Table 1.
Primer sequence of q RT-PCR.
Fig 1.
Extraction and identification of green fluorescent protein-labeled bone marrow mesenchymal stem cells.
A Schematic diagram of cell extraction. B Cell morphology under an optical microscope (x100 magnification). Scar bar: 100μm. C Cell morphology under a fluorescent microscope (x100 magnification). Scar bar: 100μm. D The surface markers of BMSCs were identified by cellular immunofluorescence. CD90 and CD44 showed red fluorescence as antigen-positive expression; CD34 did not show red fluorescence as antigen-negative expression. Scar bar: 50μm. E Statistical results on the expression of surface markers CD90, CD44 and CD34 in BMSCs. Dates are presented as mean ± SD (n = 5).
Fig 2.
A Schematic diagram of cell transfection. B Expression of miR-31 in bone marrow mesenchymal stem cells in each group after transfection was detected by qRT-PCR. C The status of miR-31agomir and miR-31antagomir transfection is detected by immunofluorescence, and successful transfection emits red fluorescence. Scar bar: 100μm. D Statistics of the results of miR-31agomir and miR-31antagomir transfection efficiency. E Morphology of the cells under optical microscopy after transfection. Scar bar: 100μm. Dates are presented as mean ± SD (n = 9,15). Statistical analysis: compared to control, **P<0.01, ***P<0.001.
Fig 3.
Effect of miR-31 on BMSCs proliferation.
A To analyze the effect of miR-31 on the proliferation of BMSCs. The OD values of miR-31agomir, miR-31antagomir and control at 450m for 24, 48 and 72 hours were detected by CCK-8. B Detection of the cell cycle distribution of each group of cells by flow cytometry. Dates are presented as mean ± SD (n = 5). Statistical analysis: within group compared to control, *P<0.05, **P<0.01, ***P<0.001; between groups compared to 24 hours miR-31agomir, #P<0.05, ###P<0.001.
Fig 4.
Analysis of the effect of miR-31 on the migration of BMSCs.
A The promoted effect of miR-31 on the horizontal migration of BMSCs tested by bidirectional wound-healing assay at 0, 12, 24 and 48 hours. Scar bar: 100μm. B Percentage cell migration area result statistics for control, miR-31agomir and miR-31antagomir at 0, 12, 24 and 48 hours. C Schematic diagram of the vertical cell migration tested by Transwell chamber assay. D Cells that passed through the polycarbonate membrane were stained with crystal violet, observed under an inverted phase contrast microscope and counted in 200× magnification field. Scar bar: 50μm. E Statistical results of the number of cells in the lower chamber of the migration value of BMSCs. Dates are presented as mean ± SD (n = 5,15). Statistical analysis: within group compared to control, *P<0.05, **P<0.01, ***P<0.001; between groups compared to 12 hours miR-31agomir, ###P<0.001.
Fig 5.
In vitro, miR-31 upregulated CXCR4 and MMP-2 expression.
A Strip chart of protein expression. B The relative expression of CXCR4 protein in each group. C The relative expression of MMP-2 protein in each group. Dates are presented as mean ± SD (n = 3). Statistical analysis: compared to control, *P<0.05, **P<0.01, ***P<0.001.
Fig 6.
Establishment of SCI model and the effect of miR-31 on BMSCs migration in vivo.
A Schematic diagram of cell transplantation. B Relative levels of IL-6 and IL-17 in normal and spinal cord injured mice. C Assessment of functional repair after spinal cord injury in mice by BMS score. D The morphology of the normal and post-injured spinal cord was observed by HE staining. The arrow in the normal spinal cord points to the central cord and neurons; the arrow in the injured tissue points to the red blood cells and vacuoles. Scar bar: 50μm. E The migration of bone marrow mesenchymal stem cells within the spinal cord was observed by frozen sections on days 5 and 7. Scar bar: 50μm. F Statistical results of the relative fluorescence intensity of migrating BMSCs within the spinal cord. Dates are presented as mean ± SD (n = 5,6,12). Statistical analysis: Within group compared to control, *P<0.05, **P<0.01, ***P<0.001; between groups compared to 3,5 days miR-31agomir, ###P<0.01; a P<0.05 indicates miR-31antagomir compared to miR-31agomir.
Fig 7.
miR-31 upregulated the expression of CXCR4 and MMP-2.
A After cell transplantation, CXCR4 expression in tissues was detected by immunohistochemistry assays on days 5 and 7. Scar bar: 25μm. B Statistical results of CXCR4 expression in tissues. C After cell transplantation, MMP-2 expression in tissues was detected by immunohistochemistry assays on days 5 and 7. Scar bar: 25μm. D Statistical results of MMP-2 expression in tissues. E IL-17 expression in vivo after cell transplantation. F IL-6 expression in vivo after cell transplantation. G After cell transplantation, CXCR4/MMP-2 protein expression was assessed in vivo by Western blot. Compared to control, *P<0.05. Dates are presented as mean ± SD (n = 3,5,12). Statistical analysis: within group compared to control, *P<0.05, **P<0.01, ***P<0.001; between groups compared to 5 days miR-31agomir, ###P<0.001.