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Objective:To investigate the relationship between long non-coding RNA (lncRNA) DHRS4-AS1 and disease-free survival in osteosarcoma patients and the mechanisms of its effect on proliferation and migration of osteosarcoma cells in vitro.Methods:The data of DHRS4-AS1 transcriptome levels and survival status of osteosarcoma patients in GEPIA database were collected since the database was established, and the patients were divided into high DHRS4-AS1 expression group and low DHRS4-AS1 expression group based on the median DHRS4-AS1 transcriptome level, with 59 cases in each group, and the Kaplan-Meier method was used to analyze the disease-free survival of the two groups. Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression of DHRS4-AS1 in osteosarcoma cell lines MG-63, HOS, 143B, U-2OS, Saos2 and normal osteoblast cell line hFOB1.19, and the osteosarcoma cell line with the lowest DHRS4-AS1 expression level was selected for subsequent experiments. The plasmid carrying DHRS4-AS1 sequence and the plasmid carrying negative control sequence were transfected into the selected osteosarcoma cells as DHRS4-AS1 group and control group. CCK-8 method was used to detect the proliferation of each group of cells, and the absorbance value was used as the cell proliferation ability; cell scratch assay was used to detect the migration of each group of cells. The bioinformatics website starBase V2.0 was used to predict the target genes of DHRS4-AS1, and the dual luciferase reporter gene assay was used to verify the targeting relationship between DHRS4-AS1 and the target genes. The expression levels of target genes and downstream genes of osteosarcoma cells in control group and DHRS4-AS1 group were detected by qRT-PCR and Western blotting.Results:Survival analysis showed that the disease-free survival of osteosarcoma patients in the high DHRS4-AS1 expression group in GEPIA database was superior to that of the low DHRS4-AS1 expression group ( P < 0.001). Compared with normal osteoblastic hFOB1.19 cells, the expression level of DHRS4-AS1 was low in all osteosarcoma cells (all P < 0.01), with the lowest expression level of DHRS4-AS1 in U-2OS cells ( P < 0.001). Cell proliferation ability was reduced in U-2OS cells of the DHRS4-AS1 group after 1, 2, 3 and 4 d of culture compared with the control group (all P < 0.05). The migration rate of U-2OS cells in the DHRS4-AS1 group was lower than that in the control group [(31±6)% vs. (63±4)%, t = 4.38, P = 0.005]. starBase V2.0 website predicted that DHRS4-AS1 complementarily bound to miRNA-411-3p (miR-411-3p); dual luciferase reporter gene assay showed that miR-411-3p overexpression reduced the luciferase activity of the wild-type DHRS4-AS1 reporter gene ( P < 0.001), but had no effect on the luciferase activity of the mutant DHRS4-AS1 reporter gene ( P > 0.05). qRT-PCR showed that the relative expression of miR-411-3p in U-2OS cells of the DHRS4-AS1 group was low (0.22±0.06 vs. 1.06±0.23, t = 3.55, P = 0.012) and the relative expression of metastasis suppressor MTSS1 mRNA was high (5.58±1.03 vs. 1.06±0.22, t = 4.28, P = 0.005) compared with the control group; Western blotting showed that MTSS1 expression was elevated, and the expression levels of cell proliferation phenotype proteins CDK3 and cyclin C and cell migration phenotype proteins ZEB2 and KLF8 were low. Conclusions:Osteosarcoma patients with high expression of lncRNA DHRS4-AS1 have better disease-free survival, and its expression is low in osteosarcoma cell lines. DHRS4-AS1 may promote MTSS1 gene expression and inhibit cell proliferation and migration by targeting and down-regulating miR-411-3p expression in osteosarcoma cells.
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BACKGROUND: SSh as a Hedgehog signal protein can promote bone development, growth and remodeling.OBJECTIVE: To investigate the effect of bone marrow mesenchymal stem cells (BMSCs) combined with Shh modified nano-hydroxyapatite/collagen (nHAC) in the repair of femoral defects in rats.METHODS: Forty-eight Sprague-Dawley rats were randomly divided into four groups, and the model of femoral defects was established in these rats. At 14 days after modeling, experimental group was implanted with the BMSCs/Shh modified nHAC, scaffold group was implanted with simple nHAC, cell scaffold group was implanted with BMSCs/nHAC,and blank control group was without any implantation. At 3, 6, 9, 12 weeks after repair, X-ray examination, bone density measurement and bone biopsy in bone defect area were performed.RESULTS AND CONCLUSION: (1) X-ray examination: The Lane-Sandhu X-ray score and bone mineral density value in the experimental group at different time points after operation were significantly higher than those in the other three groups (P < 0.05). (2) Hematoxylin-eosin staining: 12 weeks after repair, a small amount of bone tissues but no bone marrow formed in the scaffold group; a small amount of bone tissues with absence of bone marrow formed in the cell scaffold group, and the residual scaffold was visible; in the experimental group, the scaffold was completely absorbed,and mature bone and medullary cavity formed with presence of bone marrow. (3) Scanning electron microscope observation: 12 weeks after repair, irregular arrangement of bone fibers and a large number of bone fossae were observed in the scaffold group; the cell scaffold group showed a large number of osteoblasts, but bone fibers still arranged irregularly; in the presence of the Haversian system, a large number of regularly arranged bone trabeculae were detective in the experimental group. These results elucidate that the Shh modified nHAC/BMSCs complex can promote the repair of bone defects.
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BACKGROUND: Polylactic acid copolymer bone scaffold has excellent biodegradability, and it is easy to be shaped and can promote the formation and growth of bone tissue and blood vessel.OBJECTIVE: To observe the effects of adipose-derived stem cells(ADSCs)/poly(lactic-co-glycolic acid) (PLGA) complex on the biomechanical properties after fracture healing in osteoporotic bone.METHODS: Sixty Sprague-Dawley rats were randomly divided into four groups: blank control group received no treatment; the bilateral tibial fracture model was made after 3 months of bilateral ovarian resection in model group; the bilateral tibial fracture model was made and ADSCs were implanted into the bone after 3 months of bilateral ovarian resection in cell therapy group; the bilateral tibial fracture model was made and the PLGA/ADSCs complex was implanted after 3 months of bilateral ovarian resection in combined treatment group.The bone mineral density, callus thickness, biomechanical parameters and the microstructure of the trabecular bone were detected.RESULTS AND CONCLUSION: (1) The bone density: The bone density of the model group was significantly lower than that of the blank control group (P < 0.05); the bone mineral density of the cell therapy group and the combined treatment group was higher than that of the model group (P < 0.05), but lower than that of the control group (P < 0.05); and the bone mineral density of the combination treatment group was higher than that of the cell therapy group (P < 0.05). (2)Thickness of the callus: The thickness of the callus in the cell therapy group and combined treatment group was higher than that of the model group and blank control group (P < 0.05); moreover, the thickness of the callus in the combined treatment group was higher than that of the cell therapy group (P < 0.05). (3) Biomechanical test: The failure load, stress and shear strength, elastic modulus were decreased in the model group compared with the blank control group (P < 0.05), while the shear strain increased (P < 0.05). Compared with the model group, the failure load, ultimate stress, shear strength, elastic modulus were increased in the cell therapy group and combined treatment group (P < 0.05), and the shear strain was decreased (P < 0.05). Moreover, the combined treatment group showed more changes in these biomechanical parameters (P < 0.05). (4) The trabecular bone microstructure: The model group presented with trabecular derangement, spacing increases, and even fracture and lacuna. After ADSCs or ADSCs/PLGA transplantation,the trabecular bones increased in number, thickness, and spacing, and the number of lacunae reduced. In conclusion,ADSCs combined with PLGA in the treatment of osteoporotic fracture can significantly improve the biomechanical parameters of bone tissue after healing.