism of miR-152 in osteoblast differentiation via regulation of RICTOR. Materials and methods: The expression of miR-152 and RICTOR were tested in ovariectomized rat models of osteoporosis. Primary osteoblasts and MC3T -E1 cells were assigned into four groups, namely Control, miR-152 inhibitor, miR-control and miR-152 inhibitor + siRICTOR groups. qRT PCR and Western blot were performed to detect the expressions of miR-152 and RICTOR, respectively. MTT assay was used to evaluate cell viability, and ALP activity determination and mineralization analyses were also conducted. Results: In ovariectomy-induced osteoporotic rats, miR-152 (3.06 ± 0.35) in femoral tissues increased significantly, while RICTOR (0.31 ± 0.04) decreased. Compared with Control group, miR-152 inhibitor group presented appreciable reduction of miR-152 in primary osteoblasts and MC3T3-E1 cells, as well as remarkable increases in RICTOR, p-Akt(s473)/Akt ratio, and osteogenesis-related genes, with enhanced cell viability, ALP activity and mineralization. In comparison with cells in the miR-152 inhibitor group, those in the miR-152 inhibitor + siRICTOR group had no observable difference in miR-152, but were dramatically up-regulated in RICTOR, as well as the corresponding opposite tendencies of other factors. Conclusion: Inhibiting miR-152 promoted osteoblasts differentiation and alleviated osteoporosis by up-regulating RICTOR. Therefore, miR-152 may be an essential mediator of osteoblast differentiation and a new therapeutic strategy for osteoporosis.


Assuntos
Diferenciação Celular/genética , Regulação da Expressão Gênica , MicroRNAs/genética , Osteoblastos/metabolismo , Osteoporose/metabolismo , Proteína Companheira de mTOR Insensível à Rapamicina/metabolismo , Animais , Densidade Óssea , Modelos Animais de Doenças , Feminino , Fêmur/metabolismo , Fêmur/patologia , Osteoblastos/patologia , Osteoporose/genética , Osteoporose/patologia , Ovariectomia , Proteína Companheira de mTOR Insensível à Rapamicina/genética , Ratos , Ratos Sprague-Dawley
11.
Mol Immunol ; 114: 251-259, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31398664

RESUMO

BACKGROUND: Fracture healing is a complex process, and patients with fracture will undergo non-union or compromised regeneration. MicroRNA (miR)-342-5p is a Notch downstream molecule, and its roles in fracture healing remain unclear. We aimed to explore the functional roles of miR-342-5p in osteoblasts as well as the underlying mechanisms. METHODS: The expression of miR-342-5p in differentiation of MC3T3-E1 cells or hMSCs was examined by quantitative reverse transcription PCR (qRT-PCR). The effects of aberrantly expressed miR-342-5p on cell proliferation, apoptosis, migration, and expressions of proteins associated with proliferation and osteogenic differentiation were determined by Cell Counting Kit-8, trypan blue staining, flow cytometry, Transwell assay, Western blot and qRT-PCR assays, respectively. The downstream factor and the target genes of miR-342-5p as well as the involvements of the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway were finally assessed. RESULTS: miR-342-5p level was decreased during differentiation of MC3T3-E1 cells or hMSCs. After cell transfection, miR-342-5p overexpression significantly reduced cell viability, induced apoptosis, inhibited proliferation, migration and differentiation, and down-regulated Bmp7 expression. Subsequent experiments showed the effects of miR-342-5p inhibition on MC3T3-E1 cells were abrogated by Bmp7 knockdown. Additionally, COL4A6 and Bmp2 were predicated as target genes of miR-342-5p. Finally, phosphorylated levels of MEK and ERK were increased by miR-342-5p inhibition via up-regulating Bmp7 expression. CONCLUSION: miR-342-5p inhibition promoted proliferation, migration and differentiation of osteoblasts via regulating Bmp7, along with activation of the MEK/ERK pathway.


Assuntos
Proteína Morfogenética Óssea 7/genética , Diferenciação Celular/genética , Movimento Celular/genética , Proliferação de Células/genética , MicroRNAs/genética , Osteoblastos/patologia , Células 3T3 , Adulto , Animais , Apoptose/genética , Linhagem Celular , Sobrevivência Celular/genética , Regulação para Baixo/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Camundongos , Pessoa de Meia-Idade , Proteínas Quinases Ativadas por Mitógeno/genética , Osteogênese/genética , Transdução de Sinais/genética , Regulação para Cima/genética , Adulto Jovem
12.
Int J Mol Sci ; 20(16)2019 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-31405099

RESUMO

Bone metastasis is a common clinical complication in several cancer types, and it causes a severe reduction in quality of life as well as lowering survival time. Bone metastases proceed through a vicious self-reinforcing cycle that can be osteolytic or osteoblastic in nature. The vicious cycle is characterized by cancer cells residing in bone releasing signal molecules that promote the differentiation of osteoclasts and osteoblasts either directly or indirectly. The increased activity of osteoclasts and osteoblasts then increases bone turnover, which releases growth factors that benefit metastatic cancer cells. In order to improve the prognosis of patients with bone metastases this cycle must be broken. Radium-223 dichloride (radium-223), the first targeted alpha therapy (TAT) approved, is an osteomimetic radionuclide that is incorporated into bone metastases where its high-linear energy transfer alpha radiation disrupts both the activity of bone cells and cancer cells. Therefore, radium-223 treatment has been shown preclinically to directly affect cancer cells in both osteolytic breast cancer and osteoblastic prostate cancer bone metastases as well as to inhibit the differentiation of osteoblasts and osteoclasts. Clinical studies have demonstrated an increase in survival in patients with metastatic castration-resistant prostate cancer. Due to the effectiveness and low toxicity of radium-223, several novel combination treatment strategies are currently eliciting considerable research interest.


Assuntos
Antineoplásicos/uso terapêutico , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/secundário , Rádio (Elemento)/uso terapêutico , Animais , Neoplasias Ósseas/diagnóstico , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/patologia , Humanos , Terapia de Alvo Molecular , Osteoblastos/efeitos dos fármacos , Osteoblastos/patologia , Osteoclastos/efeitos dos fármacos , Osteoclastos/patologia , Prognóstico , Radioisótopos/uso terapêutico
13.
Pathol Res Pract ; 215(10): 152568, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31383536

RESUMO

The present study aimed to explore the potential anti-tumor effect of ERß overexpression and investigate its related mechanism in osteosarcoma. Cell cycle and apoptosis rates were measured by flow cytometry. Cell proliferation and formation of autophagosome were assessed by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and dansylcadaverine (MDC) staining assay. Cell migration and invasion were detected by wound healing assay and transwell assay. Western blot analysis was designed to detect the protein expressions of surviving, Bax, LC-3 П, Beclin-1, ERß, TßRⅠ, TßRⅡ, Smad2, Smad3 and Smad7. Real-Time fluorogenic PCR was designed to examine the mRNA expressions of surviving, Bax, ERß, TßRⅠ, TßRII, Smad2, Smad3 and Smad7. The results showed that ERß overexpression inhibited cell proliferation, migration and invasion, blocked cell cycle, and induced apoptosis and autophagy. Additionally, ERß overexpression significantly inhibited the expression of surviving, TßRⅠ, TßRⅡ, Smad2 and Smad3. Meanwhile, the expressions of Bax, LC-3 П, Beclin-1 and Smad7 were dramatically upregulated by ERß overexpression. In conclusion, ERß overexpression could inhibit cell proliferation, migration and invasion, block cell cycle, and promote apoptosis and autophagy in OS by downregulating TNG-ß signaling pathway.


Assuntos
Neoplasias Ósseas/metabolismo , Proliferação de Células/genética , Receptor beta de Estrogênio/metabolismo , Osteossarcoma/metabolismo , Transdução de Sinais/genética , Fator de Crescimento Transformador beta/metabolismo , Apoptose/genética , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Sobrevivência Celular/genética , Receptor beta de Estrogênio/genética , Humanos , Osteoblastos/metabolismo , Osteoblastos/patologia , Osteossarcoma/genética , Osteossarcoma/patologia , Fator de Crescimento Transformador beta/genética
14.
Oncogene ; 38(44): 6959-6969, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31409900

RESUMO

Bone metastatic prostate cancer provokes extensive osteogenesis by driving the recruitment and osteoblastic differentiation of mesenchymal stromal cells (MSCs). The resulting lesions greatly contribute to patient morbidity and mortality, underscoring the need for defining how prostate metastases subvert the MSC-osteoblast differentiation program. To gain insights into this process we profiled the effects of co-culture of primary MSCs with validated bone metastatic prostate cancer cell line models. These analyses revealed a cast of shared differentially induced genes in MSC, including betaglycan, a co-receptor for TGFß. Betaglycan has not been studied in the context of bone metastatic disease previously. Here we report that loss of betaglycan in MSC is sufficient to augment TGFß signaling, proliferation and migration, and completely blocks the MSC-osteoblast differentiation program. Further, betaglycan was revealed as necessary for prostate cancer-induced osteogenesis in vivo. Mechanistically, gene expression analysis revealed betaglycan controls the expression of a large repertoire of genes in MSCs, and that betaglycan loss provokes >60-fold increase in the expression of Wnt5a that plays important roles in stemness. In accord with the increased Wnt5a levels, there was a marked induction of canonical Wnt signaling in betaglycan ablated MSCs, and the addition of recombinant Wnt5a to MSCs was sufficient to impair osteogenic differentiation. Finally, the addition of Wnt5a neutralizing antibody was sufficient to induce the expression of osteogenic genes in betaglycan-ablated MSCs. Collectively, these findings suggest a betaglycan-Wnt5a circuit represents an attractive vulnerability to ameliorate prostate cancer-induced osteogenesis.


Assuntos
Células-Tronco Mesenquimais/patologia , Osteoblastos/patologia , Osteogênese , Neoplasias da Próstata/patologia , Proteoglicanas/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Humanos , Masculino , Proteoglicanas/genética , Receptores de Fatores de Crescimento Transformadores beta/genética , Proteína Wnt-5a/metabolismo
15.
Biomed Res Int ; 2019: 8043510, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31428646

RESUMO

The aim was to analyze histologically the bone repair in a mandibular osteotomy model with different gaps between the segments. Nine male rabbits who underwent osteotomies on the mandibular body were fixed with a 1.5 system plate and no bone graft; group 1 (2 mm gap between segments), group 2 (5 mm gap between segments), and group 3 (8 mm gap between segments) were included. After 8 weeks they were euthanized and the sample was processed for histological analysis. Group 1 showed advanced bone repair with cartilaginous tissue and cancellous bone, showing osteoblasts and type III collagenous fibers. In group 2, a more delayed ossification was observed, with an extensive area of peripheral ossifying cartilage and chondrocytes in greater number at the center of the defect; group 3 showed no evidence of ossification with fibrous tissue, a very low level of chondrocytes, and some bone sequestrate. We can conclude that, in this animal model, 2 or 5 mm gap in the osteotomy could be repaired as bone when fixation is used. The size of the gap is an important factor for the use of bone grafts considering endochondral ossification. This model can be used for graft analysis and related technologies.


Assuntos
Placas Ósseas , Transplante Ósseo , Mandíbula , Osteotomia Mandibular , Osteoblastos , Osteogênese , Aloenxertos , Animais , Osso Esponjoso/metabolismo , Osso Esponjoso/patologia , Osso Esponjoso/cirurgia , Humanos , Masculino , Mandíbula/metabolismo , Mandíbula/patologia , Mandíbula/cirurgia , Osteoblastos/metabolismo , Osteoblastos/patologia , Coelhos
16.
Mol Med Rep ; 20(4): 3019-3026, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31432111

RESUMO

Estradiol (E2) serves an important role in the changes of postmenopausal bone turnover rate and the development of osteoporosis. The present study aimed to investigate the effects of E2 on high glucose (HG)­induced osteoblast injury. Cell Counting Kit­8 was used to determine cell viability. Reverse transcription­quantitative PCR (RT­qPCR) and western blotting was used to analyze the mRNA and protein expression levels of osteocalcin, Runt­related transcription factor 2 (Runx2), nuclear factor E2­related factor 2 (Nrf2) and heme oxygenase­1 (HO1). Flow cytometry was performed to analyze apoptosis. The results revealed that cell viability was lower in cells treated with HG (100, 200 or 300 mg/dl) compared with the control group. Cell viability was decreased in cells treated with 200 mg/dl HG on days 3, 5 and 7. In addition, cell viability was increased by 0.1 µM E2. E2 with HG co­treatment increased cell viability, osteocalcin and Runx2 mRNA expression levels and nuclear Nrf2 and HO1 protein expression levels compared with the HG­only group. All these changes, with the exception of Runx2, were reversed by silencing Nrf2 expression using small interfering (si)RNA (siNrf2). Additionally, apoptosis was reduced by E2 in HG­treated cells, which was reversed by siNrf2 transfection. These results demonstrated that E2 may prevent HG­induced osteoblast injury by activating Nrf2/HO1 signaling pathways.


Assuntos
Apoptose/efeitos dos fármacos , Estradiol/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Glucose/farmacologia , Osteoblastos/metabolismo , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Subunidade alfa 1 de Fator de Ligação ao Core/biossíntese , Heme Oxigenase-1/biossíntese , Proteínas de Membrana/biossíntese , Camundongos , Fator 2 Relacionado a NF-E2/biossíntese , Osteoblastos/patologia , Osteoporose/metabolismo , Osteoporose/patologia
17.
Mol Med Rep ; 20(4): 3265-3275, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31432117

RESUMO

Patients with diabetes tend to have an increased incidence of osteoporosis, which may be associated with hyperglycemia; however, the pathogenic mechanisms governing this interaction remain unknown. The present study sought to investigate whether elevated extracellular glucose levels of bone mesenchymal stem cells (BMSCs) could influence osteoblastic differentiation and whether the intracellular Sonic hedgehog (Shh) pathway could adjust the effects. Furthermore, to verify the results in vivo, a rat tooth extraction model was constructed. BMSCs were incubated in eight types of culture medium, including low glucose (LG), LG + lentivirus (Lenti), LG + Lenti­small interfering RNA (Lenti­siRNA), LG + Lenti­Shh, high glucose (HG), HG + Lenti, HG + Lenti­siRNA and HG + Lenti­Shh. The lentiviral transfection efficiency was observed using a fluorescence microscope; protein and mRNA expression was detected by western blotting and reverse transcription­quantitative polymerase chain reaction (RT­qPCR). The matrix mineralization and alkaline phosphatase (ALP) activity of BMSCs were examined by Alizarin red staining and ALP activity assays, respectively. The expression of osteogenesis­related genes in BMSCs were quantified by RT­qPCR. The alveolar ridge reduction was measured and histological sections were used to evaluate new bone formation in the tooth socket. With high concentrations of glucose, Shh expression, matrix mineralization nodules formation, ALP activity and the levels of bone morphogenic protein 4 (BMP4), bone sialoprotein (BSP) and osteopontin (OPN) expression were greatly reduced compared with LG and corresponding control groups. Whereas activated Shh signaling via Lenti­Shh could increase the number of matrix mineralization nodules, ALP activity, and the expression levels of BMP4, BSP and OPN in BMSCs. Additionally, in vivo assays demonstrated that Lenti­Shh induced additional bone formation. Collectively, the results of the present study indicated that HG inhibited the Shh pathway in osteoblasts and resulted in patterning defects during osteoblastic differentiation and bone formation, while the activation of Shh signaling could suppress these deleterious effects.


Assuntos
Glucose/farmacologia , Proteínas Hedgehog/biossíntese , Lentivirus , Osteoblastos/metabolismo , Osteogênese/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Animais , Proteínas Hedgehog/genética , Masculino , Osteoblastos/patologia , Osteogênese/genética , Ratos , Ratos Sprague-Dawley , Transdução Genética
18.
Biofactors ; 45(5): 803-817, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31317567

RESUMO

Telomerase is a specialized reverse transcriptase/terminal transferase enzyme that adds telomeric repeat sequences at the extreme end of a newly replicated chromosome. Apart from telomere lengthening, telomerase has many extracurricular activities. Telomerase is known to regulate the expression of many genes and helps in cancer progression and epithelial-to-mesenchymal transitions (EMTs). We have previously reported that human telomerase reverse transcriptase (hTERT) regulates the expression of plasminogen activator such as urokinase-type plasminogen activator (uPA) in cancer cells following a genome-wide transcriptomic study. Here, we present data substantiating these results in terms of real-time assays, western blots, and immunofluorescence. Another aim of this study is to find out the possible mechanism by which hTERT regulates the expression of plasminogen activators. We have used some molecular biology techniques such as quantitative real-time polymerase chain reaction, western blotting, and immunofluorescence and some assays such as wound healing assay and colony formation assay to solve this question. In this study, we show a positive association between hTERT and uPA. We also demonstrate that hTERT enhances uPA expression concomitant with EMT. Knocking down of hTERT reduces uPA expression as well as reverses EMT in cancer cells. We have also found that uPA is a transforming growth factor beta (TGF-ß)-induced protein. Our observations establish that TGF-ß-induced uPA expression is hTERT dependent.


Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Membrana/genética , Osteoblastos/efeitos dos fármacos , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , Telomerase/genética , Fator de Crescimento Transformador beta/farmacologia , Células A549 , Antígenos CD/genética , Antígenos CD/metabolismo , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Células HeLa , Humanos , Proteínas de Membrana/metabolismo , Osteoblastos/metabolismo , Osteoblastos/patologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Transdução de Sinais , Telomerase/antagonistas & inibidores , Telomerase/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Vimentina/genética , Vimentina/metabolismo , beta Catenina/genética , beta Catenina/metabolismo
19.
Oxid Med Cell Longev ; 2019: 4101738, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31281574

RESUMO

Oxidative stress (OS) induces osteoblast apoptosis, which plays a crucial role in the initiation and progression of osteoporosis. Although OS is closely associated with mitochondrial dysfunction, detailed mitochondrial mechanisms underlying OS-induced osteoblast apoptosis have not been thoroughly elucidated to date. In the present study, we found that mitochondrial abnormalities largely contributed to OS-induced osteoblast apoptosis, as evidenced by enhanced production of mitochondrial reactive oxygen species; considerable reduction in mitochondrial respiratory chain complex activity, mitochondrial membrane potential, and adenosine triphosphate production; abnormality in mitochondrial morphology; and alteration of mitochondrial dynamics. These mitochondrial abnormalities were primarily mediated by an imbalance in mitochondrial fusion and fission through a protein kinase B- (AKT-) glycogen synthase kinase 3ß- (GSK3ß-) optic atrophy 1- (OPA1-) dependent mechanism. Hydroxytyrosol (3,4-dihydroxyphenylethanol (HT)), an important compound in virgin olive oil, significantly prevented OS-induced osteoblast apoptosis. Specifically, HT inhibited OS-induced mitochondrial dysfunction by decreasing OPA1 cleavage and by increasing AKT and GSK3ß phosphorylation. Together, our results indicate that the AKT-GSK3ß signaling pathway regulates mitochondrial dysfunction-associated OPA1 cleavage, which may contribute to OS-induced osteoblast apoptosis. Moreover, our results suggest that HT could be an effective nutrient for preventing osteoporosis development.


Assuntos
GTP Fosfo-Hidrolases/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Mitocôndrias/metabolismo , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteoporose/metabolismo , Álcool Feniletílico/análogos & derivados , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Osteoblastos/patologia , Osteoporose/patologia , Estresse Oxidativo/fisiologia , Álcool Feniletílico/farmacologia , Transdução de Sinais , Transfecção
20.
Cell Biochem Funct ; 37(6): 432-442, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31318458

RESUMO

Advanced glycation end products (AGEs) are naturally occurring molecules that start to accumulate from embryonic developmental stages and form as part of normal ageing. When reducing sugars interact with and modify proteins or lipids, AGE production occurs. AGE formation accelerates in chronic hyperglycemic conditions, and high AGE levels have been associated with the pathogenesis of various diseases. In addition, enhanced levels of AGEs have been linked to delayed wound healing as seen in patients with diabetes mellitus. Research has provided numerous ways in which a high AGE concentration results in impaired wound healing, including oxidative stress, structural and functional changes to proteins important in wound repair, an enhanced inflammatory response by activation of transcription factors, and possible exaggerated apoptosis of cells necessary to the wound repair process. Apoptosis is a naturally occurring cell death process that is significant for normal tissue functioning and plays an important role in wound repair by preventing a prolonged inflammatory response and excessive scar formation. Abnormal apoptosis affects wound healing, resulting in slow healing wounds. This review will summarize the role of AGEs in wound healing, focusing on the mechanisms by which AGEs lead to apoptosis in various cell types. The review provides the way forward for medical research and molecular studies as it focuses on the mechanisms by which AGEs induce apoptosis in various cell types, including fibroblasts, osteoblasts, neuronal cells, and endothelial cells. Reviewing the mechanisms of AGE-linked apoptosis is important in understanding the impact of high AGE levels in delayed wound healing in diabetic patients due to abnormal apoptosis of cells necessary to the wound healing process.


Assuntos
Apoptose , Produtos Finais de Glicação Avançada/metabolismo , Cicatrização , Animais , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Neurônios/metabolismo , Neurônios/patologia , Osteoblastos/metabolismo , Osteoblastos/patologia
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