RESUMEN
OBJECTIVE: Long noncoding RNAs (lncRNAs) have been indicated to play an important role in many different diseases. Osteoarthritis (OA) is a disease which causes a change of morphology and function in articular cartilage and synovium, leading to cartilage degradation. Synovitis is a common pathological feature of OA, owing to the proliferation of synoviocytes. In this research, we want to verify the role of lncRNA ANRIL in osteoarthritis. PATIENTS AND METHODS: qRT-PCR was used to detect the expression of lncRNA ANRIL in normal synoviocytes and osteoarthritis synoviocytes. The cell proliferation in normal synoviocytes and osteoarthritis synoviocytes after transfection with lncRNA-NC or lncRNA-ANRIL were tested. The apoptosis rate and cell cycle in normal synoviocytes and osteoarthritis synoviocytes were detected by the Flow Cytometry analysis. Western blot was used to analyze the possible mechanism that ANRIL regulated the cells' proliferation in osteoarthritis. RESULTS: We indicated that the expression of ANRIL was significantly improved in OAS compared to NS. The expression of ANRIL was decreased and the cell proliferation was reduced in OAS after transfected with siRNA. And the cell cycle was suspended in G0/G1 phase and the cell apoptosis was improved in OAS after transfected with siRNA. Moreover, ANRIL could regulate the proliferation and apoptosis of OAS via miR-122-5p/DUSP4 axis. CONCLUSIONS: We suggest that lncRNA ANRIL was closely related to osteoarthritis. ANRIL may be involved in the development and progression of osteoarthritis and become a potential target for diagnosis and treatment in OA.
Asunto(s)
Apoptosis , Osteoartritis/metabolismo , ARN Largo no Codificante/metabolismo , Sinoviocitos/metabolismo , Proliferación Celular , Células Cultivadas , Humanos , Osteoartritis/patología , ARN Largo no Codificante/genética , Sinoviocitos/patologíaRESUMEN
OBJECTIVE: This study aims to assess the effect and mechanism of genetically modified adipose-derived mesenchymal stem cells (ASCs) with recombinant lentiviruses mediated knockdown of miR-140-5p in ASCs' osteogenesis in vitro and atrophic nonunion rat model. MATERIALS AND METHODS: This study included 36 male adult Sprague-Dawley (SD) rats weighing 400 g to 450 g from the experimental animal facility of our university. Approval was obtained from the University Animal Care Committee before the study. Rats' ASCs were prepared and genetically modified with lentivirus (Lv)-empty (NC) or Lv-miR-140-5p-TuD (inhibitors). After that, the expressions of RUNX2 and osteocalcin (OCN) were detected in the ASCs. To confirm the mechanisms of miR-140-5p in ASCs, we predicted the target genes by bioinformatics analysis and then the target genes were verified by luciferase reporting assay. The artificial atrophic nonunion was created in the rat's femoral bone. Animals were randomly divided into three groups according to the material implanted into bone defects space: AT scaffolds (AT group, n=12), AT scaffold with Lv-NC modified (AT+ASCs+Lv-NC group, n=12), AT scaffold with the Lv-miR-140-5p-TuD modified ASCs (AT+ASCs+Lv-miR-140-5p-TuD group, n=12). After four weeks, the rats were euthanized for the following radiographic examination, histologic study and biomechanical testing. RESULTS: MiR-140-5p was down-regulated during osteogenic differentiation of ASCs, and inhibition of MiR-140-5p promoted osteogenesis of ASCs in vitro. Inhibition of MiR-140-5p promoted osteogenesis of ASCs and enhanced fracture in the atrophic nonunion rat model: AT+ASCs+Lv-NC group, AT+ASCs+Lv-miR-140-5p-TuD group resulted in a better bone formation and higher BMD and BMC than AT group, while excellent bone formation and the highest BMD and BMC were observed in AT+ASCs+Lv-miR-140-5p-TuD group. Both AT+ASCs+Lv-NC group and AT+ASCs+Lv-miR-140-5p-TuD group presented more mature characteristics in the micro-architecture than AT group, whereas AT+ASCs+Lv-miR-140-5p-TuD group presented the highest BV/TV, Tb.Th and Tb.N as well as the lowest Tb.Sp. The peak load of the operated femur increased by 94.43% AT+ASCs+Lv-miR-140-5p-TuD group, 50.68% in AT+ASCs+Lv-NC group compared to the control AT group, respectively. The result of luciferase reporting assay showed that miR-140-5p could directly target TLR4 and BMP2. CONCLUSIONS: This study demonstrates that lentiviruses-mediated knockdown of miR-140-5p can significantly promote osteogenesis of ACSs by directly regulating its' target genes, TLR4 and BMP2, and that combined adipose scaffold with genetically modified ASCs can significantly enhance fracture-healing and bone formation in the atrophic nonunion rat model.
Asunto(s)
Tejido Adiposo/citología , Proteína Morfogenética Ósea 2/genética , Fracturas no Consolidadas/terapia , MicroARNs/genética , Receptor Toll-Like 4/genética , Tejido Adiposo/metabolismo , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Regulación hacia Abajo , Curación de Fractura , Fracturas no Consolidadas/genética , Células HEK293 , Humanos , Masculino , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Osteogénesis , Ratas , Ratas Sprague-DawleyRESUMEN
OBJECTIVE: Osteoarthritis (OA) is one of the most common chronic joint diseases, caused by lesions in articular cartilage and synovial membranes. Synovitis is a major characteristic of OA, due to the proliferation of synoviocytes. Long noncoding RNAs (lncRNAs) have implicated to play an important role in many different diseases. The aim of this study was to verify the role of lncRNA gastric cancer-associated transcript 3 (GACAT3) in osteoarthritis. MATERIALS AND METHODS: We utilized the qRT-PCR to detect the expression of lncRNA GACAT3 in osteoarthritis synoviocytes (OAS) and normal synoviocytes (NS). The cell proliferation in NS and OAS after transfection with lncRNA-NC or lncRNA-GACAT3 was detected. The cell cycle and apoptosis rate in NS and OAS were measured by the Flow cytometry analysis. Western blot was used to analyze the possible related mechanism that GACAT3 regulated the cells proliferation in osteoarthritis. RESULTS: We found that GACAT3 expression was significantly increased in OAS compared with NS. GACAT3 expression was decreased in OAS after transfection with siRNA and the cell proliferation in OAS after transfection with siRNA was significantly inhibited. The cell cycle was arrested in G0/G1 phase and the apoptosis rate was increased in OAS after transfection with siRNA. Moreover, GACAT3 could impact the proliferation of OAS by interleukin-6/signal transducer and activator of transcription-3 (IL-6/STAT3) signaling pathway. CONCLUSIONS: In this study, we found that lncRNA GACAT3 was closely related to the osteoarthritis. GACAT3 may be involved in the development and progression of osteoarthritis and become a potential target for treating.
Asunto(s)
Proliferación Celular/fisiología , Interleucina-6/biosíntesis , Osteoartritis/metabolismo , ARN Largo no Codificante/biosíntesis , Factor de Transcripción STAT3/biosíntesis , Sinoviocitos/metabolismo , Cartílago Articular/metabolismo , Humanos , Interleucina-6/genética , Osteoartritis/genética , ARN Largo no Codificante/genética , Factor de Transcripción STAT3/genética , Transducción de Señal/fisiología , Membrana Sinovial/metabolismoRESUMEN
The development of intensity-modulated radiotherapy (IMRT) has created a clear need for a dosimeter that can accurately and conveniently measure dose distributions in three dimensions to assure treatment quality. PRESAGE is a new three dimensional (3D) dosimetry material consisting of an optically clear polyurethane matrix, containing a leuco dye that exhibits a radiochromic response when exposed to ionizing radiation. A number of potential advantages accrue over other gel dosimeters, including insensitivity to oxygen, radiation induced light absorption contrast rather than scattering contrast, and a solid texture amenable to machining to a variety of shapes and sizes without the requirement of an external container. In this paper, we introduce an efficient method to investigate the basic properties of a 3D dosimetry material that exhibits an optical dose response. The method is applied here to study the key aspects of the optical dose response of PRESAGE: linearity, dose rate dependency, reproducibility, stability, spectral changes in absorption, and temperature effects. PRESAGE was prepared in 1 x 1 x 4.5 cm3 optical cuvettes for convenience and was irradiated by both photon and electron beams to different doses, dose rates, and energies. Longer PRESAGE columns (2 x 2 x 13 cm3) were formed without an external container, for measurements of photon and high energy electron depth-dose curves. A linear optical scanning technique was used to detect the depth distribution of radiation induced optical density (OD) change along the PRESAGE columns and cuvettes. Measured depth-OD curves were compared with percent depth dose (PDD). Results indicate that PRESAGE has a linear optical response to radiation dose (with a root mean square error of -1%), little dependency on dose rate (-2%), high intrabatch reproducibility (< 2%), and can be stable (-2%) during 2 hours to 2 days post irradiation. Accurate PRESAGE dosimetry requires temperature control within 1 degrees C. Variations in the PRESAGE formulation yield corresponding variations in sensitivity, stability, and density. CT numbers in the range 100-470 were observed. In conclusion, the small volume studies presented here indicate PRESAGE to be a promising, versatile, and practical new dosimetry material with applicability for radiation therapy.