The regulatory roles of MicroRNAs in bone remodeling and osteoporosis

In bone field, microRNAs (miRNAs) have been described as key factors regulating bone formation, remodeling, and homeostasis. The identification of miRNAs involved in skeletal function will be essential to the development of miRNA-based therapeutic strategies for bone disorders. As with other regulatory molecules, miRNAs are frequently subject to change during the development of human diseases. In this regard, we identified a subset of miRNAs with altered expression in osteoporotic bone and demonstrate the functional involvement of some of those miRNAs in the regulation of bone formation and the pathways regulating the progression of osteoporosis. We also have depicted an overview of miRNAs in the human bone tissue and in primary bone cells. Furthermore, we have identified genetic variants in human osteoblast-related miRNAs associated with bone mineral density and this association was functionally demonstrated in bone and osteoblast samples. This work has provided evidences of the marked complexity behind this regulatory system and opens novel prospect for research and therapy.En l’àmbit de l’estudi de l’òs, els microRNAs (miRNAs) han estat descrits com factors claus en la regulació de la formació, remodelatge i homeòstasis de l’ òs. La identificació de miRNAs implicats en la funció esquelètica és imprescindible pel desenvolupament de noves estratègies terapèutiques, basades en miRNAs, dirigides al tractament de malalties òssies. Com en el cas d’altres molècules reguladores, els miRNAs poden patir modificacions durant el desenvolupament de malalties humanes. En aquest sentit, hem identificat un grup de miRNAs amb una expressió alterada en l’òs osteoporòtic i hem demostrat la implicació funcional d’algun d’aquests miRNAs en la regulació de la formació òssia i els mecanismes pels quals es produiria l’osteoporosi. Alhora, també hem ofert una visió general dels miRNAs presents en el teixit ossi humà i en les cèl·lules òssies. També hem identificat variants genètiques dins de les seqüències de miRNAs expressats en osteoblasts, que han estat associades amb la densitat mineral òssia. A més a més, aquesta associació ha estat funcionalment demostrada en òs i osteoblasts. Aquest treball reflexa l’elevada complexitat que hi ha darrera del sistema regulador per miRNAs i obre nous camins per la recerca i la teràpia

The regulatory roles of MicroRNAs in bone remodeling and osteoporosis

In bone field, microRNAs (miRNAs) have been described as key factors regulating bone formation, remodeling, and homeostasis. The identification of miRNAs involved in skeletal function will be essential to the development of miRNA-based therapeutic strategies for bone disorders. As with other regulatory molecules, miRNAs are frequently subject to change during the development of human diseases. In this regard, we identified a subset of miRNAs with altered expression in osteoporotic bone and demonstrate the functional involvement of some of those miRNAs in the regulation of bone formation and the pathways regulating the progression of osteoporosis. We also have depicted an overview of miRNAs in the human bone tissue and in primary bone cells. Furthermore, we have identified genetic variants in human osteoblast-related miRNAs associated with bone mineral density and this association was functionally demonstrated in bone and osteoblast samples. This work has provided evidences of the marked complexity behind this regulatory system and opens novel prospect for research and therapy.En l’àmbit de l’estudi de l’òs, els microRNAs (miRNAs) han estat descrits com factors claus en la regulació de la formació, remodelatge i homeòstasis de l’ òs. La identificació de miRNAs implicats en la funció esquelètica és imprescindible pel desenvolupament de noves estratègies terapèutiques, basades en miRNAs, dirigides al tractament de malalties òssies. Com en el cas d’altres molècules reguladores, els miRNAs poden patir modificacions durant el desenvolupament de malalties humanes. En aquest sentit, hem identificat un grup de miRNAs amb una expressió alterada en l’òs osteoporòtic i hem demostrat la implicació funcional d’algun d’aquests miRNAs en la regulació de la formació òssia i els mecanismes pels quals es produiria l’osteoporosi. Alhora, també hem ofert una visió general dels miRNAs presents en el teixit ossi humà i en les cèl·lules òssies. També hem identificat variants genètiques dins de les seqüències de miRNAs expressats en osteoblasts, que han estat associades amb la densitat mineral òssia. A més a més, aquesta associació ha estat funcionalment demostrada en òs i osteoblasts. Aquest treball reflexa l’elevada complexitat que hi ha darrera del sistema regulador per miRNAs i obre nous camins per la recerca i la teràpia

Pro-osteoporotic miR-320a impairs osteoblast function and induces oxidative stress

MicroRNAs (miRNAs) are important regulators of many cellular processes, including the differentiation and activity of osteoblasts, and therefore, of bone turnover. MiR-320a is overexpressed in osteoporotic bone tissue but its role in osteoblast function is unknown. In the present study, functional assays were performed with the aim to elucidate the mechanism of miR-320a action in osteoblastic cells. MiR-320a was either overexpressed or inhibited in human primary osteoblasts (hOB) and gene expression changes were evaluated through microarray analysis. In addition, the effect of miR-320a on cell proliferation, viability, and oxidative stress in hOB was evaluated. Finally, matrix mineralization and alkaline phosphatase activity were assessed in order to evaluate osteoblast functionality. Microarray results showed miR-320a regulation of a number of key osteoblast genes and of genes involved in oxidative stress. Regulation of osteoblast differentiation and ossification appeared as the best significant biological processes (PANTHER P value = 3.74E-05; and P value = 3.06E-04, respectively). The other enriched pathway was that of the cellular response to cadmium and zinc ions, mostly by the overexpression of metallothioneins. In hOBs, overexpression of miR-320a increased cell proliferation and oxidative stress levels whereas mineralization capacity was reduced. In conclusion, overexpression of miR-320a increased stress oxidation levels and was associated with reduced osteoblast differentiation and functionality, which could trigger an osteoporotic phenotype

Effect of the tumor suppressor miR-320a on viability and functionality of human osteosarcoma cell Llnes compared to primary osteoblasts

The miR-320a regulates a number of genes involved in various physiological processes. In particular, it has been reported as a tumor suppressor in several types of human cancers and involved in osteoporotic fracture and osteoblast function. Hence, the role of miR-320a has been evaluated in tumor cells and in primary cells in a separated context, but its effect has never been explored in a comparative manner. The present study aims to evaluate the cellular effects of miR-320a on human osteosarcoma cell lines (MG-63 and U2OS) compared to that on primary human osteoblasts (hOBs). miR-320a was either overexpressed or inhibited in all cell lines, and cell proliferation and viability were analyzed. Additionally, the effects of miR-320a on matrix mineralization, alkaline phosphatase activity, and oxidative stress were also evaluated in order to assess osteoblast functionality. In osteosarcoma cells, miR-320a overexpression reduced cell viability and proliferation, while in hOB cell viability was not affected and proliferation even was increased. The overexpression of miR-320a in both osteosarcoma cells and hOBs reduced the mineralization capacity. Finally, an increased oxidative stress was detected in all cells after miR-320a overexpression mainly in osteosarcoma. In conclusion, the overexpression of miR-320a increased stress oxidation levels, which could be involved in the reduced osteoblast performance, even though the cell viability was only affected in osteosarcoma cells.This research was supported by the CIBER on Frailty and Healthy Ageing (CIBERFES; grant number: CB16/10/00245), the CIBERER (grant number: U720), FEDER funds, and grants from the Science and Innovation Ministry (ISCIII; grant numbers: PI16/01860 and PI13/00116; SAF2016-75948-R). L.D.-U. was granted with a PFIS predoctoral fellowship from the ISCIII

Estudio del patrón de expresión de microRNAs en el hueso osteoporótico

Objetivos: Identificar microRNAs (miRNAs) diferencialmente expresados en muestras óseas con fractura osteoporótica respecto a huesos sanos. Material y métodos: Se extrajo RNA total a partir de hueso trabecular fresco del cuello femoral de mujeres sometidas a reemplazo de cadera, ya sea debido a fractura osteoporótica (n=6) o por artrosis en ausencia de osteoporosis (según la DMO) (n=6). Las muestras se hibridaron en un array de miRNAs y se realizaron diagramas de PCA y de mapa de calor. Para la comparación de los niveles de expresión, se fijó como significativo un umbral de cambio de >1,5 veces y un valor p<0,05 en la t de Student (corregido para múltiples pruebas). Resultados: Tanto los análisis de PCA como el mapa de calor mostraron una agrupación de las muestras según si eran de fractura o no. Se detectaron 790 miRNAs en las muestras de hueso, 82 de los cuales estaban alterados en las muestras osteoporóticas. Tras la validación en otro panel de 6 muestras osteoporóticas y 6 no osteoporóticas mediante PCR a tiempo real de los miRNAs más significativos, y para los que existía un ensayo disponible, se confirmaron los miRNAs miR-320a y miR-22-3p. Estos dos miRNAs se detectaron en cultivos de osteoblastos primarios, aunque no mantenían el mismo patrón de expresión que en las muestras de hueso total. Conclusiones: Hemos demostrado que existen diferencias en la expresión de miRNAs en muestras con fractura osteoporótica, lo que abre nuevas perspectivas para la investigación y diseño de nuevas terapias.Objectives: To identify microRNAs (miRNAs) differentially expressed in bone samples with osteoporotic fracture compared with healthy bones. Methods: Total RNA was extracted from fresh trabecular bone of the femoral neck of women undergoing hip replacement surgery, either because to osteoporotic fracture (n=6) or in the absence of osteoarthritis osteoporosis (based on BMD) (n=6). The samples were hybridized on an array of miRNAs and PCA diagrams and heat map were made. To compare expression levels, >1.5 times and a value p<0.05 Student's T test (corrected for multiple testing) was set as a threshold of significant change. Results: Both PCA analysis and the heat map showed a samples grouping whether there was fracture or not. 790 were detected miRNAs in bone samples, 82 of which were altered in the osteoporotic samples. After validation in another panel of 6 samples 6 osteoporotic and non-osteoporotic by PCR real time of the most significant miRNAs, and for which there was a test available, the miRNAs, miR-320a and miR22-3p were confirmed. These two miRNAs were detected in cultures of primary osteoblasts, although they did not maintain the same pattern of expression in total bone samples. Conclusions: We have shown that there are differences in the expression of miRNAs in samples with osteoporotic fracture. This opens prospects for research and design of new therapies.Este trabajo ha sido financiado por la Red Temática de Investigación Cooperativa en Envejecimiento y Fragilidad (RETICEF; RD12/0043/0022), y la ayuda FIS PI13/00116 (Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación) y los fondos FEDE

Estudio del patrón de expresión de microRNAs en el hueso osteoporótico

Objetivos: Identificar microRNAs (miRNAs) diferencialmente expresados en muestras óseas con fractura osteoporótica respecto a huesos sanos. Material y métodos: Se extrajo RNA total a partir de hueso trabecular fresco del cuello femoral de mujeres sometidas a reemplazo de cadera, ya sea debido a fractura osteoporótica (n=6) o por artrosis en ausencia de osteoporosis (según la DMO) (n=6). Las muestras se hibridaron en un array de miRNAs y se realizaron diagramas de PCA y de mapa de calor. Para la comparación de los niveles de expresión, se fijó como significativo un umbral de cambio de >1,5 veces y un valor p<0,05 en la t de Student (corregido para múltiples pruebas). Resultados: Tanto los análisis de PCA como el mapa de calor mostraron una agrupación de las muestras según si eran de fractura o no. Se detectaron 790 miRNAs en las muestras de hueso, 82 de los cuales estaban alterados en las muestras osteoporóticas. Tras la validación en otro panel de 6 muestras osteoporóticas y 6 no osteoporóticas mediante PCR a tiempo real de los miRNAs más significativos, y para los que existía un ensayo disponible, se confirmaron los miRNAs miR-320a y miR-22-3p. Estos dos miRNAs se detectaron en cultivos de osteoblastos primarios, aunque no mantenían el mismo patrón de expresión que en las muestras de hueso total. Conclusiones: Hemos demostrado que existen diferencias en la expresión de miRNAs en muestras con fractura osteoporótica, lo que abre nuevas perspectivas para la investigación y diseño de nuevas terapias.Objectives: To identify microRNAs (miRNAs) differentially expressed in bone samples with osteoporotic fracture compared with healthy bones. Methods: Total RNA was extracted from fresh trabecular bone of the femoral neck of women undergoing hip replacement surgery, either because to osteoporotic fracture (n=6) or in the absence of osteoarthritis osteoporosis (based on BMD) (n=6). The samples were hybridized on an array of miRNAs and PCA diagrams and heat map were made. To compare expression levels, >1.5 times and a value p<0.05 Student's T test (corrected for multiple testing) was set as a threshold of significant change. Results: Both PCA analysis and the heat map showed a samples grouping whether there was fracture or not. 790 were detected miRNAs in bone samples, 82 of which were altered in the osteoporotic samples. After validation in another panel of 6 samples 6 osteoporotic and non-osteoporotic by PCR real time of the most significant miRNAs, and for which there was a test available, the miRNAs, miR-320a and miR22-3p were confirmed. These two miRNAs were detected in cultures of primary osteoblasts, although they did not maintain the same pattern of expression in total bone samples. Conclusions: We have shown that there are differences in the expression of miRNAs in samples with osteoporotic fracture. This opens prospects for research and design of new therapies.Este trabajo ha sido financiado por la Red Temática de Investigación Cooperativa en Envejecimiento y Fragilidad (RETICEF; RD12/0043/0022), y la ayuda FIS PI13/00116 (Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación) y los fondos FEDE

Expression profiling of microRNAs in human bone tissue from postmenopausal women

Bone tissue is composed of several cell types, which express their own microRNAs (miRNAs) that will play a role in cell function. The set of total miRNAs expressed in all cell types configures the specific signature of the bone tissue in one physiological condition. The aim of this study was to explore the miRNA expression profile of bone tissue from postmenopausal women. Tissue was obtained from trabecular bone and was analyzed in fresh conditions (n = 6). Primary osteoblasts were also obtained from trabecular bone (n = 4) and human osteoclasts were obtained from monocyte precursors after in vitro differentiation (n = 5). MicroRNA expression profiling was obtained for each sample by microarray and a global miRNA analysis was performed combining the data acquired in all the microarray experiments. From the 641 miRNAs detected in bone tissue samples, 346 (54%) were present in osteoblasts and/or osteoclasts. The other 46% were not identified in any of the bone cells analyzed. Intersection of osteoblast and osteoclast arrays identified 101 miRNAs shared by both cell types, which accounts for 30-40% of miRNAs detected in these cells. In osteoblasts, 266 miRNAs were detected, of which 243 (91%) were also present in the total bone array, representing 38% of all bone miRNAs. In osteoclasts, 340 miRNAs were detected, of which 196 (58%) were also present in the bone tissue array, representing 31% of all miRNAs detected in total bone. These analyses provide an overview of miRNAs expressed in bone tissue, broadening our knowledge in the microRNA field.This work was supported by the Red Temática de Investigación Cooperativa en Envejecimiento y Fragilidad (RETICEF; RD12/0043/0022) and Centro de Investigación Biomédica en Red de Fragilidad y Envejecimiento Saludable (CIBERFES; CB16/10/00245), and the Grants PI10/01537, PI13/00116, and PI13/00444 from FIS (Carlos III Health Institute, Science and Innovation Ministry); SAF2016-75948-R, from Ministerio de Economia y Competitividad, and 2014SGR-932 from Generalitat de Catalunya. FEDER funds also supported this study

MiRNA profiling of whole trabecular bone: identification of osteoporosis-related changes in MiRNAs in human hip bones.

BACKGROUND: MicroRNAs (miRNAs) are important regulators of gene expression, with documented roles in bone metabolism and osteoporosis, suggesting potential therapeutic targets. Our aim was to identify miRNAs differentially expressed in fractured vs nonfractured bones. Additionally, we performed a miRNA profiling of primary osteoblasts to assess the origin of these differentially expressed miRNAs. METHODS: Total RNA was extracted from (a) fresh femoral neck trabecular bone from women undergoing hip replacement due to either osteoporotic fracture (OP group, n = 6) or osteoarthritis in the absence of osteoporosis (Control group, n = 6), matching the two groups by age and body mass index, and (b) primary osteoblasts obtained from knee replacement due to osteoarthritis (n = 4). Samples were hybridized to a microRNA array containing more than 1900 miRNAs. Principal component analysis (PCA) plots and heat map hierarchical clustering were performed. For comparison of expression levels, the threshold was set at log fold change > 1.5 and a p-value < 0.05 (corrected for multiple testing). RESULTS: Both PCA and heat map analyses showed that the samples clustered according to the presence or absence of fracture. Overall, 790 and 315 different miRNAs were detected in fresh bone samples and in primary osteoblasts, respectively, 293 of which were common to both groups. A subset of 82 miRNAs was differentially expressed (p < 0.05) between osteoporotic and control osteoarthritic samples. The eight miRNAs with the lowest p-values (and for which a validated miRNA qPCR assay was available) were assayed, and two were confirmed: miR-320a and miR-483-5p. Both were over-expressed in the osteoporotic samples and expressed in primary osteoblasts. miR-320a is known to target CTNNB1 and predicted to regulate RUNX2 and LEPR, while miR-483-5p down-regulates IGF2. We observed a reduction trend for this target gene in the osteoporotic bone. CONCLUSIONS: We identified two osteoblast miRNAs over-expressed in osteoporotic fractures, which opens novel prospects for research and therapy.This work was supported by grant FIS PI10/01537 and the Red Temática de Investigación Cooperativa en Envejecimiento y Fragilidad (RETICEF) (Carlos III Health Institute, Science and Innovation Ministry), and FEDER funds. Grant SAF2011-25431 and PIB2010AR-00473 (Science and Innovation Ministry), and the support from the Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER, an initiative of ISCIII) are also acknowledged. Grants from the Generalitat de Catalunya (DIUE; 2009 SGR 818, 2009 SGR 971) also supported this work