Neuroscience Letters 494 (2011) 89–93
Contents lists available at ScienceDirect
Neuroscience Letters
journal homepage: www.elsevier.com/locate/neulet
Early changes of microRNAs expression in the dorsal root ganglia following rat
sciatic nerve transection
Songlin Zhou 1 , Bin Yu 1 , Tianmei Qian, Dengbing Yao, Yongjun Wang, Fei Ding ∗ , Xiaosong Gu ∗
Jiangsu Key Laboratory of Neuroregeneration, Nantong University, 19 Qixiu Road, Nantong, JS 226001, PR China
a r t i c l e
i n f o
Article history:
Received 21 December 2010
Received in revised form 19 February 2011
Accepted 23 February 2011
Keywords:
Sciatic nerve injury
microRNA expression
Regeneration
a b s t r a c t
MicroRNAs (miRNAs) are a novel class of small non-coding RNAs that regulate gene expression at the
post-transcriptional level. Here we report early alterations of miRNAs expression following rat sciatic
nerve injury using microarray analysis. We harvested dorsal root ganglia (DRG) tissues and identified
19 miRNAs that showed significant changes at four early time points after sciatic nerve transection.
Subsequently, miR-188 and miR-500 microarray results were verified by real-time quantitative reverse
transcriptase polymerase chain reaction (qRT-PCR). The bioinformatics analysis indicated that the potential targets for these miRNAs were involved in the intracellular signaling cascade, the regulation of signal
transduction, the regulation of cellular process and the response to cAMP that were known to play important roles in mobilizing the inherent capacity for neurite outgrowth and promoting regeneration during
the early phase of sciatic nerve injury. Our results show that abnormal expression of miRNAs may contribute to illustrate the molecular mechanisms of nerve regeneration and miRNAs are potential targets
for therapeutic interventions that may enhance intrinsic regenerative ability.
© 2011 Elsevier Ireland Ltd. All rights reserved.
The peripheral nervous system (PNS), differing from the central
nervous system (CNS), has the intrinsic capacity to regenerate. Previous studies have demonstrated that severed peripheral nerves
are able to re-grow and re-connect to their targets, even if their
previous functions were seriously compromised [5]. The sciatic
nerve is a commonly used model for regeneration studies, comprising a mixed population of motor and sensory axons. As we know,
nerve regeneration is a complex biological phenomenon incorporating multiple cells, growth factors and an extracellular matrix
[6,27,28]. Although sciatic nerve regeneration has been studied
for many decades, many studies have attached importance to the
roles of Schwann cells or how peripheral nerves repair after crush
injuries [23,33], and much less is understood about nerve regeneration after complete transection. In particular, the crucial regulators
that determine neural survival and trigger axon regeneration during the early phase of sciatic nerve injury remain largely unclear.
Hence, dissecting these events is key not only to the development
of therapeutic strategies for the improvement of nerve regeneration, but also to the understanding of basic principles governing the
biology of tissue development.
MiRNAs are attractive candidates as upstream regulators,
because miRNAs can post-transcriptionally regulate the entire set
∗ Corresponding authors. Tel.: +86 513 85051801; fax: +86 513 85511585.
E-mail addresses: [email protected] (F. Ding), [email protected]
(X. Gu).
1
Both authors contributed equally to this work.
0304-3940/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.neulet.2011.02.064
of genes [14]. MiRNAs are endogenous, non-coding 21- to 23nucleotide small RNA molecules that regulate gene expression by
binding to the 3 untranslated region of target mRNAs, leading to
their translational inhibition or degradation [11]. A number of miRNAs were found in the mammalian CNS and PNS, such as the brain,
spinal cord and DRG, where they play key roles in neuronal development [8,10,21]. Recently, several studies have suggested the
possibility of miRNA involvement in neurological diseases [2,11]. To
date, however, no reports are available on early alterations of miRNAs in DRG after sciatic nerve injury. To obtain new insights into the
effects of miRNAs, this study was designed to investigate whether
miRNAs are capable of mobilizing the intrinsic capacity for neurite
outgrowth and promoting axon regeneration through analyzing the
early changes in miRNA expression in the DRG, following sciatic
nerve transection, using microarray and bioinformatics analysis.
Thirty-six adult, male Sprague–Dawley (SD) rats (180–220 g,
supplied by the Experimental Animal Center of Nantong University)
were randomly divided into six groups of six rats each. Each animal was anaesthetized by an intraperitoneal injection of complex
narcotics (85 mg/kg trichloroac etaldehyde monohydrate, 42 mg/kg
magnesium sulfate, 17 mg/kg sodium pentobarbital), and the sciatic nerve was exposed and lifted through an incision on the lateral
aspect of the mid-thigh of the left hind limb. A 1 cm long segment of
sciatic nerve was then resected at the site just proximal to the division of tibial and common peroneal nerves, and the incision sites
were then closed. To minimize the discomfort and possible painful
mechanical stimulation, the rats were housed in large cages with
sawdust bedding after the surgery. L4-6 DRGs were collected at
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S. Zhou et al. / Neuroscience Letters 494 (2011) 89–93
Table 1
Altered microRNA expression following sciatic nerve resection.
(A) 1 h
miRNA
Upregulated
miR-188
miR-134
miR-194
miR-376a*
miR-29a*
miR-499
Downregulated
miR-328
(B) 3 h
Fold change
P value
miRNA
Fold change
P value
10.2
5.6
3.9
2.2
1.9
1.6
<0.001
<0.01
<0.001
<0.05
<0.05
<0.05
miR-188
miR-134
miR-135a
miR-499
9.9
5.5
3.0
1.5
<0.001
<0.001
<0.05
<0.05
1.4
<0.01
miR-30c-1*
miR-500
4.9
1.3
<0.01
<0.05
(C) 6 h
(D) 9 h
miRNA
Fold change
P value
miRNA
Fold change
P value
Upregulated
miR-499
1.5
<0.001
miR-188
miR-134
miR-493
miR-212
miR-145
miR-376a
miR-324-3p
14.0
10.3
6.4
2.2
1.8
1.5
1.5
<0.001
<0.001
<0.01
<0.05
<0.05
<0.01
<0.05
Downregulated
miR-296*
miR-500
6.2
1.4
<0.05
<0.05
miR-30c-1*
miR-500
miR-542-3p
miR-142-3p
let-7e
8.3
1.5
1.4
1.3
1.3
<0.05
<0.05
<0.05
<0.05
<0.05
0 h, 1 h, 3 h, 6 h, 9 h and 12 h after injury, respectively. The experiment was repeated three times. All the experimental procedures
involving animals were conducted in accordance with Institutional
Animal Care guidelines and approved ethically by the Administration Committee of Experimental Animals, Jiangsu Province, China.
For miRNA microarray assay, total RNA was extracted using the
mirVanaTM miRNA Isolation Kit (Ambion, Austin, TX) according
to the manufacturer’s instructions. The labeling and hybridization
were performed at the Shanghai Biochip Company, according to
the protocols in the Agilent miRNA microarray system. Agilent
Scan Control software was used for scanning the microarray slides,
and Agilent Feature Extraction software version 9.5.3 was used for
image analysis. Microarray data were analyzed using GeneSpring
GX v11.0 software (Agilent Technologies, Santa Clara, CA). The
data were analyzed statistically using the two-sample independent groups t test, and differences were considered statistically
significant at P < 0.05.
Real-time qRT-PCR assays were performed using a TaqMan
miRNA assay kit (Applied Biosystems, Foster City, CA) on RNA from
the DRG of rats killed at 0 h, 1 h, 3 h, 6 h, 9 h and 12 h after injury.
TaqMan microRNA assays (Applied Biosystems) that included specific RT primers and TaqMan probes were used to quantify the
expression of mature miRNAs. Real-time qRT-PCR was performed
with the 7300 real-time PCR system (Applied Biosystems). The
relative expression of each miRNA was calculated using the comparative 2−Ct method and was normalized using RNU6B mature
miRNA. All data were expressed as means ± S.D.
Two types of miRNA target prediction software, TargetScan (http://www.targetscan.org), and miRanda (http://www.
microrna.org/microrna/home.do), were used to predict the target
genes of two specifically expressed miRNAs. The intersection of
these two datasets was used as the prediction results of the target
genes of two miRNAs. Using GO to validate the target prediction
is one of the most biologically relevant approaches for indicating
the functional coherence of target genes. The GO package in R
http://www.r-project.org/ was used to annotate the functions of
the miRNA targets. In detail, two-sided Fisher’s exact test was used
to classify the GO category, and the type I error was calculated to
correct the P value.
We examined the expression of 350 Rattus norvegicus-miRNAs
based on Version 10.0 of the Sanger miRBase (Sanger Institute,
Cambridge, U.K.; http://microrna.sanger.ac.uk/sequences) in the
DRG after sciatic nerve amputation. Microarray analysis revealed
that a total of 19 miRNAs showed significant expressional changes
between the experimental specimens (at 1 h, 3 h, 6 h and 9 h) and
the control group (at 0 h) (Table 1), 6 miRNAs were upregulated and
1 miRNAs was downregulated at 1 h (Table 1A), 4 miRNAs upregulated and 2 miRNAs downregulated at 3 h (Table 1B), 1 miRNA
upregulated and 2 miRNAs downregulated at 6 h (Table 1C), 7 miRNAs upregulated and 5 miRNAs downregulated at 9 h (Table 1D).
Notably, miR-188 was significantly upregulated at most time
points, while, in contrast, miR-500 was visibly downregulated at
most time points. To validate the microarray platform, we assessed
the expression of one downregulated (miR-500) and one upregulated (miR-188) miRNA by real-time qRT-PCR. The results showed
the time course of changes in the expression of miR-188 and miR500 (Fig. 1). MiR-188 was promptly elevated at 1 h after sciatic
nerve injury, and then leveled off with significantly higher values,
compared to that for the control group, throughout a period of 12 h.
In contrast, miR-500 declined at 3 h after sciatic nerve injury, showing significantly lower values than those for control group over
a period of 12 h. Theses results suggest that the microarray data
were reliable to warrant further analysis. Although the change in
the expression of miR-188 was not identified at 6 h, compared to
the control group, in microarray analysis, this was likely the result
of false-negatives in microarray hybridization in that the result
of real-time qRT-PCR clearly indicated the obviously upregulated
expression of miR-188.
To analyze the roles of miR-188 and miR-500 following rat
sciatic nerve transection, potential downstream targets for them
S. Zhou et al. / Neuroscience Letters 494 (2011) 89–93
91
Fig. 1. Summary of qRT-PCR analysis of the expression of mature miR-188 and miR-500. (A) The expression of miR-188 significantly increased at 1 h after sciatic nerve injury,
and then leveled off with significantly higher values compared to that for control group throughout a period of 12 h. (B) The expression of miR-500 significantly decreased
at 3 h after sciatic nerve injury and remained significantly lower than that in the control group thereafter. Error bars indicate standard deviation.
were predicted by integrating two public databases (TargetScan
and miRanda). Subsequently, GO function enrichments were performed by importing the predicted 240 and 217 target genes from
miR-500 and miR-188, respectively. The top five important GO
functions (regulation of protein kinase cascade; positive regulation
of cellular process; intracellular signaling cascade; positive regulation of signal transduction; positive regulation of transcription
from the RNA polymerase II promoter) were significantly enriched
by the targets of miR-188. The five most important GO functions
(SMAD protein signal transduction; intracellular signaling cascade;
2-oxoglutarate metabolic process; positive regulation of biological process; response to cAMP) were significantly enriched by the
targets of miR-500 (Table 2).
Recent evidence suggests that the expression of at least 20–30%
of human protein-coding genes is modulated by miRNAs. A single miRNA has the potential to target hundreds of distinct mRNA
molecules, and one mRNA molecule can be regulated by multiple
miRNAs [13]. Although many studies have indicated that miRNAs
are associated with multiple human cancers and can be used in
cancer diagnosis [16], the effects of miRNA in non-oncologic diseases are not yet understood. The related research has just begun.
Two very recent studies have demonstrated that the miRNA expression profile were significantly altered in the spinal cord injury (SCI)
model of adult rats [15,19]. MiR-124a expression was significantly
decreased at 1 d after SCI. In contrast, the expression of miR-223
was significantly increased at 6 and 12 h after SCI. The expression of
muscle-specific miRNAs are significantly altered after sciatic nerve
denervation and reinnervation, and miR-206 determines the fiber
type after peripheral nerve regeneration [9]. Intriguingly, miR-206
also delays amyotrophic lateral sclerosis progression by sensing
motor neuron injury and promoting the compensatory regeneration of neuromuscular synapses in mice [35]. It is, therefore,
important to uncover the alteration in early expression of a large set
of miRNAs following peripheral nerve injury and elucidate the role
of miRNAs in mobilizing intrinsic capacity for neurite outgrowth
and promoting the regeneration of peripheral nerves, which has
not been reported previously.
In this study, high-throughput miRNA microarray technology
with 350 miRNAs was used to detect miRNA expression in DRG
tissues following sciatic nerve injury. Real-time qRT-PCR analysis
verified the results of the microarray study and showed that the
microarray data were consistent and reliable. We demonstrated
that 19 miRNAs were significantly deregulated after sciatic nerve
injury, 12 upregulated and 7 downregulated (Table 1). These altered
miRNAs with a diversity of functions may affect a large number of
neuronal genes [15].
Based on the results of the miRNA microarray and real-time
qRT-PCR, we selected the two successively deregulated miRNAs
(miR-188 and miR-500) for further study. Until now, very little
research has been available with regard to these two, small noncoding RNAs. MiR-188 was first characterized in the cardiovascular
diseases, where it was documented that miR-188 was involved
in homocysteine-induced cardiac remodeling and downregulated
by the homocysteine-induced oxidative stress [17]. Furthermore,
miR-188 in the smooth muscle cells of the human airway is downregulated by a proinflammatory stimulus and thought to act as
a regulator of the inflammatory response [12]. Intriguingly, miR188 could affect TNF-related apoptosis-inducing ligand (TRAIL)
induced apoptotic pathways through blocking caspase-3 activation
[4,26], and TNF-alpha/TNF-alpha receptor is an important mediator of apoptosis, which is upregulated in the DRG after peripheral
nerve injury [25]. In our experiment, miR-188 was significantly
upregulated at most time points after sciatic nerve injury, which
implied that DRG neurons could regulate apoptosis, keep neuronal survial, and mobilize growth potential, thus establishing an
important prerequisites for regenaration during the early phase of
sciatic nerve injury [18,20]. MiR-500 has been reportedly expressed
specifically in the central nervous system and is considered a key
player in the development of the left hand side [34]. In addition, miR-500 significantly downregulates neurokinin-1 receptors
in bladder pain syndrome patients [29]. Briefly, miR-188 and miR500 may play key roles in nervous system development and the
regulation of some cellular process, but its function remains largely
unclear.
Table 2
Top five significant GO functions for microRNA targets.
GO ID
miR-188
GO:0010627
GO:0048522
GO:0007242
GO:0009967
GO:0010552
miR-500
GO:0060395
GO:0007242
GO:0006103
GO:0048518
GO:0051591
GO terms
P value
Corrected P value
Regulation of protein kinase cascade
Positive regulation of cellular process
Intracellular signaling cascade
Positive regulation of signal transduction
Positive regulation of specific transcription from RNA polymerase II promoter
3.19E−06
3.56E−06
1.18E−05
3.29E−05
3.77E−05
0.003
0.003
0.007
0.013
0.013
SMAD protein signal transduction
Intracellular signaling cascade
2-oxoglutarate metabolic process
Positive regulation of biological process
Response to cAMP
8.76E−06
1.17E−05
2.17E−05
3.13E−05
8.75E−05
0.009
0.009
0.011
0.012
0.023
92
S. Zhou et al. / Neuroscience Letters 494 (2011) 89–93
Furthermore, targets of miR-188 and miR-500 were mapped
to gene function databases. This approach clearly illustrated that
specifically expressed miRNAs and their targets perform integrated
regulatory functions. The analysis indicated that the potential
targets for these miRNAs were mainly involved in the intracellular signaling cascade, the regulation of signal transduction, the
regulation of cellular process and the response to cAMP. For example, the “positive regulation of cellular process and intracellular
signaling cascade” could be affected by processes that modulate the extent of oxidative stress and inflammatory response.
Intriguingly, previous studies have demonstrated that stimulating
cAMP signaling increased the intrinsic growth capacity of injured
sensory axons [22]. In addition, Smads have been identified as
mediators of intracellular signal transduction by members of the
transforming growth factor-beta superfamily. Following receptor
activation, Smads are subsequently translocated into the nucleus,
where they are thought to play an important role in gene transcription [7]. Moreover, Smads are upregulated in the DRG and
denervated Schwann cells after sciatic nerve injury [3,24,31]. Taken
together, these results imply that the deregulation of miR-188 and
miR-500 may elucidate molecular mechanisms of injured sensory
axons mobilizing the intrinsic growth capacity at an early stage
after sciatic nerve injury. Further studies are, therefore, necessary to test the predicted targets, such as c-jun and calmodulin 1,
that are known to play important roles in mobilizing the inherent capacity for neurite outgrowth and promoting regeneration
during the early phase of sciatic nerve injury [1,32], which are
respectively within the predicted targets of miR-188 and miR500.
The miRNA-based array screening revealed additional 17 deregulated miRNAs besides miR-188 and miR-500; however their
functions have not been fully elucidated by previous studies. Out
of these miRNAs, miR-145 and miR-134 were significantly upregulated at one and three time points after sciatic nerve injury,
respectively. MiR-145, as well as miR-188, is known to affect TRAIL
induced apoptotic pathways through blocking caspase-3 activation [4,26]. MiR-134 is distributed in a punctate pattern within
dendrites and near synapses. Its overexpression cause significant
decrease in spine volumes whereas its inhibition result in an
opposite effect. Moreover, miR-134 targets lim-domaincontaining
kinase 1 mRNA, a direct regulator of actin filament dynamics that
is important for spine remodeling [10,30]. Perhaps, these altered
miRNAs might be responsible for the common morphological and
functional changes in neurons after axotomy, such as cell survival
and neurite remodeling, through different target molecules and distinct pathways. The underlying mechanism, however, needs to be
further explored.
To our knowledge, this is the first study of early changes in
miRNA expression in DRG after transection of the sciatic nerve;
however, the functions and cell-type distributions of most of the
miRNAs were not clear. These will be studied in detail in the future.
These findings nonetheless suggest that miRNAs may critically contribute to the triggering of nerve regeneration and may, therefore,
be potential targets for therapeutic intervention following sciatic
nerve injury. Mechanism studies of miRNAs regulating the intrinsic capacity for neurite outgrowth may contribute to identifying
the triggers of nerve regeneration and elucidating the molecular
mechanisms responsible for nerve regeneration.
Acknowledgments
This study was supported by the Hi-Tech Research and Development Program of China (863 Program, grant no. 2006AA02A128),
the National Natural Science Foundation of China (grant no.
30870811), the Jiangsu Provincial Natural Science Foundation
(grant no. BK2008010), and the Basic Research Program of jiangsu
Education Department (grant no. 08KJA310002). We thank professor Jie. Liu for help in manuscript revision.
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