The present study was approved by the Administration Committee of Experimental Animals in (Jiangsu, China; no. 20170302-016). Sprague Dawley (SD) rats were obtained from the Experimental Animal Center (animal license nos. SCXK (Su) 2014-0001 and SYXK (Su) 2012-0031). A total of 30 adult male SD rats (weight, 180–220 g; age, 2 months) were randomly separated into 5 groups (at 0, 1, 4, 7 and 14 days after nerve injury) with 6 rats/group, and used for sciatic nerve transection as previously described (24). Briefly, after anesthesia, 10 mm of rat sciatic nerve was exposed, lifted, and resected at the site proximal to the division of tibial and common peroneal nerves. After the surgical incisions were closed, animals were housed in temperature- and humidity-controlled environment, maintained under a 12-h light/dark cycle, and were allowed free access to water and food. Rats were sacrificed by decapitation at 0, 1, 4, 7 and 14 days after nerve transection, and 5 mm of proximal sciatic nerve segments were collected for subsequent reverse transcription-quantitative polymerase chain reaction (RT-qPCR) experiments. A total of 60 neonatal male SD rats were used for Schwann cell isolation as previously described (25). Briefly, after anesthesia, sciatic nerve was exposed, and an 8 mm of nerve segment was collected for subsequent cell isolation.

Primary Schwann cells were isolated from sciatic nerve segments using trypsin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Isolated cells were purified by the treatment of anti-Thy1.1 antibody (1:1,000; cat. no. M7898; Sigma-Aldrich; Merck KGaA) and rabbit complement (Sigma-Aldrich; Merck KGaA), as previously described (25). Purified Schwann cells were grown in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific Inc.), 1% penicillin and streptomycin (Invitrogen; Thermo Fisher Scientific Inc.), 2 µM forskolin (Sigma-Aldrich; Merck KGaA), and 10 ng/ml human heregulin-β1 (HRG; Sigma; Merck KGaA). Schwann cells were cultured at 37°C in a humidified atmosphere containing 5% CO₂, and were passaged for no more than three times prior to use. Cultured Schwann cells had a purity >98% confirmed via immunostaining with S100β (1:200; cat. no. ab52642; Abcam, Cambridge, MA, USA), a marker of Schwann cells. For cell transfection, Schwann cells were transfected with a miR-sc6 mimic (sense, 5′-UGGGGGACAGGGCAAGGUGG-3′ and antisense, 5′-CCACCUUGCCCUGUCCCCCA-3′), mimic (sense, 5′-UUCUCCGAACGUGUCACGU-3′ and antisense, 5′-ACGUGACACGUUCGGAGAA-3′), miR-sc6 inhibitor (sequence, 5′-CCACCUUGCCCUGUCCCCCA-3′) or inhibitor control (sequence, 5′-ACGUGACACGUUCGGAGAA-3′) (Guangzhou RiboBio Co., Ltd., Guangzhou, China) using Lipofectamine^® RNAiMAX transfection reagent (Invitrogen; Thermo Fisher Scientific Inc.), according to the manufacturer's protocols. Briefly, the miR-sc6 mimic, mimic control, miR-sc6 inhibitor or inhibitor control was diluted in Opti-MEM I Medium (Gibco; Thermo Fisher Scientific Inc.). Lipofectamine^® RNAiMAX was then added into the corresponding diluted complexes and incubated for 10–20 min at room temperature. The transfection mixture containing the reagent and miRNA diluted in Opti-MEM was then added onto the Schwann cells at 30–50% confluence, which were then incubated at 37°C under 5% CO₂ for 24 h. The experiment was repeated three times.

Schwann cells were suspended and seeded at a density of 2×10⁵ cells/ml onto 96-well plates precoated with 0.01% poly-L-lysine (Sigma; Merck KGaA), and transfected with miR-sc6 mimic, mimic control, miR-sc6 inhibitor or inhibitor control for 36 h. EdU (Guangzhou RiboBio Co., Ltd., Guangzhou, China) was then added into culture media and cells were cultured for another 24 h. The cells were then fixed with 4% paraformaldehyde (PFA; Xilong Scientific, Guangzhou, China) for 30 min at room temperature, stained with Hoechst 33342 (Beyotime Institute of Biotechnology, Haimen, China) for 10 min at room temperature, and observed with a DMR fluorescence microscope (magnification, ×200; Leica Microsystems GmbH, Wetzlar, Germany). The proliferation rate was calculated by dividing the number of EdU-positive cells to the number of total cells. The experiment was repeated three times.

Schwann cells were transfected with miR-sc6 mimic, mimic control, miR-sc6 inhibitor or inhibitor control. Following 36 h of transfection, the cells were suspended in DMEM, and seeded at a density of 3×10⁵ cells/ml onto the top chamber of a 6.5 mm transwell insert with 8 µm pores (Costar; Corning Incorporated; Corning, NY, USA). A total of 500 µl DMEM supplemented with 10% FBS were added to the bottom chamber of transwell pre-coated with 10 µg/ml fibronectin. Schwann cells left on the top chamber were wiped off by a cotton swab after a 24 h incubation at 37°C with 5% CO₂. The Schwann cells that migrated to the lower chamber were fixed with 4% paraformaldehyde for 30 min at room temperature, stained with 0.1% crystal violet for 10 min at room temperature and then observed with a DMR inverted microscope (magnification, ×200; Leica Microsystems GmbH). Crystal violet staining the Schwann cells was dissolved by acetic acid. The migratory ability of Schwann cells was calculated by the optical density value of the dissolved crystal violet dye. The experiment was repeated three times.

The 3′-UTR of netrin-1 (Ntn1), erbB2 receptor tyrosine-protein kinase 4 (ErbB4) or protein kinase C α (Prkcα) was amplified and subcloned into the downstream region of the luciferase gene stop codon in the luciferase reporter vector to generate the p-Luc-UTR reporter plasmid (Promega Corporation, Madison, WI, USA). A total of 120 ng constructed p-Luc-UTR reporter plasmid was combined with 20 pmol miR-sc6 mimic and 20 ng Renilla luciferase vector pRL-CMV (Promega Corporation) to generate a mixture. On the day of the assay 293 cells were seeded onto 24-well plates and then transfected with the mixture using the Lipofectamine 2000 transfection system (Invitrogen; Thermo Fisher Scientific, Inc.), and subsequently cultured for an additional 24 h. The firefly and Renilla luciferase activities were measured using the dual-luciferase reporter assay system (Promega Corporation).

RNA samples were isolated from proximal sciatic nerve segments using TRIzol (Life technologies; Thermo Fisher Scientific, Inc). Total RNA was reverse transcribed into cDNA using Omniscript^® Reverse Transcription Kit (Qiagen GmbH, Hilden, Germany). RT-qPCR experiments were conducted using an Applied Biosystems StepOne real-time PCR System and QuantiNova™ SYBR^® Green PCR Kit (Qiagen GmbH). The following primer pairs were used for qPCR: ErbB4 forward, 5′-GCATGTGATGGAATCGGCAC-3′ and reverse, 5′-TCCCCATGAATGCCAGTGAC-3′. The thermocycling conditions were as follows: Initial denaturation for 5 min at 95°C; 40 cycles of denaturation for 30 sec at 95°C, annealing for 45 sec at 60°C, and extension for 30 sec at 72°C; and final extension for 5 min at 72°C. The expression levels of ErbB4 were determined using the 2^−ΔΔCq method (26), and were normalized to the internal reference gene GAPDH. The relative expression levels of ErbB4 at 1, 4, 7 and 14 days after sciatic nerve transection were compared with its expression levels at 0 day.

The 3′-UTRs of all genes in the rat genome were downloaded, and sequence alignment was conducted using Basic Local Alignment Search Tool (BLAST) (24). The candidate target genes of miR-sc6 were predicted using TargetScan. Functional genes associated with cell proliferation and migration were examined using Database for Annotation, Visualization, and Integrated Discovery (DAVID) (27) bioinformatic resources. The minimum free energy of hybridization between miR-sc6 and the candidate target genes was determined using the RNAhybrid software (bibiserv.cebitec.uni-bielefeld.de/rnahybrid) (28).

Statistical analyses were conducted using GraphPad Prism 5.0 (GraphPad Software, Inc., La Jolla, CA). Three independent experiments were performed, and the results were presented as mean ± standard error of the mean. Student's t-test or one-way analysis of variance followed by Dunnett's multiple comparisons test was used for statistical comparison. P<0.05 was considered to indicate a statistically significant difference.

Schwann cells are the main cell types in the sciatic nerve stumps and play important roles during peripheral nerve regeneration (29), therefore, the effect of miR-sc6 expression on Schwann cell proliferation was investigated. Transfection using a miR-sc6 mimic or miR-sc6 inhibitor significantly increased or decreased the expression of miR-sc6, respectively (Fig. 1A). EdU assay was used to determine the effect of miR-sc6 upregulation/downregulation on the proliferative ability of Schwann cells. Schwann cells transfected with miR-sc6 mimic after 36 h exhibited a higher ratio of the number of EdU-positive cells to the number of total cells, compared with the negative control group (Fig. 1B). The results revealed that transfection using miR-sc6 mimics led to a significantly elevated proliferation rate (Fig. 1B). Schwann cells were also transfected with a miR-sc6 inhibitor or its corresponding negative control. Transfection with the miR-sc6 inhibitor led to a significantly reduced proliferation rate compared with control (Fig. 1C).

To examine the effect of miR-sc6 on Schwann cell migration, Transwell migration assay was performed. Schwann cells transfected with miR-sc6 mimic showed an increased number of migrated cells compared with those transfected with mimic control, suggesting that an increased expression of miR-sc6 promoted the migration of Schwann cells (Fig. 2A). However, transfection with the miR-sc6 inhibitor significantly decreased the migration of Schwann cells (Fig. 2B).

Considering that miRNAs perform their biological functions through the direct regulation of their target genes (30), the candidate target genes of miR-sc6 were predicted. Bioinformatic analysis suggested that ErbB4, Ntn1 and Prkcα were potential target genes of miR-sc6. The sequences of ErbB4, Ntn1 and Prkcα in the seed region were also completely complementary to that of miR-sc6 (Fig. 3A). All three candidate target genes displayed negative free energy as determined by using the RNAhybrid software (Fig. 3A). The results revealed that ErbB4 has a free energy between −20 and −30 kcal/mol, indicating that ErbB4 mRNA may bind with miR-sc6.

The expression association between miR-sc6 and its potential target genes ErbB4, Ntn1 and Prkcα were determined using previously obtained Solexa sequencing and microarray data (24,31). The heatmap of the temporal expression patterns of miR-sc6 revealed upregulation, while, the expression of ErbB4, Ntn1 and Prkcα were downregulated after sciatic nerve transection. The results obtained on day 7 revealed that the expression pattern of miR-sc6 may be negatively associated with that of ErbB4, Ntn1 and Prkcα (Fig. 3B).

Renilla luciferase assay was performed to investigate further whether miR-sc6 directly targets the 3′-UTR of ErbB4, Ntn1 and Prkcα. Transfection using miR-sc6 mimic and p-Luc-UTR reporter plasmid containing the 3′-UTR of ErbB4 showed a significantly reduced relative luciferase activity compared with cells transfected with its corresponding non-targeting negative control (Fig. 4A). In contrast, 293 cells transfected with miR-sc6 mimic and p-Luc-UTR reporter plasmid containing the 3′-UTR of Ntn1 or Prkcα did not show an altered relative luciferase activity (Fig. 4A).

The expression levels of ErbB4 in the proximal nerve segments at 0, 1, 4, 7 and 14 days after sciatic nerve transection were measured to investigate further the association between miR-sc6 and its candidate target gene ErbB4. Previous results from Solexa sequencing showed that miR-sc6 was upregulated after sciatic nerve transection, reaching a peak value at 7 days after nerve injury (24). The temporal expression patterns of ErbB4 at different timepoints after sciatic nerve transection displayed opposite trends. The expression levels of ErbB4 were decreased at 1, 4, 7 and 14 days after nerve injury, reaching the lowest value at 7 days (Fig. 4B). These results collectively suggest that ErbB4 might be the direct target of miR-sc6.