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, 2019, 9610687
eCollection

MicroRNA-219 Inhibits Proliferation and Induces Differentiation of Oligodendrocyte Precursor Cells After Contusion Spinal Cord Injury in Rats

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MicroRNA-219 Inhibits Proliferation and Induces Differentiation of Oligodendrocyte Precursor Cells After Contusion Spinal Cord Injury in Rats

Fang Li et al. Neural Plast.

Abstract

MicroRNA-219 (miR-219) regulates the proliferation and differentiation of oligodendrocyte precursor cells (OPCs) during central nervous system (CNS) development. OPCs only differentiate into oligodendrocytes (OLs) in the healthy CNS, but can generate astrocytes (As) after injury. We hypothesized that miR-219 may modulate OPC proliferation and differentiation in a cervical C5 contusion spinal cord injury (SCI) model. After injury, we observed a decrease in the miR-219 level and quantity of OLs and an increase in the number of OPCs and As. Silencing of miR-219 by its antagomir in vivo produced similar results, but of greater magnitude. Overexpression of miR-219 by its agomir in vivo increased the number of OLs and suppressed generation of OPCs and As. Luxol fast blue staining confirmed that SCI caused demyelination and that the extent of demyelination was attenuated by miR-219 overexpression, but aggravated by miR-219 reduction. Monocarboxylate transporter 1 (MCT-1) may be implicated in the regulation of OPC proliferation and differentiation mediated by miR-219 following contusion SCI. Collectively, our data suggest that miR-219 may mediate SCI-induced OPC proliferation and differentiation, and MCT-1 may participate in this process as a target of miR-219.

Figures

Figure 1
Figure 1
Expression of miR-219 in different groups. (a, b) Relative expression level of miR-219 detected by qRT-PCR over the time course in all groups. Each bar represent the means ± SEM for triplicate experiments. p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. NC: negative control.
Figure 2
Figure 2
Dynamic changes in NG2+ with BrdU+ OPCs, MBP+ oligodendrocytes, and GFAP+ astrocytes after contusion SCI. (a, c) Representative immunostained images of NG2 positive (red) with BrdU positive (green) cells, MBP positive (red) cells, and GFAP positive (green) cells, respectively, between the sham and SCI group. (b, d) Quantitative data of NG2+ with BrdU+ OPCs, MBP+ oligodendrocytes, and GFAP+ astrocytes, respectively, between the sham and SCI group. ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. Scale bar = 50 μm. OPCs: oligodendrocyte precursor cells; MBP: myelin basic protein; GFAP: glial fibrillary acidic protein; BrdU: bromodeoxyuridine.
Figure 3
Figure 3
The effects of miR-219 on the proliferation and differentiation of OPCs (typical images at 7 days). (a, c) Typical images of NG2+/BrdU+ OPCs, MBP+ oligodendrocytes, and GFAP+ astrocytes in the SCI, antagomir-219, agomir-219, antagomir-NC, and agomir-NC groups, with NG2 (red), BrdU (green), DAPI (blue), MBP (red), and GFAP (green). (b, d) The quantitative data of NG2+/BrdU+ OPCs, MBP+ oligodendrocytes, and GFAP+ astrocytes, respectively, at days 3, 7, 10, and 14 in the SCI, antagomir-219, agomir-219, antagomir-NC, and agomirNC groups. p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. Scale bar = 50 μm. OPCs: oligodendrocyte precursor cells; MBP: myelin basic protein; GFAP: glial fibrillary acidic protein; BrdU: bromodeoxyuridine; NC: negative control.
Figure 4
Figure 4
Typical images of Luxol fast blue (LFB) staining for assessment of myelination following contusion SCI. (a) LFB staining for evaluation of myelination indicated demyelination after contusion SCI. (c) The results of LFB staining revealed partial restoration of myelination in animals treated with agomir-219 and aggravated demyelination in the antagomir-219 group (representative images at 3 days after SCI). In addition, no significant group differences were observed between the antagomir-NC or agomir-NC and SCI groups. (b, d) Quantification results of LFB staining. p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. NC: negative control.
Figure 5
Figure 5
Effect of miR-219 on forelimb movement during behavioral tests after C5 hemicontusion. (a) Compared to preinjury, the rats in the SCI group produced marked deficits in rearing performance on days 7, 10, and 14 after SCI (p < 0.001). Rats on days 10 and 14 after SCI in the antagomir-219 group exhibited a more profound decline in usage of the affected paw, whereas a significant increase on day 14 after SCI was observed in the agomir-219 group. (b) A more severe grooming deficit of the ipsilateral paw was observed after contusion injury (p < 0.001). At days 10 and 14 postinjury, animals in the antagomir-219 group achieved lower scores than did those in the SCI group, whereas only on day 14 postinjury the rats in the agomir-219 group achieved higher scores than did the SCI group. Error bars indicate SEM. p < 0.05. NC: negative control.
Figure 6
Figure 6
Expression of MCT-1 in different groups. (a, b) Relative expression level of MCT-1 mRNA detected by qRT-PCR over the time course in all groups. (c, d) MCT-1 protein level of different groups at all time points was detected by western blotting. Each bar represent the means ± SEM for triplicate experiments. p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. MCT-1: monocarboxylate transporter 1; NC: negative control.

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