RESUMO
The occurrence and progress of osteoporosis (OP) are partially caused by impaired osteoblast differentiation. Interleukin-I receptor antagonist (IL1RN) is an immune modulatory molecule that commonly functions by means of competing the binding site of IL-1R with IL-1. Although it was recently reported that IL1RN is involved in osteoblast differentiation, the role of IL1RN in osteogenesis remains unclear. In this work, we first investigated the expression pattern of IL1RN in ovariectomy mice and in vitro osteogenic induction of MC3T3-E1 and C3H10T1/2 cells. To verify the exact role of IL1RN in osteoblast differentiation, we established IL1RN-downregulated/upregulated cell lines. The results indicated that IL1RN was constantly expressed in MC3T3-E1 and C3H10T1/2 cells. Interestingly, an increase of IL1RN expression in osteoblasts occurred when osteoblasts were cultured in osteogenic medium (OM). As expected, silencing of IL1RN attenuated the osteogenic effect of OM, while IL1RN overexpression increased the osteogenic staining and promoted the expression of osteogenic markers, including alkaline phosphatase, osterix, and osteocalcin. In addition to evaluating the function of IL1RN in osteoblasts, we also investigated the molecular mechanism of the role of IL1RN in osteoblasts. We found that IL1RN interacts with integrin ß3 to activate ß-catenin signaling, which finally regulates osteoblast differentiation. Taken together, this study provides the framework that IL1RN, as a novel regulator of osteogenesis, may be a potential therapeutic target for the treatment of OP.
Assuntos
Diferenciação Celular , Integrina beta3/metabolismo , Proteína Antagonista do Receptor de Interleucina 1/metabolismo , Osteoblastos/metabolismo , Osteoporose/metabolismo , Animais , Linhagem Celular , Integrina beta3/genética , Proteína Antagonista do Receptor de Interleucina 1/genética , Camundongos , Osteoblastos/patologia , Osteoporose/genética , Osteoporose/patologiaRESUMO
Understanding neurite outgrowth, orientation, and migration is important for the design of biomaterials that interface with the neural tissue. However, the molecular signaling alternations have not been well elucidated to explain the impact of hydrogels on cell morphology. In our previous studies, a silk fibroin peptide (SF16) hydrogel was found to be an effective matrix for the viability, morphology, and proliferation of PC12 rat pheocrhomocytoma cells. We found that PC12 cells in the peptide hydrogel exhibited adhesive morphology compared to those cultured in agarose or collagen. Moreover, we identified that cell adhesion molecules (E- and N-cadherin) controlled by mTOR signaling were highly induced in PC12 cells cultured in the SF16 peptide hydrogel. Our findings suggest that the SF16 peptide might be suitable to be a cell-adhesion material in cell culture or tissue engineering, and mTOR/cadherin signaling is required for the cell adhesion in the SF16-peptide hydrogel.
Assuntos
Caderinas/metabolismo , Adesão Celular , Proliferação de Células , Fibroínas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/enzimologia , Peptídeos/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Alicerces Teciduais , Animais , Adesão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Forma Celular , Hidrogéis , Neurônios/efeitos dos fármacos , Células PC12 , Inibidores de Proteínas Quinases/farmacologia , Ratos , Transdução de Sinais , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidoresRESUMO
BACKGROUND/AIMS: Transplantation of bone-marrow-derived mesenchymal stem cells (MSCs) promotes neural cell regeneration after spinal cord injury (SCI). Recently, we showed that suppression of microRNA-383 (miR-383) in MSCs increased the protein levels of glial cell line derived neurotrophic factor (GDNF), resulting in improved therapeutic effects on SCI. However, the overall effects of miR-383 suppression in MSCs on SCI therapy were not determined yet. Here, we addressed this question. METHODS: We used bioinformatics tools to predict all miR-383-targeting genes, confirmed the functional bindings in a dual luciferase reporter assay. The effects of alteration of candidate genes in MSCs on cell proliferation were analyzed by MTT assay and by Western blotting for PCNA. The effects on angiogenesis were assessed by HUVEC assay. The effects on SCI in vivo were analyzed by transplantation of the modified MSCs into nude rats that underwent SCI. RESULTS: Suppression of miR-383 in MSCs not only upregulated GDNF protein, but also increased vascular endothelial growth factor A (VEGF-A) and cyclin-dependent kinase 19 (CDK19), two other miR-383 targets. MiR-383-suppression-induced increases in CDK19 resulted in a slight but significant increase in MSC proliferation, while miR-383-suppression-induced increases in VEGF-A resulted in a slight but significant increase in MSC-mediated angiogenesis. CONCLUSIONS: Upregulation of CDK19 and VEGF-A by miR-383 suppression in MSCs further improve the therapeutic potential of MSCs in treating SCI in rats.
Assuntos
Regulação da Expressão Gênica , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/genética , Traumatismos da Medula Espinal/terapia , Adulto , Animais , Proliferação de Células , Células Cultivadas , Regulação para Baixo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/citologia , Neovascularização Fisiológica , Ratos , Ratos Nus , Traumatismos da Medula Espinal/genética , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
BACKGROUND/AIMS: Transplantation of bone-marrow-derived mesenchymal stem cells (MSCs) has been used to treat spinal cord injury (SCI) to enhance tissue repair and neural cell regeneration. Glial cell line derived neurotrophic factor (GDNF) is an identified neural growth and survival factor. Here, we examined whether modification of GDNF levels in MSCs may further increase the potential of MSCs in promoting neural cell regeneration and subsequently the therapeutic outcome. METHODS: We examined the mRNA and protein levels of GDNF in human MSCs by RT-qPCR and Western blot, respectively. Bioinformatics analyses were done to predict microRNAs (miRNAs) that target GDNF in MSCs. The functional binding of miRNAs to GDNF mRNA was examined by a dual luciferase reporter assay. MSCs were transduced with adeno-associated virus (AAV) carrying null or antisense for miR-383 (as-miR-383), which were transplanted into nude rats that underwent SCI. The intact tissue, cavity volume, and recovery of locomotor activity were assessed. RESULTS: MSCs expressed very low GDNF protein, but surprisingly high levels of GDNF mRNA. Bioinformatics analyses showed that miR-383 inhibited protein translation of GDNF, through binding to the 3'-UTR of the GDNF mRNA. MSCs transduced with AAV-as-miR-383 further increased the intact tissue percentage, decreased cavity volume, and enhanced the recovery of locomotor activity in nude rats that underwent SCI, compared to MSCs. CONCLUSIONS: Suppression of miR-383 may increase the therapeutic potential of human bone-marrow-derived MSCs in treating SCI via augmentation of GDNF protein levels.
Assuntos
Células da Medula Óssea/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/biossíntese , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/biossíntese , Traumatismos da Medula Espinal , Regiões 3' não Traduzidas , Adulto , Animais , Modelos Animais de Doenças , Células HEK293 , Xenoenxertos , Humanos , Masculino , Ratos , Ratos Nus , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/terapiaRESUMO
OBJECTIVE: The critical role of circular RNAs (circRNAs) in osteoporosis (OP) has been highlighted. We tried to explore the role of circPVT1 in OP in relation to microRNA-30d-5p (miR-30d-5p) and ITGB3. METHODS: After bone marrow collection, bone marrow mesenchymal stem cells (BMSCs) were isolated and identified. Then, Pearson coefficient was used to analyze the correlation among circPVT1, miR-30d-5p and ITGB3, and the binding sites were predicted and verified. Gain- and loss-of function assays in circPVT1, miR-30d-5p and ITGB3 were performed to analyze their effect on osteogenic differentiation of BMSCs. RESULTS: The osteogenic differentiation of BMSCs from OP patients was significantly decreased, and reduced circPVT1 expression was found in the BMSCs from OP patients. Overexpression of circPVT1 stimulated the formation of calcified nodules, increased alkaline phosphatase activity, and enhanced the expression of osteogenic marker genes in the BMSCs from OP patients. Additionally, circPVT1 expression was negatively correlated with miR-30d-5p, and miR-30d-5p was negatively correlated with ITGB3 in OP patients. Mechanically, circPVT1 regulated the osteogenic differentiation potential of BMSCs by relieving the inhibition of miR-30d-5p on ITGB3 through the competitive endogenous RNA mechanism. CONCLUSION: Our study highlighted a circPVT1/miR-30d-5p/ITGB3 axis in regulating osteogenic differentiation potential of BMSCs from OP patients.
Assuntos
Células-Tronco Mesenquimais , MicroRNAs , Osteoporose , Células da Medula Óssea/metabolismo , Diferenciação Celular/genética , Células Cultivadas , Humanos , Integrina beta3/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Osteogênese/genética , Osteoporose/genéticaRESUMO
Emerging evidence indicates that extracellular vesicle (EV)-encapsulated circRNAs have the potential diagnostic and prognostic values for malignancies. However, the role of circNRIP1 in osteosarcoma remains unclear. We herein investigated the therapeutic potential of circNRIP1 delivered by bone marrow mesenchymal stem cell-derived EVs (BMSC-EVs) in osteosarcoma. The expression of circNRIP1 was examined in the clinical tissue samples of osteosarcoma patients, after which the downstream genes of circNRIP1 were bioinformatically predicted. Gain- and loss-of function assays were then performed in osteosarcoma cells with manipulation of circNRIP1 and miR-532-3p expression. EVs isolated from BMSCs were characterized and co-cultured with osteosarcoma cells to examine their effects on cell phenotypes, as reflected by CCK-8 and Transwell assays. Further, a mouse model of tumor xenografts was established for in vivo substantiation. circNRIP1 was upregulated in osteosarcoma tissues and cells. Overexpression of circNRIP1 promoted the proliferative, migratory, and invasive potential of osteosarcoma cells. Co-culture data showed that BMSC-EVs could transfer circNRIP1 into osteosarcoma cells where it competitively bound to miR-532-3p and weakened miR-532-3p's binding ability to AKT3. By this mechanism, the PI3K/AKT signaling pathway was activated and the malignant characteristics of osteosarcoma cells were stimulated. In vivo experimental results unveiled that circNRIP1-overexpressing BMSC-EVs in nude mice resulted in enhanced tumor growth. In conclusion, the BMSC-EV-enclosed circNRIP1 revealed a new molecular mechanism in the pathogenesis of osteosarcoma, which might provide a novel therapeutic target for osteosarcoma.
RESUMO
BACKGROUND: Osteoarthritis (OA) is characterized by chondrocyte injury. Circular RNAs (circRNAs) are involved in the pathogenesis of various diseases, including OA. The purpose of this study was to determine the potential role of circATRNL1 in OA pathology in vitro. METHODS: Human chondrocytes were isolated and treated with interleukin-1 beta (IL-1ß) to mimic OA in vitro. High-throughput RNA sequencing was performed to identify differentially expressed circRNAs, miRNAs and mRNAs between IL and 1ß-treated chondrocytes and normal chondrocytes. The expression of circATRNL1, miR-153-3p and KLF5 was measured using quantitative real-time polymerase chain reaction (qRT-PCR). For functional analyses, cell apoptosis was assessed using a flow cytometry assay. Extracellular matrix (ECM) degradation was monitored by measuring the levels of ECM-associated proteins by Western blot. The potential target miRNAs of circATRNL1 were screened by bioinformatics analysis and verified by dual-luciferase reporter assay. RESULTS: The expression of circATRNL1 was decreased in IL-1ß-treated chondrocytes. CircATRNL1 overexpression ameliorated cell apoptosis and ECM degradation, which were promoted by IL-1ß treatment. Mechanistic analysis revealed that circATRNL1 directly targeted miR-153-3p and that miR-153-3p could reverse the inhibitory effects of circATRNL1 overexpression on inflammatory responses, cell apoptosis and ECM degradation. KLF5 is a target of miR-153-3p. CONCLUSION: Taken together, the results in this study suggested that circATRNL1 might ameliorate the development and progression of OA through regulating miR-153-3p/KLF5 axis. Our study increased the understanding of circRNAs as therapeutic targets in the treatment of OA.
Assuntos
Condrócitos/metabolismo , Fatores de Transcrição Kruppel-Like/antagonistas & inibidores , MicroRNAs/antagonistas & inibidores , Osteoartrite/metabolismo , RNA Circular/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Apoptose/fisiologia , Células Cultivadas , Condrócitos/patologia , Matriz Extracelular , Humanos , Interleucina-1beta/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , MicroRNAs/genética , Osteoartrite/genética , Osteoartrite/patologia , Osteoartrite/prevenção & controle , RNA Circular/genética , Transdução de SinaisRESUMO
OBJECTIVE: To evaluate the effect on increasing bone cement-bone interface micro-gomphosis intensity with bone cement oscillator. METHODS: One hundred femoral bones of adult pig were randomly divided into 6 groups: oscillating group (A1) and control group (A2) of anti-tensile force, oscillating group (B1) and control group (B2) of anti-pressure (n = 20 in each group), oscillating group (C1) and control group (C2) of imaging (n = 10 in each group). Mechanics and CT test was performed, micro-gomphosis intensity of bone cement-bone interface between oscillating group and control group was compared. RESULTS: Mechanics and CT test showed bone cement-bone interface micro-gomphosis intensity in oscillating group was significantly stronger than control group (P < 0.01). CONCLUSION: Bone cement oscillator can significantly increase micro-gomphosis intensity of bone-cement interface, and reduce long-term aseptic loosening of artificial prostheses.
Assuntos
Cimentos Ósseos , Animais , Cimentação , Desenho de Equipamento , Fêmur , Prótese Articular , Masculino , Teste de Materiais , Mecânica , Distribuição Aleatória , Suínos , VibraçãoRESUMO
OBJECTIVE: To study the correlation between single nucleotide polymorphism (SNP) of hsa-miR-124a and risk and prognosis of osteosarcoma (OS). METHODS: OS patients (n = 174) hospitalized at The Second Affiliated Hospital of Harbin Medical University from January 2010 to March 2012 were selected as case group by inclusion and exclusion criteria, and healthy people (n = 150) receiving physical examination at the same duration were recruited as control group. Polymerase chain reaction-ligase detection reaction (PCR-LDR) was performed for genotyping of hsa-miR-124a rs531564. RESULTS: There were significant differences in the frequency distribution of genotypes and alleles of hsa-miR-124a rs531564 in the case and control group (all P < 0.05); the individuals carrying with CG + GG genotype showed significantly decreased risk for OS. The clinical pathological characteristics were significantly different in the patients with CC genotype and CG + GG genotype, including tumor size, tumor differentiation grading, Enneking staging, operation manner, time of chemotherapy and metastasis (all P < 0.05). The 5-year survival rate of the cases with CC genotype was significantly lower than that of the ones with CG + GG genotype (P < 0.05). CG + GG genotype, Enneking staging and operation manner were independent risk factors for prognosis of OS (all P < 0.05). CONCLUSIONS: CG +$ GG genotype of hsa-miR-124a rs531564 had decreased risk for OS and affected prognosis of OS.
Assuntos
Neoplasias Ósseas/genética , Neoplasias Ósseas/mortalidade , Predisposição Genética para Doença , MicroRNAs/genética , Osteossarcoma/genética , Osteossarcoma/mortalidade , Polimorfismo de Nucleotídeo Único , Adolescente , Adulto , Idoso , Alelos , Neoplasias Ósseas/diagnóstico , Neoplasias Ósseas/terapia , Estudos de Casos e Controles , Criança , Feminino , Estudos de Associação Genética , Genótipo , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Metástase Neoplásica , Estadiamento de Neoplasias , Razão de Chances , Osteossarcoma/diagnóstico , Osteossarcoma/terapia , Prognóstico , Adulto JovemRESUMO
This study investigated the biological effects of microRNA-126 overexpression in human MG63 osteosarcoma cells. A recombinant plasmid expressing microRNA-126, pcDNA6.2-microRNA-126, was constructed and transfected into MG63 cells. Using real-time fluorogenic quantitative polymerase chain reaction, the microRNA-126 expression was measured in microRNA-126-MG63 group, Ctrl-MG63 group, and blank group. Cell proliferation, cell cycle distribution, cell migration, and invasion were analyzed using methyl thiazolyl tetrazolium assay, flow cytometer, wound-healing assay, and transwell assay, respectively. As expected, microRNA-126 expression was higher in microRNA-126-MG63 group than in Ctrl-MG63 group and blank group (both P < .05). After 48/72 hours of transfection, cell proliferation in microRNA-126-MG63 group was significantly reduced compared to blank group (both P < .05). Compared to blank group, cell population in G0/G1 stage was significantly higher in microRNA-126-MG63 group, accompanied by lower cell numbers in the S and G2/M phases and decreased proliferation index (all P < .05). Wound-healing assay showed a wider scratch width in microRNA-126-MG63 group and reduced cell migration than blank group (both P < .05). Cells overexpressing microRNA-126 exhibited reduced ADAM9 expression levels compared to other 2 groups (all P < .05), suggesting ADAM9 is a target of microRNA-126. Cell proliferation, migration, and invasion rates were reduced in microRNA-126 group after 48/72 hours of transfection, compared with blank group (all P < .05). Cotransfection of pcDNA6.2-microRNA-126 and pMIR-ADAM9 into MG63 cells led to higher cell proliferation, invasion, and migration rates, compared with transfection of pcDNA6.2-microRNA-126 alone (all P < .05). In summary, our data show that microRNA-126 inhibits cell proliferation, migration, and invasion in human osteosarcoma cells by targeting ADAM9.
Assuntos
Neoplasias Ósseas/genética , Expressão Gênica , MicroRNAs/genética , Osteossarcoma/genética , Regiões 3' não Traduzidas , Proteínas ADAM/genética , Apoptose , Ciclo Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Genes Reporter , Humanos , Proteínas de Membrana/genética , Interferência de RNA , TransfecçãoRESUMO
Modern cementing techniques aim to improve the interlock between bone and cement and to establish a durable interface. Cement penetration is generally believed to influence interface failure, but current methods for improving the cement-bone interface are inadequate. Oscillation is the reciprocated movement of an object through its balanced position, or the quantum physics of systematic fluctuation back and forth near an average value (or trimmed value). To increase the interlock strength at the cement-bone interface, we designed a cement oscillator according to the principles of vibrational mechanics. To evaluate the effect of oscillation on the quality of interlock strength at the cement-bone interface, we randomly divided 156 femoral bones of adult pigs into 2 groups, oscillated and control, and performed mechanical tests to assess interlock strength at the cement-bone interface. The filling effect of bone cement was observed and analyzed under a stereomicroscope, and then each oscillated femur was compared with a control femur. The interlock strength at the cement-bone interface in the oscillated group was significantly greater than in the control group (P<.05), and the filling effect in the oscillated group was also better than that in the control group (P<.05). Our findings show that oscillation of bone cement significantly increases interlock strength at the cement-bone interface, point the way for clinicians to develop a high-performance and pragmatic fixation technique for prostheses to increase interlock strength, and will be of considerable practical importance in helping to prevent aseptic loosening of cemented prostheses.