Neurol Sci DOI 10.1007/s10072-014-1940-0

REVIEW ARTICLE

Identifying the role of microRNAs in spinal cord injury Jun Dong • Meng Lu • Xijing He • Junkui Xu Jie Qin • Zhijian Cheng • Baobao Liang • Dong Wang • Haopeng Li

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Received: 5 April 2013 / Accepted: 6 August 2014 Ó Springer-Verlag Italia 2014

Abstract Spinal cord injury (SCI) is medically and socioeconomically debilitating, and effective treatments are lacking. The elucidation of the pathophysiological mechanisms underlying SCI is essential for developing effective treatments for SCI. MicroRNAs (miRNAs) are small non-coding RNA molecules (18–24 nucleotides long) that regulate gene expression by interacting with specific target sequences. Recent studies suggest that miRNAs can act as post-transcriptional regulators to inhibit mRNA translation. Bioinformatic analyses indicate that the altered expression of miRNAs has an effect on critical processes of SCI physiopathology, including astrogliosis, oxidative stress, inflammation, apoptosis, and neuroplasticity. Therefore, the study of miRNAs may provide new insights into the molecular mechanisms of SCI. Current studies have also provided potential therapeutic clinical applications that involve targeting mRNAs to treat SCI. This review summarizes the biogenesis and function of miRNAs and the roles of miRNAs in SCI. We also discuss the

J. Dong  M. Lu  X. He (&)  J. Qin  Z. Cheng  D. Wang  H. Li The Orthopedics Department, The Second Affiliated Hospital of Xi’an Jiaotong University, no. 157, West Five Road, Xi’an 710004, China e-mail: [email protected] J. Xu The Orthopedics Department, Honghui Hosptial of Xi’an Jiaotong University, no. 76, Nan Guo Road, Xi’an 710054, China B. Liang The Plastic Surgery Department, Second Affiliated Hospital of Xi’an Jiaotong University, no. 157, West Five Road, Xi’an 710004, China

potential therapeutic applications of miRNA-based interventions for SCI. Keywords Spinal cord injury  MicroRNA  Pathophysiology

Introduction Spinal cord injury (SCI) is a major cause of morbidity, with adverse impacts on families and society [1]. The pathogenic mechanism of SCI is commonly separated into primary and secondary mechanisms [1, 2]. Astrogliosis, oxidative stress, inflammation, apoptosis and neuroplasticity are critical elements of SCI pathophysiology that contribute to the functional deficiency of SCI patients [2, 3]. Although the complex pathophysiological processes that follow SCI have been elucidated in recent years, the treatment of SCI remains insufficient [4], and many fatalities occur each year. Nearly 12,000 new cases of paraplegia and quadriplegia resulting from SCI occur in the United States annually; of these, 4,000 die before reaching the hospital, and 1,000 die during hospitalization [2]. The elucidation of the pathophysiological mechanisms underlying SCI is essential for the development of effective SCI treatments. MicroRNAs (miRNAs) are small, non-coding RNA molecules (18–24 nucleotides long) that regulate gene expression by interacting with specific sequences of target mRNAs or promoters [5–7]. miRNAs play vital roles in the homeostatic processes of cell proliferation or cell death [8] and modulate 20–30 % of human protein-coding genes [9]. Nearly 2,000 human genes with miRNA target sites have been reported [10]. Recent studies suggest that miRNAs can act as post-transcriptional regulators to inhibit mRNA translation [11], particularly in the nervous system [12].

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Dysregulation of miRNAs in the central nervous system (CNS) is associated with neurodegenerative disorders, including traumatic injury [13–15]. Bioinformatic analyses indicate that the altered expression of miRNAs has an effect on critical processes of SCI physiopathology [11]. Thus, studies of miRNA function may provide additional insights into the molecular mechanisms of SCI. In this review, we summarize the biogenesis and function of miRNAs and the roles of these small molecules in different SCI-related processes. Current studies of miRNAs for the treatment of SCI are also discussed. Finally, we analyze potential therapeutic applications of miRNA-based interventions for SCI.

The biogenesis and function of miRNA In 1993, Lee et al. [16] discovered that lin-4, a small 21-nucleotide RNA molecule, contained sequences complementary to a repeated sequence element in the 30 untranslated region (30 -UTR) of lin-14 mRNA and that lin4 played a role in lin-14 translation. Similarly, Wightman et al. [17] demonstrated that lin-4 was a heterochronic gene that could regulate the lin-14 30 -UTR, producing a temporal gradient of Lin-14 protein expression in Caenorhabditis elegans. However, another 20 years passed before the importance of these small molecules, miRNAs, was recognized [18]. In 2007, Berezikov and Ruby reported the production and behavior of miRNAs [19, 20]. An *70 nt pri-miRNA is liberated by an RNase ribonuclease, Drosha, together with an interacting partner, DGCR8 [21], to form a pre-miRNA. Mirtrons, the pre-miRNAs encoded in introns, can avoid the above pathway and are exported

into the cytoplasm by exportin V [20, 22]. With the help of the RNase Dicer, the pre-miRNA becomes a mature 18- to 24-nt duplex and is loaded into the RNA-induced silencing complex (RISC) [12]. The duplex then contacts the target mRNAs via base pairing, resulting in cleavage of the targeted mRNA or translational inhibition [12] (Fig. 1).

Pathophysiology and altered gene expression following SCI The pathophysiology of SCI is commonly separated into primary and secondary mechanisms [2]. The primary mechanism encompasses the initial injury caused by impact with persistent compression or impact alone with transient compression, whereas the secondary mechanism comprises a cascade of biochemical and cellular processes that are initiated by the primary process and result in the expansion of the injury site and the limitation of restorative processes. Ultimately, all of these mechanisms lead to demyelination, ischemia, necrosis, and apoptosis of spinal cord tissue, among other processes [1]. Finally, a fluid-filled cavity forms in the center of the spinal cord and is surrounded by hypertrophic astrocytes and macrophages. These and other cells secrete extracellular matrix and inhibitory molecules to form a glial scar, which acts as a barrier to regeneration. Previous studies have demonstrated that altered gene expression significantly contributes to the pathogenesis of secondary SCI [23]. The expression of miRNAs is temporally altered after CNS injury [24]; temporal differences in miRNA expression at different stages of SCI may also occur.

miRNAs regulate critical processes of SCI pathophysiology

Fig. 1 The process of the generation and function of miRNAs. In the nucleus, Drosha/DGCR8 processes the pri-miRNA into the premiRNA. The pre-miRNA is then transported into the cytoplasm via exportin V and is cut into a 21–22 nt duplex by Dicer. After binding to RNA-induced silencing complex (RISC), miRNA base-pairs to the target (mRNA), resulting in cleavage or translational inhibition of the target mRNA

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Damage to neurons and axons after SCI leads to perpetual disability [1, 2]. Because the primary injury is unpredictable and uncontrollable, treatments for SCI generally focus on alleviating the secondary injury to the spinal cord. The secondary mechanism is characterized by astrogliosis, oxidative stress, inflammatory responses, apoptosis, and neuroplasticity [2, 3]. Because miRNAs regulate gene expression after SCI, they might provide new molecular insights as well as potential therapeutic tools to regulate the pathways of neuronal cell death and survival. We will discuss the role of some miRNAs that interact with key processes of the pathophysiology of SCI below. miRNAs regulate astrogliosis following SCI Because astrocytes are one of the primary types of glial cells and are distributed widely in various regions of the

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spinal cord [25], the investigation of the changes in miRNAs in astrocytes may lead to the development of novel interventions that promote recovery. The response of astrocytes following SCI, which is known as astrogliosis, involves two processes: hyperplasia and hypertrophy [26]. Hypertrophy promotes the repair of the damaged blood– brain barrier, whereas hyperplasia facilitates the development of gliosis. These two processes, which are characterized by profound morphological, molecular, and functional changes in astrocytes, contribute to the damage to the spinal cord following the primary injury [27, 28]. Recently, a protective effect of miRNA-21 (miR-21) was revealed for SCI [29–31]. Bhalala et al. [30] observed that astrocytes express a high level of miR-21 in the injured area but a low level in the intact area after SCI. Overexpression of miR-21 in astrocytes reduced the hypertrophic response to SCI, and expression of an miR-21 sponge exacerbated the hypertrophic phenotype. Further, BMPR1a and BMPR1b induce opposite effects on astrocytic hypertrophy via miR-21 [29]. BMPR1a signaling decreases the level of miR-21, which in turn downregulates GFAP levels in astrocytes [29]. Because miR-21 promotes the shift from hypertrophy to hyperplasia in astrocytes, blocking this transition by enhancing BMPR1a signaling or inhibiting miR-21 may be beneficial. miR-146a was reported to be a key regulator of the innate immune response in the modulation of astrocytemediated inflammation [32]. Similar to miR-21, which plays different roles in various diseases, miR-146a is important in other disorders [33]. Gene ontology analysis predicted that miR-21 and miR-146a regulate cell proliferation pathways. The two miRNAs are transiently induced following SCI and act primarily to inhibit cell proliferation and apoptosis via the regulation of transcription and translation [4]. miR-146a also plays a key role in controlling astrocyte activation and function in cell proliferation and apoptosis [34]. Iyer et al. [32] observed greatly increased expression of miR-146a in focal cortical dysplasia (FCD). Three target genes of miR-146a, IRAK-1, IRAK-2, and TRAF-6, were downregulated in association with the overexpression of miR-146a. Increased mRNA levels of IRAK-1, IRAK-2, and TRAF-6 were detected after transfection of LNA anti-miR-146a [32]. Thus, miRNA function provides an opportunity to develop novel therapeutic strategies for neurological disorders. However, the change in miRNA expression in astrocytes is species specific and varies under similar conditions. Mor et al. [35] reported that miRNA function might be more influential on the mechanism controlling astrocyte activation in rodents than in primates, possibly because the pathway of astrocyte activation is different, e.g., lipopolysaccharide (LPS) or INF-c [36, 37]. Species-specific immune systems also contribute to this phenomenon. Some

miRNAs are shared in other immune-related systems between mice and humans, while others are mouse and rat specific [35]. For example, the miR-351 gene, a member of the miR-125 family that performs various roles in inflammation, is found only in mice and rats. In addition, miRNA-gene-identifying algorithms cannot locate the miR-351 gene in any other animal species [35]. miRNAs regulate endogenous antioxidant systems after SCI The increase in reactive oxygen species (ROS) after SCI amplifies the secondary injury by actively recruiting macrophages to the lesion [38]. Altering the microenvironment of the lesion after SCI is thought to stimulate the endogenous repair system to attenuate the secondary injury [39, 40]. The endogenous antioxidant system, which includes glutathione, ascorbic acid, and ROS-scavenging-related enzymes, is modulated by ROS [41]. Neurogenic differentiation 6 (NeuroD6), a factor that has been suggested to be involved in the development and maintenance of the mammalian nervous system [42, 43], regulates thioredoxinlike 1 and glutathione peroxidase 3 to scavenge ROS and thus reduce the oxidization of the lesion after SCI [44]. Infusion of exogenous NeuroD6 into SCI lesions effectively blocked apoptosis, indicating its neuroprotective capacity. Recently, miR-486 was found to be unregulated in motor neurons of spinal cord lesions. Jee et al. [41] determined that the major target of miR-486 in the motor neuron was NeuroD6. They identified a novel miR-486 that could repress NeuroD6 in a mouse model and observed a large increase in the expression of NeuroD6 after injury; the functional deficit was notably decreased after knockdown of miR-486 expression. Thus, inhibition of the expression of miR-486 after SCI might represent a therapeutic strategy [41]. Neurogenin 1 (Ngn1) is essential for neuronal differentiation during embryogenesis [45], and Ngn1-induced neuronal precursor cells express high levels of NeuroD [46]. In another study, Jee et al. [47] focused on miR-20a and found that the re-expression of Ngn1, a key target gene of miR-20a, was apparent in the lesion area of SCI animals after blocking miR-20a, suggesting that miR20a might be a potential target for therapeutic intervention following SCI. Superoxide dismutase (SOD), the antioxidant enzyme defense system, is crucial for the defense against ROS [48]. Several miRNAs that are upregulated in SCI patients, such as miR-1, miR-206, miR-152, and miR-214, are upstream of the SOD1 gene [48, 49]. SOD2 has been reported to be a therapeutic target of miR-145 for remyelination after injury [50]. Bioinformatics analysis has revealed that the rat miR145 sequence contains a complementary 8-bp targeting site in the 30 -UTR of rat SOD2 [51]. Because SOD2 is known

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to play a role in antioxidant defense, its expression after SCI might promote cell survival.

miRNAs involved in inflammation and apoptosis after SCI The inflammatory response after SCI, which involves the activation of resident microglia and the infiltration of neutrophils, monocytes, and lymphocytes [52], leads to secondary damage to the core and penumbral regions of the injury site [53]. The traumatic environment during the acute phase of SCI is dominated by the presence of proinflammatory cytokines, e.g., IL-1 and IL-6. However, SCI also induces the expression of anti-inflammatory cytokines. Bioinformatics analyses have revealed that miRNAs are involved in inflammation after SCI via the targeting of mRNAs that mediate the inflammatory response [54]. Certain miRNAs targeting pro-inflammatory mRNAs, such as miR-181a, miR-411, and miR-99a, are downregulated after SCI [49]. By contrast, miR-221 and miR-1, which target anti-inflammatory mRNAs, are upregulated [49]. miR-223 regulates neutrophils in the early stage after spinal cord injury [55]. The expression of miR-223 is time dependent, and two peaks in expression have been detected at 12 h and 3 days after SCI. However, overall miR-223 expression decreases significantly from the 1st day to the 7th day after SCI [56]. Neutrophil recruitment is delayed because neutrophils appear at the primary lesion site at 4–6 h after injury, peak in number at 12–24 h, and disappear within 5 days [57]. Thus, the change in miR-223 expression may be associated with the inflammatory response and may reflect cell death. IL-6 and Cox-2, two major inflammatory mediators, have been reported to be associated with the activation of the innate immune response under various pathological conditions. miR-146a has been reported to regulate these two inflammatory mediators during chronic inflammation [32]. A high level of miR-146a inhibits the expression of these two mediators, while a low level of miR-146a increases the expression of IL-6 and COX-2 [32]. Apoptosis is an active form of cell death that occurs in neurons, oligodendrocytes, and microglial cells following CNS injury, resulting in neuronal atrophy, post-injury demyelination, and microglial nodule formation [58, 59]. Several miRNAs play roles in apoptosis via inhibition of the expression of pro-apoptotic and anti-apoptotic proteins [60]. In general, after SCI, multiple miRNAs targeting proapoptotic genes are downregulated, and miRNAs targeting anti-apoptotic genes are upregulated [49]. Increasing evidence has revealed that miR-21 is a strong anti-apoptotic factor [60]. The expression of FasL and PTEN, which increases apoptosis, is upregulated when miR-21

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expression is knocked down in a SCI rat model [31]. miR21 is regarded as an attractive therapeutic molecule for the treatment of stroke because overexpression of miR-21 protects against ischemic neuronal death by targeting the Fas ligand gene (FASLG) [61]. The miRNA Let-7a, a proapoptotic factor, positively affects the expression of the anti-apoptotic genes RAS and MYC [62]. Both miR-133b and miR-15b act as pro-apoptotic mediators of cell death by reducing the expression of the anti-apoptotic factor Bcl2 and increasing the expression of caspases 3, 8, and 9 [63, 64]. The abnormal expression of these miRNAs following SCI contributes to inflammation and apoptosis during secondary SCI. Therefore, these miRNAs could be potential targets for therapeutic intervention following SCI. miRNAs regulate neuroplasticity after SCI Studies have demonstrated the occurrence of neuroplasticity in the adult nervous system after SCI [3]. These plasticity changes occur at multiple levels, including in the spinal cord [65]. The process of plasticity involves changes in several factors, such as cytokines and growth factors, and the sprouting of new neural connections, resulting in axon regeneration. miRNAs represent novel targets for promoting repair and regeneration, which are crucial for neural plasticity [66]. Some miRNAs have been reported to play a role in neuroplasticity after SCI by targeting phosphatase and tensin homolog deleted on chromosome ten (PTEN)/mammalian target of rapamycin (mTOR) and RhoA. miRNAs regulate axon regeneration by targeting PTEN/mTOR mTOR, a key factor in intracellular signaling pathways, plays an important role in cell metabolism. Downregulation of mTOR activity has been associated with increasing age. CNS injury has also been shown to lead to a decrease in mTOR activity [67–69]. PTEN, a newly discovered cancer suppressor gene [70], has also recently been proposed as a negative regulator of mTOR. A role of PTEN/mTOR in promoting axon sprouting in neuroplasticity has been demonstrated [71]. Kyungsuk et al. [71] recently demonstrated that the loss of neuronal mTOR activity was a major cause of the lack of regeneration in optic nerve axons after injury and that genetic activation of mTOR could promote successful optic axon regeneration. Liu and Lu established that the PTEN/mTOR pathway regulates the regenerative capacity of corticospinal neurons, and in a PTEN knockout mouse model, axons sprouted from the lesion site of spinal cord [72]. Therefore, modulating the activity of PTEN/ mTOR has been proposed as a potential therapeutic strategy for SCI. Recently, Houle et al. [73] reported that

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exercise contributes to plasticity after SCI via regulation of the expression of miRNAs that specifically target PTEN and mTOR. For example, miR-21 and miR-199-3p expression increased significantly after exercise in a rat model of SCI [73]. Increased expression of miR-21 and decreased expression of miR-199-3p would significantly affect the PTEN/mTOR pathway [73]. In another study, cycling exercise was shown to increase the expression of apoptosis-associated miRNAs such as PTEN, PDCD4, and RAS mRNA [64]. These results indicate that rehabilitation in the form of exercise is an effective method to promote functional recovery after SCI. However, experimental results suggest that extended exercise has no effect on the expression of miRNAs associated with neuroplasticity [74]. Based upon extensive clinical experience, the promotion of functional recovery via simple exercise requires further optimization. miR-133b improves spinal cord function by inhibiting RhoA expression miRNA targets have been identified in fish as well as in humans [75]. Several zebrafish tissues are capable of regenerating after injury [76], and thus, zebrafish are now used as a CNS regeneration model [77]. During regeneration, miR-133b enhances muscle regeneration and reduces the formation of fibrotic tissue [76]. However, it is unclear if miR-133b has the same function in the nervous system. Yu et al. [75] were the first to demonstrate that miR-133b promotes tissue regeneration in the nervous system. miR133b can increase axonal plasticity and promote neurite remodeling in the rat ischemic boundary zone (IBZ) [78]. During the recovery process, miR-133b restrains the expression of the GTPase RhoA, an inhibitor of axonal growth, and regulates the expression of connective tissue growth factor (CTGF) [78]. Thus, the application of miR133b to reduce RhoA protein expression via interaction with its mRNA might promote the recovery of spinal cord function after SCI. In addition, the study by Yu et al. [75] demonstrated that RhoA mRNA is a target of miR-133b. These results indicate potential directions to improve the functional recovery of SCI patients.

Conclusions and perspectives Spinal cord injury dramatically alters the cellular landscape of impacted tissue through the activation of inflammatory pathways, ultimately resulting in further insult by contributing to the pathogenesis of the secondary SCI [4]. Cell death resulting from secondary injury reduces the number of surviving cells adjacent to the core and also leads to substantial biochemical changes far from the site of

primary impact [79]. The dynamic process of the pathological mechanism is accompanied by alterations in gene expression after SCI [23]. Vast evidence that abnormal expression of miRNAs contributes to the pathogenesis of SCI has already been provided [49]. During the progression of SCI, various miRNAs participate in different pathological processes and play important roles in recovering homeostasis [64]. Thus, miRNAs could be used as therapeutic targets as well as biomarkers of the pathological process of SCI. Changes in miRNA expression after SCI are classified into three categories: upregulation, downregulation, and early upregulation, at 4 h, followed by downregulation from the 1st to the 7th day post-SCI [49]. In addition, Yunta and colleagues [11] observed extended miRNA dysregulation, which primarily consisted of increasing numbers of downregulated miRNAs, with only a few upregulated miRNAs. A similar increase in the number of downregulated miRNAs up to 14 days after spinal cord injury was also reported in the study by Strickland [4]. Thus, the alterations in the expression of these miRNAs suggest temporal differences in their expression. Several miRNAs can bind to a specific mRNA, and a single miRNA can bind to several mRNAs. Structural studies have estimated that most miRNAs can act on *200 mRNAs [80]. Thus, most miRNAs regulate more than one target and induce distinct effects on different pathological processes and cell types. For example, miR-21 plays an anti-apoptotic role by targeting FASLG and promoting axon regeneration by targeting PTEN/mTOR. A protective role of miR-21 against ischemic injury in the heart via antiapoptosis has also been demonstrated [81]. Similarly, miR133b enhances cell regeneration in muscular tissue and promotes axonal plasticity in the nervous system. miR133b also improves spinal cord function by inhibiting RhoA expression. However, miR-133b has been demonstrated to be both anti-apoptotic and pro-apoptotic after SCI [64]. We analyzed the involvement of some miRNAs in SCI and summarized their target genes or predicted target genes from various studies (Table 1). However, many of the predicted mRNA targets of identified miRNAs still require verification. The function of some miRNAs also requires further study. For example, the expression of miR-455 is downregulated after SCI [35], but there is a lack of detail regarding the function of this miRNA. Because one miR-455 target is calreticulin (Calr), which modulates hypertrophic growth in pathologic heart [82], miR-455 may also play a role in astrogliosis by targeting Calr following SCI, but this hypothesis requires confirmation. A role of stem cells in the treatment of SCI has been demonstrated [83]. Researchers are primarily focusing their attention on how to promote stem cell differentiation into

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Neurol Sci Table 1 miRNAs function and regulation in SCI Function

miRNA

(Predicted) target gene

Regulation

Animal model

References

Therapeutic target

miR-1

TNFR1 signaling pathwaya

Apoptosis, inflammation, cell motility, cell cycle initiation

SD rat

[4]

MAPK signaling pathwaysa Unidentified

Neuroplasticity

SD rat

[4]

Astrocytic GFAP

Mouse

[29]

Unidentified

Astrogliosis

Transgenic mouse

[30]

FasLa, TNF-aa, PTENa

Apoptosis

Rat

[31]

PTEN/mTORa

Axon regeneration

Rat

[73]

miR-486

NeuroD6a

NeuroD6 expression

Mouse

miR-20a

Neurogenin 1 (Ngn1)a

Motor neuron degeneration

miR-223

Unidentified

Regulate neutrophils

Adult male C57BL/6 mouse

[55]

miR-199a-3p

PTEN/mTORa

Axon regeneration

Rat

[73]

miR-133b

RhoAa

Promoting tissue regeneration

Zebrafish

[75]

SD rat

[64]

Human embryonic stem cell

[72]

Embryonic day 16 (E16) Wistar rat

[86]

miR-124 miR-21

Biomarker

a

a

miR-21

PTEN , PDCD4

Apoptosis

Let-7a

RASa, MYCa

Inhibit RAS and MYC expression

miR-15b miR-16

Bcl-2a

Regulate Bcl-2 expression

miR-145 miR199a-5p

C11Orf9

Oligodendrocyte maturation and myelin production

miR-214

Mobp

Providing the proper structural properties of myelin

miR-205

Cldn11 30 -UTR

Encoding for a transmembrane protein

miR-184

BCL2-like 1a

Regulate astrocyte

miR-1183

Eif2b5

Facilitate astrocyte differentiation

miR-126

Hoxa

Regulate HOXA3

miR-31

Unidentified

Maintaining NSCs in an undifferentiated state

[41] [47]

Currently mirNRA can be used as therapeutic target and biomarker in SCI. The table lists the target gene or predicted target gene and regulation of miRNAs in different animal model SD rat Sprague–Dawley rat a

Validated target gene of miRNA

neurons, repair damaged axons, improve the production of neurotrophic factors and anti-inflammatory mediators, and inhibit the expression of pro-inflammatory mediators. As the mechanisms by which miRNAs interact with mRNA during cell growth, proliferation, and differentiation have become clearer, miRNAs have been reported to intervene in the directional differentiation process of stem cells [72]. Letzen et al. [72] analyzed the expression of miRNAs in oligodendrocytes matured from human embryonic stem cells, revealing that miRNA profiles are key markers of the differentiation process (Table 1). Thus, these miRNAs provide targets to prompt stem cell differentiation after transplantation. Similarly, olfactory ensheathing cell transplantation (OECT) is regarded as an ideal procedure for the treatment

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of SCI. Although there is no report of miRNAs regulating olfactory ensheathing cells (OECs), regulating related miRNAs in transplanted OECs might represent a novel strategy to augment the benefit of transplantation to the injured spinal cord. The study of miRNAs has provided new molecular information, and miRNAs represent a potential therapeutic tool that may be used to regulate the functions of neuronal death and survival at the genetic level [84]. Based on the specific interactions of miRNAs with their target genes, RNA-based technologies are potential therapeutic strategies. Although many studies have provided encouraging results, therapeutic strategies for SCI treatment involving the modulation of these small molecules should be developed with caution because clinical

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trials of gene therapy have proven unsatisfactory thus far, and complications of key techniques remain to be overcome [85]. Strict ethical review and oversight are indeed necessary.

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