Entrapment neuropathy results in different microRNA expression patterns from denervation injury in rats

Abstract

Background: To compare the microRNA (miRNA) expression profiles in neurons and innervated muscles after sciatic nerve entrapment using a non-constrictive silastic tube, subsequent surgical decompression, and denervation injury.

Methods: The experimental L4-L6 spinal segments, dorsal root ganglia (DRGs), and soleus muscles from each experimental group (sham control, denervation, entrapment, and decompression) were analyzed using an Agilent rat miRNA array to detect dysregulated miRNAs. In addition, muscle-specific miRNAs (miR-1, -133a, and -206) and selectively upregulated miRNAs were subsequently quantified using real-time reverse transcription-polymerase chain reaction (real-time RT-PCR).

Results: In the soleus muscles, 37 of the 47 miRNAs (13.4% of the 350 unique miRNAs tested) that were significantly downregulated after 6 months of entrapment neuropathy were also among the 40 miRNAs (11.4% of the 350 unique miRNAs tested) that were downregulated after 3 months of decompression. No miRNA was upregulated in both groups. In contrast, only 3 miRNAs were upregulated and 3 miRNAs were downregulated in the denervated muscle after 6 months. In the DRGs, 6 miRNAs in the entrapment group (miR-9, miR-320, miR-324-3p, miR-672, miR-466b, and miR-144) and 3 miRNAs in the decompression group (miR-9, miR-320, and miR-324-3p) were significantly downregulated. No miRNA was upregulated in both groups. We detected 1 downregulated miRNA (miR-144) and 1 upregulated miRNA (miR-21) after sciatic nerve denervation. We were able to separate the muscle or DRG samples into denervation or entrapment neuropathy by performing unsupervised hierarchal clustering analysis. Regarding the muscle-specific miRNAs, real-time RT-PCR analysis revealed an approximately 50% decrease in miR-1 and miR-133a expression levels at 3 and 6 months after entrapment, whereas miR-1 and miR-133a levels were unchanged and were decreased after decompression at 1 and 3 months. In contrast, there were no statistical differences in the expression of miR-206 during nerve entrapment and after decompression. The expression of muscle-specific miRNAs in entrapment neuropathy is different from our previous observations in sciatic nerve denervation injury.

Conclusions: This study revealed the different involvement of miRNAs in neurons and innervated muscles after entrapment neuropathy and denervation injury, and implied that epigenetic regulation is different in these two conditions.

Figure 1

Hierarchical cluster analysis. Using a hierarchical method, a clustering graph was created from those miRNAs with increased (red) or decreased (blue) fold of expression from 3 soleus muscles (left graph) and dorsal root ganglia (right graph) in the group of rats that sustained denervation for 6 months, entrapment for 6 months, or decompression for 3 months against those from the sham control group.

Figure 2

In situ hybridization analysis. In comparison with the negative control (Figure 2A), in situ hybridization analysis revealed that the positive control probe rno-U6 was abundantly and diffusely expressed in the perinuclear region of the neurons in the denervated dorsal root ganglia (DRGs) (Figure 2B). Intense signals for miR-21 in the perinuclear region of the neurons were also observed in the tissue sections at 1 month after denervation injury by using digoxigenin-labeled miR-21 probes (Figure 2C). Real time RT-PCR revealed that the expression of miR-21 in the DRGs was increased by ~6 fold; it was detected 1 week after denervation and lasted for up to 6 months (Figure 2D). Bars represent means ± standard error of 6 independent experiments; *, P < 0.05 vs. sham control.

Figure 3

Expression of muscle-specific miRNAs using quantitative real-time PCR. After entrapment, the expression of miR-1 and miR-133 was significantly decreased to ~50% of those observed in the sham control group at 3 and 6 months after entrapment. After decompression, miR-1 and miR-133a levels were unchanged and sustained a significant decrease at 1 and 3 months later, respectively. There were no statistical differences in the expression of miR-206 at the indicated time points after nerve entrapment and after decompression. Bars represent means ± standard error of 6 independent experiments; *, P < 0.05 vs. sham control.