miR-30c promotes Schwann cell remyelination following peripheral nerve injury

Abstract

Differential expression of miRNAs occurs in injured proximal nerve stumps and includes miRNAs that are firstly down-regulated and then gradually up-regulated following nerve injury. These miRNAs might be related to a Schwann cell phenotypic switch. miR-30c, as a member of this group, was further investigated in the current study. Sprague-Dawley rats underwent sciatic nerve transection and proximal nerve stumps were collected at 1, 4, 7, 14, 21, and 28 days post injury for analysis. Following sciatic nerve injury, miR-30c was down-regulated, reaching a minimum on day 4, and was then upregulated to normal levels. Schwann cells were isolated from neonatal rat sciatic nerve stumps, then transfected with miR-30c agomir and co-cultured in vitro with dorsal root ganglia. The enhanced expression of miR-30c robustly increased the amount of myelin-associated protein in the co-cultured dorsal root ganglia and Schwann cells. We then modeled sciatic nerve crush injury in vivo in Sprague-Dawley rats and tested the effect of perineural injection of miR-30c agomir on myelin sheath regeneration. Fourteen days after surgery, sciatic nerve stumps were harvested and subjected to immunohistochemistry, western blot analysis, and transmission electron microscopy. The direct injection of miR-30c stimulated the formation of myelin sheath, thus contributing to peripheral nerve regeneration. Overall, our findings indicate that miR-30c can promote Schwann cell myelination following peripheral nerve injury. The functional study of miR-30c will benefit the discovery of new therapeutic targets and the development of new treatment strategies for peripheral nerve regeneration.

Keywords: Schwann cells; dedifferentiation; in vitro; in vivo; miR-30c; miRNAs; myelination; nerve regeneration; neural regeneration; peripheral nerve injury; peripheral nerve regeneration; sciatic nerve.

Figure 1

Changes in miRNA expression in proximal nerve stumps following sciatic nerve transaction (A) The expression patterns of six miRNAs in pattern 9 were obtained after bioinformatic analysis at 0, 1, 4, 7, and 14 days post sciatic nerve transection. Red: miR-181c; purplish red: miR-20b-5p; light green: miR-30e; dark green: miR-30c; sky blue: miR-301a; dark blue: miR-338. (B) The regulatory network among miRNAs in pattern 9 and their 347 target genes. Green and red nodes indicate miRNA and mRNA, respectively. (C) Quantitative real-time PCR of miR-30c expression showing initial downregulation in proximal nerve stumps following sciatic nerve transaction and then gradual upregulation to near normal levels. miRNA: MicroRNA; d: day(s).

Figure 2

Immunostaining of miR-30c-mediated myelination of Schwann cells (A) Transfection with miR-30c agomir elevated myelin basic protein expression in co-cultured dorsal root ganglia and Schwann cells. Quantified results of myelinated segments in co-cultured cells transfected with miR-30c agomir (miR-30c) or agomir control (AC) are shown in the histogram. (B) Transfection with miR-30c inhibitor decreased myelin basic protein expression in co-cultured dorsal root ganglia and Schwann cells. Quantified results of the myelinated segments in co-cultured cells transfected with miR-30c inhibitor (anti-miR-30c) or inhibitor control (anti-con) are shown in the histogram. Blue shows Hoechst 33342 staining of cell nuclei and red represents myelin basic protein immunostaining. Bars: 50 μm. n = triplicate wells from three independent assays. **P < 0.01 (Student's t-test).

Figure 3

miR-30c promoted myelination of injured sciatic nerve in vivo. (A) Schematic depiction of the in vivo miR-30c study. (B) Western blot analysis of P0 and MAG protein levels (myelin-associated proteins) in the sciatic nerve of rats injected with miR-30c agomir (miR-30c) or negative control (AC). (C) Immunohistochemistry of P0 and MAG proteins. Green indicates P0 and red indicates MBP. Bars: 50 μm. (D) Representative transmission electron microscopy photomicrographs and analysis of myelinated nerve fibers. The lower two figures are higher magnifications of the upper two figures. Bars: 5.0 μm in the upper two figures, bars: 500 nm in the lower two figures. *P < 0.05, **P < 0.01 (Student's t-test). MAG: Myelin-associated glycoprotein; GAPDH: glyceraldehyde-3-phosphate dehydrogrnase.

Figure 4

Identification of candidate miR-30c targets. (A) Heatmap of down-regulated mRNAs in response to miR-30c over-expression. The expression levels of mRNAs were determined by cDNA sequencing and mRNAs that were down-regulated more than 2-fold in Schwann cells transfected with miR-30c are listed. (B) The intersection of down-regulated mRNAs and predicted target genes of miR-30c. (C) The list of candidate miR-30c targets.