Figure 1
miR-130b is reduced in chondrogenic differentiation and enhanced in osteoarthritis chondrocytes. A, Morphology of bone marrow mesenchymal stem cells (BMSCs) observed by electron microscope. B, Alizarin red staining, oil red O staining, and toluidine blue staining were used to measure the osteogenic, lipogenic, and chondrogenic abilities of BMSCs (scale bar, 50 μm). C, Col II and Col X expression in BMSCs determined by immunofluorescence assay (scale bar, 50 �m). D, Flow cytometry identified BMSCs. E, RT-qPCR measured miR-130b expression in chondrogenic differentiation of BMSCs. F, RT-qPCR measured miR-130b expression in interleukin-1β-treated chondrocytes. The data in panels E and F were analyzed using one-way ANOVA, and the data in panel C were analyzed using two-way ANOVA. *P<0.05, **P<0.01 compared to control. Experiments were repeated 3 times independently.
Figure 2
Inhibition of miR-130b promotes chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and chondrocyte growth. A, The transfection efficiency of miR-130b inhibitor was verified by RT-qPCR. B, Western blot analysis measured the expression of two cartilage markers in BMSCs. C, The synthesis of extracellular matrix during chondrogenesis of BMSCs was examined by toluidine blue staining (scale bar, 50 μm). D, MTT method was used to measure the viability of interleukin (IL)-1β-stimulated chondrocytes. E, IL-6 and tumor necrosis factor (TNF)-α expression in IL-1β-treated chondrocytes was determined by RT-qPCR. F, Levels of apoptosis-related proteins in IL-1β-treated chondrocytes were measured by western blot analysis. G, Apoptosis rate in IL-1β-treated chondrocytes was measured by TUNEL assay (scale bar, 50 �m). *P<0.05, compared to control; #P<0.05 compared to the NC inhibitor group. The data in panels A and B were analyzed using one-way ANOVA, and the data in panels D, E, F, and G were analyzed using two-way ANOVA. Experiments were repeated 3 times independently. NC: negative control.
Figure 3
miR-130b targeted SOX9. A, Potential binding site of miR-130b to SOX9. B, RT-qPCR measured the transfection efficiency of miR-130b mimic. C, RT-qPCR measured the effect of miR-130b mimic on SOX9 expression. D, Luciferase activity of 293T cells co-transfected with wild type (WT) and mutant (MUT)-SOX9 reporter plasmids, miR-130b mimic, and their controls measured by dual-luciferase reporter assay. E, SOX9 expression during chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) detected by RT-qPCR. F, RT-qPCR detected SOX9 expression in chondrocytes. *P<0.05, **P<0.01 compared to control. Data in panels B, C, E, and F were analyzed using one-way ANOVA and data in panel D were analyzed using two-way ANOVA. Experiments were repeated 3 times independently. NC: negative control.
Figure 4
Overexpression of SOX9 promotes chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and chondrocyte growth. A, Transfection efficiency measured by RT-qPCR. B, Toluidine blue staining detected the effect of pcDNA-SOX9 and siRNA-SOX9 on the synthesis of extracellular matrix in the process of cartilage differentiation of BMSCs (scale bar, 50 μm). C, Effect of pcDNA-SOX9 and siRNA-SOX9 on the proliferation of interleukin (IL)-1β-treated chondrocytes measured by colony formation assay. D, RT-qPCR measured the effects of pcDNA-SOX9 and siRNA-SOX9 on the expression of IL-6 and TNF-α. E, Effect of pcDNA-SOX9 and siRNA-SOX9 on apoptosis of IL-1β-treated chondrocytes measured by flow cytometry. Data in panels A, C, and E were processed using one-way ANOVA, and data in panel D were analyzed using two-way ANOVA. *P<0.05 compared to cells transfected with pcDNA; #P<0.05 compared to cells transfected with siRNA-NC. Experiments were repeated 3 times independently.
Figure 5
SOX9 silencing weakened the inhibition of miR-130b inhibitor on chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and chondrocyte growth. A, Expression of SOX9 in BMSCs and chondrocytes was measured by RT-qPCR. B. Western blot analysis measured the protein level of aggrecan and Col II in BMSCs. C, Toluidine blue staining indicated the extracellular matrix synthesis of BMSCs (scale bar, 50 μm). D, MTT method measured the viability of chondrocytes. E, Colony formation ability of chondrocytes. F, RT-qPCR measured the expression of interleukin (IL)-6 and tumor necrosis factor (TNF)-α in chondrocytes. G, Western blot analysis measured the expression of apoptosis-related factors in chondrocytes. H, TUNEL staining examined the apoptosis rate of chondrocytes (scale bar, 50 �m). The data in panels A, E, and H were analyzed using one-way ANOVA, and the data in panels B, D, F, and G were analyzed using two-way ANOVA. *P<0.05 compared to cells transfected with NC inhibitor; #P<0.05 compared to cells transfected with miR-130b inhibitor + siRNA-NC. Experiments were repeated 3 times independently. NC: negative control.
Figure 6
Experimental mechanism diagram. The inhibition of miR-130b promotes the chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and chondrocyte growth by targeting SOX9.
Figure 1
miR-130b is reduced in chondrogenic differentiation and enhanced in osteoarthritis chondrocytes. A, Morphology of bone marrow mesenchymal stem cells (BMSCs) observed by electron microscope (scale bar 50 μm). B, Alizarin red staining (scale bar 50 μm), oil red O staining (scale bar 50 μm), and toluidine blue staining were used to measure the osteogenic, lipogenic, and chondrogenic abilities of BMSCs. C, Col II and Col X expression in BMSCs determined by immunofluorescence assay (scale bar, 50 μm). D, Flow cytometry identified BMSCs. E, RT-qPCR measured miR-130b expression in chondrogenic differentiation of BMSCs. F, RT-qPCR measured miR-130b expression in interleukin-1β-treated chondrocytes. The data in panels E and F were analyzed using one-way ANOVA, and the data in panel C were analyzed using two-way ANOVA. *P<0.05, **P<0.01 compared to control. Experiments were repeated 3 times independently.
Figure 2
Inhibition of miR-130b promotes chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and chondrocyte growth. A, The transfection efficiency of miR-130b inhibitor was verified by RT-qPCR. B, Western blot analysis measured the expression of two cartilage markers in BMSCs. C, The synthesis of extracellular matrix during chondrogenesis of BMSCs was examined by toluidine blue staining. D, MTT method was used to measure the viability of interleukin (IL)-1β-stimulated chondrocytes. E, IL-6 and tumor necrosis factor (TNF)-α expression in IL-1β treated chondrocytes was determined by RT-qPCR. F, Levels of apoptosis-related proteins in IL-1β-treated chondrocytes were measured by western blot analysis. G, Apoptosis rate in IL-1β-treated chondrocytes was measured by TUNEL assay (scale bar, 50 μm). *P<0.05, compared to control; #P<0.05 compared to the NC inhibitor group. The data in panels A and G were analyzed using one-way ANOVA, and the data in panels B, D, E, and F were analyzed using two-way ANOVA. Experiments were repeated 3 times independently. NC: negative control.
Figure 4
Overexpression of SOX9 promotes chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and chondrocyte growth. A, Transfection efficiency measured by RT-qPCR. B, Toluidine blue staining detected the effect of pcDNA-SOX9 and siRNA-SOX9 on the synthesis of extracellular matrix in the process of cartilage differentiation of BMSCs. C, Effect of pcDNA-SOX9 and siRNA-SOX9 on the proliferation of interleukin (IL)-1β-treated chondrocytes measured by colony formation assay. D, RT-qPCR measured the effects of pcDNA-SOX9 and siRNA-SOX9 on the expression of IL-6 and TNF-α. E, Effect of pcDNA-SOX9 and siRNA-SOX9 on apoptosis of IL-1β-treated chondrocytes measured by flow cytometry. The data in panels A, C, and E were processed using one-way ANOVA, and the data in panel D were analyzed using two-way ANOVA. *P<0.05 compared to cells transfected with pcDNA; #P<0.05 compared to cells transfected with siRNA-NC. Experiments were repeated 3 times independently.
Figure 5
SOX9 silencing weakened the inhibition of miR-130b inhibitor on chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and chondrocyte growth. A, Expression of SOX9 in BMSCs and chondrocytes was measured by RT-qPCR. B, Western blot analysis measured the protein level of aggrecan and Col II in BMSCs. C, Toluidine blue staining indicated the extracellular matrix synthesis of BMSCs. D, MTT method measured the viability of chondrocytes. E, Colony formation ability of chondrocytes. F, RT-qPCR measured the expression of interleukin (IL)-6 and tumor necrosis factor (TNF)-α in chondrocytes. G, Western blot analysis measured the expression of apoptosis-related factors in chondrocytes. H, TUNEL staining examined the apoptosis rate of chondrocytes (scale bar, 50 μm). The data in panels A, E, and H were analyzed using one-way ANOVA, and the data in panels B, D, F, and G were analyzed using two-way ANOVA. *P<0.05 compared to cells transfected with NC inhibitor; #P<0.05 compared to cells transfected with miR-130b inhibitor + siRNA-NC. Experiments were repeated 3 times independently. NC: negative control.
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