The skin is the largest organ of the integumentary system and possesses a vast number of functions. Due to the distinct layers of the skin and the variety of cells which populate each, a tightly regulated network of molecular signals control development and regeneration, whether due to programmed cell termination or injury. MicroRNAs (miRs) are a relatively recent discovery; they are a class of small non-coding RNAs which possess a multitude of biological functions due to their ability to regulate gene expression via post-transcriptional gene silencing. Of interest, is that a plethora of data demonstrates that a number of miRs are highly expressed within the skin, and are evidently key regulators of numerous vital processes to maintain non-aberrant functioning. Recently, miRs have been targeted as therapeutic interventions due to the ability of synthetic ‘antagomiRs’ to down-regulate abnormal miR expression, thereby potentiating wound healing and attenuating fibrotic processes which can contribute to disease such as systemic sclerosis (SSc). This review will provide an introduction to the structure and function of the skin and miR biogenesis, before summarizing the literature pertaining to the role of miRs. Finally, miR therapies will also be discussed, highlighting important future areas of research.

Introduction

Skin

The skin is the largest and one of the most varied organs of the body, and possesses a vast number of functions including thermoregulation, protection from external stimuli and pathogens, as well as water resistance. Composed of three layers, the skin acts as a protective barrier against environmental exposures. The top layer, the epidermis, is composed of proliferating basal and differentiated suprabasal keratinocytes, in addition to melanocytes, Langerhans and Merkel cells (Figure 1).

Structure of the epidermis and dermis.

Keratinocytes are the most abundant cell type located in the epidermis and possess a number of immune functions. For example, they exert a protective role via secretion of chemokines upon invasion of a pathogenic threat [1]. Production of a broad range cytokines, with both pro- and anti-inflammatory functions also occurs [2]. Expression of various Toll-like receptors (TLRs) contributes to the defence of the skin against antimicrobial pathogens [3]. In addition to their immune functions, they also secrete keratin, a key structural protein, and finally become the cornified layer once the cells flatten. Melanoblasts proliferate and colonize in the epidermis as melanocytes. Located in the basal layer, these cells produce melanin, the pigment found in skin, hair and eyes which protects against UV radiation [4]. Keratinocytes stimulate the proliferation and differentiation of melanocytes, in addition to melanogenesis. Epidermal and follicular homoeostasis, therefore, is maintained largely by interactions between these two cell types [4]. Langerhans cells are dendritic cells which process microbial antigens and are able to induce T-cell differentiation and polarization [5], in addition to activation of regulatory T cells [6]. In contrast, Merkel cells which are located in the basal epidermis, function as mechanoreceptors [7]. Homoeostasis of the epidermis is maintained by three distinct classes of stem cells: bulge stem cells of located in the hair follicle, interfollicular stem cells and sebaceous gland stem cells.

The middle layer, the dermis, is populated by macrophages, lymphocytes, mast cells and dendritic cells, in addition to fibroblasts which provide structural strength through production of collagen and elastin fibres and other extracellular matrix proteins [8]. Meissner’s and Pacinian corpuscles, in addition to nerve fibres and free endings, detect touch, pressure and pain [9], while sweat and sebaceous glands regulate temperature. Not only do capillary networks in the dermis provide the epidermis with oxygen and nutrients [8], but dermal stem cells are also able to differentiate into functional cells in the epidermis, highlighting an interdependence between the two layers [10].

The hypodermis is primarily composed of adipocytes, and is also involved in thermoregulation via its insulative properties, while production of vitamin D also occurs in this subcutaneous layer.

The large number of cell types and functional diversity of the skin necessitate tightly regulated molecular signals in order to deter aberrant growth and maintain homoeostasis. MicroRNAs (miRs) are one group of molecules which aid in such mechanisms.

MicroRNA biogenesis and function

miRs are small non-coding RNA molecules, typically ∼22 nucleotides (nt) long, involved in RNA silencing and post-transcriptional regulation of gene expression by binding to the 3′-UTR of its targets; target recognition occurs via a 6–8 nt site which matches the miR seed region. Each miR is capable of repressing hundreds of genes, and each gene can be targeted by multiple miRs, making it a powerful system for the fine-tuning of gene expression.

miR biogenesis is similar to protein-coding genes in that upon transcription factor binding, RNA polymerase II transcribes the miR gene, followed by Drosha-mediated cleavage of the primary miR transcript resulting in a number of precursor miRs (pre-miR). A 2 nt overhang at the 3′ end exists as a binding site for Exportin-5 which causes translocation to the cytoplasm. Dicer then processes the pre-miR into a mature miR strand which is incorporated into the RNA-induced silencing complex (RISC), which in turn, inhibits translation (see Figure 2). Reduction in the abundance of the target mRNA may also occur via RNA cleavage or decapping and deadenylation. The RISC can also silence genes via formation of heterochromatin at the genomic level [11]. A number of miRs are highly expressed in the skin, and possess a plethora of important functions with regard to development, homoeostasis and regeneration. The following sections serve to summarize the current literature within these areas. In addition, chemically synthesized miRs are beginning to be trialled and used as therapeutic treatments for a number of diseases, and will also be discussed.

Simplified miR biogenesis and incorporation into the RISC pathway.

Skin development and homoeostasis: role of microRNAs

During embryonic development, gastrulation occurs whereby the single-layered blastula transforms into the tri-layered gastrula. Epidermal progenitor cells of the outer layer, the ectoderm, cause hair follicles, sebaceous glands and the epidermis itself to grow. Mesenchymal cells begin to infiltrate the skin and in combination with the basal epidermal layer, begin to direct extracellular matrix proteins and growth factors. Once the formation of new keratinocyte layers (stratification) and differentiation of their properties are complete, the epidermis consists of distinct layers: a cornified layer (stratum corneum), a translucent layer (stratum lucidum), a granular layer (stratum granulosum), a spinous layer (stratum spinosum) and a basal layer (stratum basale). The inner layer of basal cells proliferate, while suprabasal cells terminally differentiate. Hair follicle morphogenesis occurs via interactions between epidermal keratinocytes and dermal fibroblasts. Primarily, the mesenchyme coordinates keratinocyte proliferation. The follicle continues to develop downwards, whereby differentiation results in the hair shaft and sebaceous gland. The epidermis, therefore, is composed of the interfollicular epidermis and a pilosebaceous unit which contains a hair follicle.

Epidermal homoeostasis is maintained by three distinct pools of stem cells located in the interfollicular epidermis, the bulge (a region located at the bottom of the hair follicle outer root sheath) and the sebaceous gland. Stem cells continuously self-renew and differentiate into various cell lineages, which is an essential process in order to replenish cells such as keratinocytes which die due to injury or programmed termination. Skin barrier renewal via spinous transition of basal cells in mature skin occurs concomitantly with changes to gene expression; for example, Keratin5/14 (K5/K14) are down-regulated, and Keratin1/10 (K1/K10) are up-regulated [12].

The molecular signals which control the development of the skin are numerous and complex, with a clear role for miRs as key regulators of a variety of processes crucial to epidermal and follicular development and homoeostasis. The importance of miRs in skin morphogenesis has been shown by progenitor cell Dicer knockdown in an embryonic mouse skin model in which hair germ cells evaginated towards the epidermis, instead of invaginating towards the dermis as usual [13]. Interestingly, lower Dicer expression has been observed in mouse embryonic epidermis than the hair follicle, suggesting differential epigenetic regulation even between two conjoining structures [14].

The functional consequence of Dicer knockdown is also significantly different between the epidermis and hair follicle; one week after birth, epidermal proliferation and apoptosis were not significantly altered; however, elevated apoptosis and attenuated cell proliferation inhibited follicular development [13,14]. In support of the postulation that differential epigenetic signals regulate development of the epidermis and hair follicle, the miR-19/20 and miR-200 families are highly expressed in the former, while conversely, the miR-199 family is exclusively expressed in the latter [13].

The transcription factor p63 is essential in epidermal development, as demonstrated by the death of p63-deficient mice immediately following birth [15], likely as it is important in maintaining stem cell proliferation [16]. The Notch pathway is a regulator of interfollicular differentiation and active primarily in suprabasal cells [17], while inhibition of this pathway results in a lack of skin barrier development [18]. In addition, the MAPK pathway regulates epidermal proliferation and differentiation as evidenced by Mek1/2 deletion which results in underdevelopment of the skin [19]. Conversely, overactivation of epidermal growth factor (EGF) signalling, involved in the MAPK pathway, causes augmented proliferation and epidermal tumour growth [20]. Consistently, attenuated interfollicular proliferation occurred as a consequence of EGF down-regulation.

miR-203 is expressed in both human adult and foetal skin, although expression was not detectable until 17 weeks [21]. Evidence suggests that miR-203 is an important regulator of keratinocyte differentiation; protein kinase C (PKC) inhibition blocked miR-203 expression following calcium-induced differentiation, while overexpression of miR-203 significantly augmented differentiation [22]. These data demonstrate that PKC pathway activation is essential for miR-203 up-regulation, and thus, keratinocyte differentiation. Furthermore, miR-203 appears to promote epidermal differentiation via decreasing proliferation. Given that p63 regulates stem cell maintenance, miR-203-induced p63 repression is a plausible mechanism by which miR-203 promotes differentiation [23]. SOCS3 is expressed basally in both human foetal and adult skin, and is also directly targeted by miR-203 [21], further supporting the role of this miR in skin development.

miR-34a, -34c, -574-3p and -720 also appear to be regulated by p63. Inhibition of miR-34a and -34c restored keratinocyte cell cycle progression, while knockdown of p63 augmented both of these miRs concomitantly with inhibition of cell cycle regulators [24]. p63 and inhibitor of apoptosis stimulating protein of p53 (iASPP) are involved in an autoregulatory feedback loop; iASPP itself negatively regulates miR-574-3p and miR-720, which could, in turn, be negatively regulating p63. Depletion of iASPP in keratinocytes promotes differentiation concomitantly with an overexpression of these two miRs. Furthermore, miR-574-3p and miR-720 expression in these cells correlate with stratification pattern [25], thereby supporting their role in epidermal differentiation, plausibly via down-regulation of p63.

Another miR-200 family member, miR-205, exhibits one of the highest levels of expression in skin stem cells. Significantly attenuated neonatal epidermal and follicular development occur as a consequence of miR-205 knockdown, resulting in thinner skin [26]. Phosphorylated protein kinase B (pAKT), previously shown to be involved in skin stem cell renewal and proliferation [27], was reported to be down-regulated in the knockout model, as miR-205 directly targets and represses negative regulators of PI3K signalling. Proliferation, therefore, of skin stem cells appears to occur via miR-205-mediated mechanisms which maintain pAKT.

In addition to p63 expression, interfollicular, sebaceous gland and hair follicle stem cells also express K5 and K14. Maintenance of miR-125b in cells expressing K14 was reported to sustain stemness, resulting in aberrant skin morphogenesis in the form of a thickened epidermis, lack of hair follicle development and enlarged sebaceous glands, plausibly via an attenuation of Blimp1 protein level. The data suggest that this dysregulated follicular development is due to an increased number of early bulge cell divisions in the outer root sheath, restricting cell lineage commitment. Interestingly however, upon restoration of miR-125b to normal levels, these effects were reversed [28]. miR-125b does not, however, influence stem cell maintenance or activation, suggesting that the function of this miR is to primarily enhance stem cell renewal. Overall, these results indicate that miR-125b possesses the ability to repress stem cell differentiation, which appears logical given that miR-125b expression is low in early stem cell progeny.

In terms of hair follicle morphogenesis, the Wnt/β-catenin pathway is an important regulator of hair follicle specification; during development, augmentation of β-catenin results in failed hair follicle differentiation [29], while similarly, Runx1 expression is also essential for follicular differentiation [30]. Furthermore, bone morphogenetic protein (BMP) ligands and antagonists are key, as evidenced by deletion of the BMP antagonist Noggin which attenuates development of the hair follicle [31]. miR-214 targets β-catenin; when overexpressed in keratinocytes, miR-214 inhibits proliferation and consequently hair follicle development due to attenuation of β-catenin and Lef-1. miR-214, therefore, is a key regulator of Wnt signalling, which itself is key in follicular morphogenesis [32].

Smad proteins, which are signal transducers of the TGF signalling pathway, are also involved in follicular development via mediation of BMP signalling and inhibition of Wnt/β-catenin signalling [33]. Interestingly, Smads also regulate miR expression through transcriptional and post-transcriptional mechanisms [34], further supporting the importance of the TGF- β superfamily and miRs in the skin. BMPs are expressed in the epidermis and mesenchyme, and are involved in skin development and tissue remodelling [35]; bone morphogenic protein 4 (BMP4) treatment resulted in significant inhibition of miR-21 expression in primary mouse keratinocytes. Similarly, overexpression of the BMP antagonist Noggin significantly augments miR-21 in a transgenic mouse model [36]. Furthermore, the BMP4-mediated inhibition of cell proliferation and migration was prevented by miR-21. Given that miR-21 is consistently overexpressed in a variety of tumour types, these findings suggest a mechanism by which the BMP pathway inhibits miR-21 in order to maintain normal function of the skin.

Embryonic RUNX1 deletion in the epidermis causes delayed hair follicle regeneration due to failure of bulge stem cell proliferation [37]. In contrast with epidermal development whereby elevated β-catenin leads to a reduction in hair follicle differentiation, during adulthood, high levels of β-catenin cause abnormal growth of hair follicles [29]. The aforementioned BMP pathway mediates bulge stem cell quiescence in adult mice, and as BMP expression reduces, bulge stem cells activate and augment hair follicle regeneration [38].

In terms of dermal homoeostasis, miR-145 is highly expressed in dermal fibroblasts; however, expression was reported to be significantly down-regulated during reprogramming to pluripotent stem cells. Inhibition of miR-145 resulted in an elevation in epithelial markers, and a reduction in mesenchymal markers, suggesting mesenchymal to epithelial transitioning, a key process in reprogramming of somatic cells to induced pluripotent stem cells. It seems clear, therefore, that the reduction in miR-145 significantly contributes to homoeostatic control of the dermis via a switch from dermal fibroblasts to stem cells [39], which themselves can be programmed to a different cell type.

The literature discussed thus far demonstrates that a large number of miRs exert significant regulatory effect on the fine tuning of a variety of signalling pathways involved in epidermal and dermal development and homoeostasis (Table 1).

Table 1
Summary of miRs involved in skin development and homoeostasis
miR Cell/Model Role 
203 Foetal/adult skin (human) [22Regulator of keratinocyte differentiation 
34a/34c Keratinocytes (mouse) [24p63-regulated repression of -34a/34c controls cell cycle progression 
574-3p/720 Neonatal keratinocytes (human) [25Negatively regulated by iASPP; depletion of iASPP = miR expression and cell differentiation 
205 Mouse models [26Neonatal epidermal and follicular development 
125b Mouse models [28Enhances stem cell renewal and inhibits stem cell differentiation 
214 Mouse models/keratinocytes [32Inhibits follicular development via targeting β-catenin 
21 Mouse models/keratinocytes [36Inhibits effects of BMP4 on cell proliferation/migration, leading to aberrant growth 
145 Human dermal fibroblasts [39Down-regulated during reprogramming of somatic cells into pluripotent stem cell 
miR Cell/Model Role 
203 Foetal/adult skin (human) [22Regulator of keratinocyte differentiation 
34a/34c Keratinocytes (mouse) [24p63-regulated repression of -34a/34c controls cell cycle progression 
574-3p/720 Neonatal keratinocytes (human) [25Negatively regulated by iASPP; depletion of iASPP = miR expression and cell differentiation 
205 Mouse models [26Neonatal epidermal and follicular development 
125b Mouse models [28Enhances stem cell renewal and inhibits stem cell differentiation 
214 Mouse models/keratinocytes [32Inhibits follicular development via targeting β-catenin 
21 Mouse models/keratinocytes [36Inhibits effects of BMP4 on cell proliferation/migration, leading to aberrant growth 
145 Human dermal fibroblasts [39Down-regulated during reprogramming of somatic cells into pluripotent stem cell 

MicroRNAs in wound healing and skin regeneration

The underlying molecular mechanisms which mediate the development and homoeostasis of the skin which have been discussed thus far all refer to processes which occur in healthy, undamaged skin. However, when injury occurs, differing signals, including miRs, coordinate the many stages of skin regeneration. The principal stage, haemostasis, is characterized by vasoconstriction which reduces blood flow, and platelet degranulation which activates fibrin, a protein involved in clotting. The second phase involves inflammation, whereby neutrophils immediately migrate to the wound and phagocytose pathogens and tissue debris. Monocyte-derived macrophages cause efferocytosis which aids in the resolution of the acute inflammatory response. Proliferation then occurs, which is characterized by a number of processes which assist in the formation of new tissue; several cell types, in particular keratinocytes, fibroblasts and endothelial cells, coordinate re-epithelialization, collagen deposition, angiogenesis and granulation tissue formation. Finally, the remodelling phase involves full formation of the epidermal barrier primarily via extracellular matrix protein synthesis and apoptosis of immune cells. Scar formation may also occur during this phase, thought to be a consequence of aberrant inflammation, re-epithelialization and collagen deposition.

Haemostasis

miR-143-145 appears to be essential during haemostasis, as evidenced by attenuated vasoconstriction, a critical event during the early stages of wound healing, and impaired vascular smooth muscle cell (VSMC) differentiation in miR-143-145 knockout animals when compared with age-matched wild-type controls [40]. Platelet-derived growth factor (PDGF) induces miR-15b, which is essential for VSMC proliferation [41]. This mimics previous findings which showed that PDGF elicited miR-221 up-regulation concomitantly with VSMC proliferation via down-regulation of c-Kit [42].

Fibrinogen, the precursor to fibrin which causes clots at the site of skin injury, is regulated by miRs with attenuation of the FGB transcript occurring due to miR-409-3p overexpression. miR-29 reduced the levels of all three fibrinogen transcripts, FGA, FGB and FGG [43] (table 2).

Table 2
Summary of miRs involved in haemostasis during skin regeneration
miR Cell/Model Role 
143-145 Mouse models [40Essential for VSMC differentiation, and functionally, vasoconstriction and vasodilation 
15b Pulmonary artery SMCs [41Induced by PDGF, which inhibits SMC-specific gene expression and promotes cell proliferation 
221 Pulmonary artery SMCs [42Induced by PDGF, which inhibits SMC-specific gene expression and promotes cell proliferation via attenuation of c-kit 
409-3p/29 HuH7 (liver) cell line [43Attenuates fibrinogen gene (FGA, FGB and FGG) expression 
18a/19a/20a HepG2 (liver) cell line [45IL-6 stimulation augmented expression of all three miRs. miR-18a increased STAT3 activation and fibrinogen protein level 
miR Cell/Model Role 
143-145 Mouse models [40Essential for VSMC differentiation, and functionally, vasoconstriction and vasodilation 
15b Pulmonary artery SMCs [41Induced by PDGF, which inhibits SMC-specific gene expression and promotes cell proliferation 
221 Pulmonary artery SMCs [42Induced by PDGF, which inhibits SMC-specific gene expression and promotes cell proliferation via attenuation of c-kit 
409-3p/29 HuH7 (liver) cell line [43Attenuates fibrinogen gene (FGA, FGB and FGG) expression 
18a/19a/20a HepG2 (liver) cell line [45IL-6 stimulation augmented expression of all three miRs. miR-18a increased STAT3 activation and fibrinogen protein level 

Each of the aforementioned stages does not necessarily occur in isolation, as evidenced by miR regulation of overlapping haemostatic and inflammatory mechanisms. The pleiotropic cytokine IL-6 is a significant inducer of fibrinogen synthesis [44], which itself is regulated by the transcription factor STAT3. Expression of the miR-17.92 family transcripts, miR-18a, miR-19a and miR20a, were elevated after 24 h of IL-6 treatment in both hepatoma cells and hepatocytes. Moreover, miR-18a was able to augment STAT3 transcriptional activity in HepG2 cells, which, in addition to fibrinogen protein up-regulation following miR-18a transfection of IL-6-stimulated cells, highlights that a positive feedback loop may exist by which miR-18a and the STAT3 pathway, and subsequent IL-6/fibrinogen activation, up-regulate one another during the acute phase response [45].

Although these data do not directly pertain to cells located in the skin, the underlying mechanisms may still be applicable however.

Inflammation

The inflammatory phase, as described above, depends on recruitment of immune cells to the site of the wound [46]. Differentiation of blood monocytes into macrophages in the injured tissue is essential in order to mount an effective phagocytic response. This process involves a complex cascade of interlinked events in which miRs have been identified. For example, activation of the macrophage colony stimulating factor receptor (M-CSFR) gene due to miR-424-mediated nuclear factor I type A (NFI-A) transcription factor down-regulation has been reported [47]. Additionally, computational analysis revealed that the transcription factors CEBPB, CREB1, ELK1, NFE2L2, RUNX1 and USF2, which are involved in monocytic differentiation, target miR-21, -424, -155 and -17-92 [48].

Furthermore, albeit using fibroblast-like synoviocytes isolated from rheumatoid arthritis (RA) patients, Nakamachi et al. [49] were able to demonstrate that miR-124a impaired monocyte chemoattractant protein 1 (MCP-1) expression which may have significant implications for monocyte migration following skin injury.

Efferocytosis of apoptotic cells by these differentiated macrophages is critical, and has been shown to induce miR-21, while this miR also augmented efferocytosis, thereby demonstrating that a positive feedback loop exists. Of functional consequence, both post-efferocytotic and experimentally induced miR-21 were able to suppress LPS-induced NF-κB activation and TNF-α expression. In addition, efferocytosis, and thus miR-21, augmented IL-10, which together indicates that miR-21 dampens pro-inflammatory mediators and enhances anti-inflammatory signalling [50].

TLRs enable inflammatory cells to recognize microbial pathogens, thereby aiding in the regulation of the innate inflammatory response. LPS, which is recognized by TLR4, has been shown to augment miR-146a and -146b in primary monocytes isolated from cord and adult blood [51], in addition to the monocytic cell line THP-1, whereby these miRs were shown to down-regulate IRAK1 and TRAF6, and thus, negatively regulate TLR signalling [52]. LPS also up-regulated miR-155.

Similarly, miR-147 was reported to be inducible by TLR stimulation, with binding of NF-κB and STAT1α to the miR-147 promoter also observed [53]. miR-147 knockdown resulted in increased TNF-α and IL-6 protein concentrations, demonstrating that this miR is a significant negative regulator of TLR-induced inflammatory responses in macrophages, thereby preventing aberrant inflammation which could be causative of scar formation.

In addition to the aforementioned LPS-induced up-regulation of miR-155 [52], the same group then reported that IFN-β also augmented miR-155 expression [54]; the functional consequence of which was not identified, in contrast with the role of miR-146a. More recently, however, Jablonski et al. [55] demonstrated that miR-155, which was up-regulated in LPS and IFN-γ treated macrophages, suppresses a number of a genes, which drive the transformation to a classically activated ‘M1’ pro-inflammatory, anti-fibrotic macrophage phenotype in vitro. miR-155-deficient mice exhibited accelerated wound closing concomitantly with increased numbers of macrophages at the wound site following punch biopsy [56]. Consistent with Jablonski et al.’s findings, treatment of these knockout mice with IL-4, the main cytokine involved in ‘M2’ or alternative macrophage phenotype activation, which is characterized by pro-fibrotic properties, induced the expression of the fibrotic protein FIZZ1. As expected, type-1 collagen deposition was elevated in this system. Combined, these data show that miR-155 inhibits M2 polarization in favour of an inflammatory phenotype, and also that an overlap exists between the inflammatory phase and the growth of new tissue in the proliferation phase. miR-155 is now also recognized as a multifunctional miR; elevated expression has been reported in activated B and T cells [57], synovial fibroblasts isolated from RA patients [58] and malignant tumours [59]. Interestingly, it also regulates BMAL1, an intrinsic component of the circadian clock, which regulates circadian rhythm and oscillations [60].

IL-10, through activation of, and also secretion by alternatively activated ‘M2’ macrophages, typically possess anti-inflammatory properties. miR-4661 transfection of RAW 264.7 macrophages resulted in augmented mRNA and protein levels of IL-10 [61]. Recently, miR-142 has also been found to be critical to appropriate wound healing through the regulation of neutrophil actin cytoskeleton gene modifiers as demonstrated by miR-142 KO mice which have abnormal wound closure rates [62].

The literature discussed above all pertains to inflammatory regulation by monocytes and macrophages; however, miRs also appear to regulate inflammatory responses in keratinocytes and in inflammatory skin disorders.

TLR2 stimulation of keratinocytes also up-regulates the expression of miR-146a, via NF-κB and MAPK, with this overexpression attenuating neutrophil chemoattraction due to down-regulated production of IL-8, TNF-α and CCL20, with the reverse occurring when miR-146a was inhibited [63]. miR-146a is also significantly associated with the inflammatory skin conditions atopic dermatitis (AD) and psoriasis. For example, miR-146a has been shown to be overexpressed in keratinocytes and skin lesions of AD patients [64], and functionally, appears to suppress NF-κB-dependent genes. Patients suffering from psoriasis exhibit miR-146a overexpression in skin lesions, which, as above, impairs neutrophil chemoattraction of keratinocytes. Genetic deficiency of miR-146a elicits an elevation in skin inflammation, in addition to earlier onset, due to epidermal hyperproliferation and augmented neutrophil infiltration [65]. Upon administration of an miR-146a mimic, IL-17-driven inflammation was suppressed, and the inflammatory response magnified upon inhibition, clearly demonstrating that miR-146a possesses a key role in the regulation of skin inflammation.

miR-155 is also associated with chronic inflammatory skin disorders, including AD and vitiligo. Overexpression of miR-155 was found in both CD4+ T cells and AD patient skin samples. A positive correlation was also observed between expression of the miR and AD disease severity, Th17 cell percentage, IL-17 mRNA expression and plasma concentrations, which was further exacerbated upon transfection of an miR mimic, while an inhibitor elicited contrasting effects [66]. Th17 cells have previously been postulated to significantly contribute to AD pathology; therefore, it appears plausible that miR-155 contributes to disease progression due to augmentation of these cells [67]. Furthermore, expression of miR-155 was reported to be elevated in the epidermis of vitiligo patients. When overexpression was induced in primary melanocytes and keratinocytes, genes associated with melanogenesis were down-regulated [68]. These data indicate that miR-155 is a key regulator in the pathogenesis of the chronic skin conditions such as AD and vitiligo.

The reviews published by Rozalski et al. [68,69] and Hawkes et al. [70] outline the extensive number of aberrations in miR expression associated with AD and psoriasis respectively, and can be referred to for further discussion on these diseases outside the remit of this review.

Altogether the literature presented in section ‘Inflammation’ demonstrates that a vast number of miRs regulate both the induction and resolution of inflammation during this stage of skin regeneration. Table 3 summarises miRs involved in the inflammation stage.

Table 3
Summary of miRs involved in inflammatory phase of skin regeneration
miR Cell/Model Role 
424 NB4 cell line [47Up-regulation during monocyte/macrophage differentiation; down-regulates NFI-A transcription factor, activating M-CSFR 
21/424/155/17-92 THP-1 cell line [48Targets of monocyte differentiation transcription factors 
124a Rheumatoid arthritis fibroblast-like synoviocytes [49Impairs MCP-1 expression 
21 Monocyte-derived macrophages (human) [50Induces and is induced by efferocytosis; suppresses LPS-induced NF-κB activation and TNF-α expression, and augments IL-10 
146a/146b Primary monocytes (cord and adult blood) [51Increased expression upon LPS stimulation 
 THP-1 cell line [52Down-regulation of IRAK1 and TRAF6, and thus, TLR signalling 
146a Keratinocytes (human) [63Up-regulation following TLR2 stimulation and attenuated neutrophil chemoattraction 
 AD skin lesions/keratinocytes [64miR-146a overexpression 
 Psoriasis skin lesions [65Overexpression in skin lesions, impairing neutrophil chemoattraction 
 KO mouse model [65Earlier disease onset, increased skin inflammation, epidermal hypoproliferation, augmented neutrophil infiltration; IL-17-driven inflammation suppressed upon administration of miR-146a mimic 
147 Peritoneal/alveolar macrophages (mouse) [53Expression induced by TLR (LPS) stimulation; miR-147 knockdown augmented IL-6 and TNF-α concentrations 
 RAW 264.7 cell line [53NF-κB and STAT1α bound to miR-147 promoter 
155 THP-1 cell line [52LPS-induced overexpression 
 Primary macrophages (mouse) [54IFN-β-induced overexpression 
 Primary macrophages (mouse) [55Up-regulated in LPS/IFN-γ-treated macrophages; drives transformation to pro-inflammatory ‘M1’ phenotype 
 KO mouse model [56Augmented wound closing correlated with increase number of infiltrating macrophages; IL-4 (M2) treatment increased FIZZ-1 expression and type 1 collagen deposition 
155 CD4+ T cells/AD skin lesions [66Overexpression of miR-155; expression correlated with disease severity, Th17 cell percentage, IL-17 mRNA and plasma concentrations 
 Vitiligo patient epidermis/melanocytes and keratinocytes [68Overexpression of miR-155 in epidermis; induced overexpression in vitro inhibited genes associated with melanogenesis 
4661 RAW 264.7 cell line [61Elevated IL-10 mRNA and protein following miR transfection 
142 KO mouse model/wound-infiltrated neutrophils (mouse) [62Impaired wound closure rate; altered neutrophil phagocytosis; actin cytoskeleton regulators Rho and Rac elevated, suggesting involvement in neutrophil migratory capacity 
miR Cell/Model Role 
424 NB4 cell line [47Up-regulation during monocyte/macrophage differentiation; down-regulates NFI-A transcription factor, activating M-CSFR 
21/424/155/17-92 THP-1 cell line [48Targets of monocyte differentiation transcription factors 
124a Rheumatoid arthritis fibroblast-like synoviocytes [49Impairs MCP-1 expression 
21 Monocyte-derived macrophages (human) [50Induces and is induced by efferocytosis; suppresses LPS-induced NF-κB activation and TNF-α expression, and augments IL-10 
146a/146b Primary monocytes (cord and adult blood) [51Increased expression upon LPS stimulation 
 THP-1 cell line [52Down-regulation of IRAK1 and TRAF6, and thus, TLR signalling 
146a Keratinocytes (human) [63Up-regulation following TLR2 stimulation and attenuated neutrophil chemoattraction 
 AD skin lesions/keratinocytes [64miR-146a overexpression 
 Psoriasis skin lesions [65Overexpression in skin lesions, impairing neutrophil chemoattraction 
 KO mouse model [65Earlier disease onset, increased skin inflammation, epidermal hypoproliferation, augmented neutrophil infiltration; IL-17-driven inflammation suppressed upon administration of miR-146a mimic 
147 Peritoneal/alveolar macrophages (mouse) [53Expression induced by TLR (LPS) stimulation; miR-147 knockdown augmented IL-6 and TNF-α concentrations 
 RAW 264.7 cell line [53NF-κB and STAT1α bound to miR-147 promoter 
155 THP-1 cell line [52LPS-induced overexpression 
 Primary macrophages (mouse) [54IFN-β-induced overexpression 
 Primary macrophages (mouse) [55Up-regulated in LPS/IFN-γ-treated macrophages; drives transformation to pro-inflammatory ‘M1’ phenotype 
 KO mouse model [56Augmented wound closing correlated with increase number of infiltrating macrophages; IL-4 (M2) treatment increased FIZZ-1 expression and type 1 collagen deposition 
155 CD4+ T cells/AD skin lesions [66Overexpression of miR-155; expression correlated with disease severity, Th17 cell percentage, IL-17 mRNA and plasma concentrations 
 Vitiligo patient epidermis/melanocytes and keratinocytes [68Overexpression of miR-155 in epidermis; induced overexpression in vitro inhibited genes associated with melanogenesis 
4661 RAW 264.7 cell line [61Elevated IL-10 mRNA and protein following miR transfection 
142 KO mouse model/wound-infiltrated neutrophils (mouse) [62Impaired wound closure rate; altered neutrophil phagocytosis; actin cytoskeleton regulators Rho and Rac elevated, suggesting involvement in neutrophil migratory capacity 

Proliferation and remodelling

The transition between inflammatory and proliferative phases is an essential aspect of wound healing and regeneration, and may be regulated by miR-132, which is induced by TGF-β1 and TGF-β2 in keratinocytes. miR-132 was shown to augment keratinocyte proliferation, and similarly to miR-146a and miR-155, attenuate their chemoattractive ability via NF-κB suppression [71]. It is likely, therefore, that miR-132 serves to mediate inflammation during progression to the proliferative phase, which is likely considering that miR-132 also suppresses NF-κB signalling.

Mobilization of hair follicle and interfollicular epidermal stem cells during the inflammatory stage, and migration and proliferation of keratinocytes, cause skin re-epithelialization. In an acute human skin wound model, miR-21 and miR-130a have been reported to delay re-epithelialization [72]. Conversely, however, TGF-β1-induced miR-21 expression was able to augment keratinocyte migration in HaCaT cells [73]. Consistently, knockdown of miR-21 decreased TGF-β1-induced keratinocyte migration. Following mouse skin punch biopsies, miR-21 expression was elevated, while miR-21 knockdown impaired re-epithelialization. Both in vitro and in vivo data from this study strongly suggest that miR-21 drives keratinocyte migration and proliferation. The data regarding miR-21 appear somewhat conflicting, however, these differences may be due to the use of human cell lines and mouse skin model in one investigation [73], and the use of an acute human skin wound model in the other [72]. Nonetheless, it is clear that miR-21 possesses a significant regulatory role in keratinocyte migration and thus, re-epithelialization.

In addition to the results reported by Yang et al. [73], Li et al. [74] showed that another transforming growth factor, TGF-β2, known to be highly expressed in skin wounds, elicits a significant elevation in the expression of miR-31 and subsequent proliferation in primary human keratinocytes, with miR-31 knockdown causing contrasting effects. Interestingly, EMP-1 appeared to mediate the effects of miR-31; a significant negative association was observed between the two, while silencing of EMP-1 exerted similar effects as miR-31 overexpression in terms of migratory capacity. In vivo, punch biopsy of a human wound healing model demonstrated that miR-31 gradually increased from the first day, and thus, the inflammatory phase, until day seven during the proliferative phase [74]. This supports not only the postulation that considerable overlap exists between the phases of skin regeneration, but also that miR-31 regulates re-epithelialization in a similar manner to miR-21.

An inverse correlation between RAN and RAPH1 and miR-203 expression was observed [75]. These two proteins, involved in cell proliferation and survival, and cytoskeleton remodelling respectively, have shown to be direct targets of miR-203. Furthermore, both silencing of these targets and overexpression of miR-203 in vitro using human neonatal epidermal keratinocytes resulted in attenuated cell proliferation and migratory capacity. In vivo, elevated miR-203 expression was reported in the suprabasal epidermal layers surrounding the wound in a mouse skin model; however, minimal expression was found in the migrating keratinocytes themselves. miR-203, could, therefore, be a possible target for therapeutic intervention, given the need for keratinocyte migration and proliferation in re-epithelialization, and the evident role miR-203 possesses in terms of inhibiting these key processes.

Reactive oxygen species released by phagocytic cells during the inflammatory phase appear to drive angiogenesis as evidenced by H2O2-induced VEGF augmentation in keratinocytes [76], in addition to impairment of angiogenesis upon antioxidant treatment in human microvascular endothelial cells [77]. In the study conducted by Shilo et al. [77], despite elevated VEGF expression following Dicer knockdown, the angiogenic response of these endothelial cells was compromised, demonstrated by attenuated tube formation and cell migration. Furthermore, Dicer knockdown in human endothelial cells elicited aberrant expression of a number of angiogenic genes concomitantly with attenuated cell proliferation [78]. Similarly, mice that lacked the first two exons of the Dicer gene exhibited under-developed blood vessels [79].

Moreover, miR-221 and -222 transfection of endothelial cells results in a reduction in c-Kit protein levels through targeting the c-Kit 3′-UTR [80]. c-Kit signalling involves Akt and Erk1/2 pathways, similarly to VEGF [81], while activation of c-Kit has been shown to also up-regulate VEGF [82]. Thus, miR-221 and -222 appear to regulate angiogenesis directly through c-Kit, which itself is the receptor for stem cell factor, and indirectly via c-Kit-dependent modulation of VEGF. Tissue hypoxia due to reduced blood supply of the damaged skin is also known to be an inducer of angiogenesis during regeneration; ETS-1, the angiogenesis-related transcription factor, in addition to MMP1 and VEGFR1, is negatively regulated by miR-200b. Expression of ETS-1 was de-repressed following hypoxia-induced down-regulation of miR-200b, which augmented angiogenic capacity [83], again, in experiments which utilized endothelial cells.

Seven days post-skin excision injury in mice, over 50 miRs exhibited altered expression of greater than 2-fold, 33 of which were up-regulated and 21 were down-regulated. The former included miR-21, -31 and -203, and the latter, miR-249. Prior to skin injury, miR-21 expression was undetectable in the epidermis; however, following excision, expression in the migrating epithelial cells was augmented greatly [84]. Furthermore, mesenchymal expression of miR-21 was also elevated in granulation tissue. Overexpression of miR-21 has been shown to inhibit granulation tissue formation in a rat wound model [72], which could have significant implications for therapeutic targeting in chronic non-healing wounds. Interestingly, TGF-β signalling not only has been robustly shown to induce miR-21 expression in keratinocytes [85], but also is a key pathway involved in the contraction of wounds [86].

In terms of collagen deposition, augmented miR-29a expression was shown to occur concurrently with a reduction in collagen type 1 α 2 (COL1α2) and VEGF-A following thermal skin injury [87]. As miR-29a began to decrease, COL1α2 and VEGF-A began to increase, suggesting that they were targets of miR-29a, which was discovered to be the case. This was further demonstrated by inhibition of miR-29a, which elicited a significant elevation in fibroblast proliferation and migration. The naturally occurring down-regulation of miR-29a during skin regeneration, therefore, appears to be a mechanism by which type 1 collagen synthesis and angiogenesis are enhanced, aiding the regenerative process. In a similar manner, Zhu et al. [88] corroborated these findings by recently reporting that miR-29a exhibited down-regulation in a murine thermal wound model; however, much more drastic attenuation of miR-29b was observed in both thermal and excisional wound models, concomitantly with a significant elevation in heat shock protein 47 (HSP47) expression and a gradual increase in COL1α1. Of note was that TGF-β1 inhibited miR-29b transcription in skin fibroblasts; given that miR-29b overexpression impairs biosynthesis of COL1α1, this further highlights the importance of TGF-β signalling in collagen deposition. Together, these data demonstrate that the miR-29 family possesses essential regulatory roles in mediating fibrotic processes and collagen deposition associated with wound contraction and scar formation. It has long been suggested that miR-29 is ‘fibromiR’ and is a critical target in fibrotic diseases where constitution of miR-29 is currently being investigated; the miR-29 family shares seed sequences complementary to conserved binding sites of multiple collagen genes [89] and down-regulation of miR-29a in systemic sclerosis (SSc), a major fibrotic disease, has also been shown [90]; experimentally, this study reported that overexpression of miR-29a caused a decrease in type I and type III collagen mRNA, with the opposite effects observed upon miR-29a knockdown. These data were corroborated, as increased miR-29a expression reversed the fibrotic SSc fibroblast phenotype due to attenuation of collagen and TIMP-1, which itself is regulated by the miR-29a target TAB1 [91]. Table 4 summarises key miRs involved in regeneration and fibrosis.

Table 4
Summary of miRs involved in proliferation and remodelling phase of skin regeneration
miR Cell/Model Role 
132 Keratinocytes (human) [71Augments proliferation and attenuate chemoattractive ability 
21 Skin punch biopsy (human/rat) [72Inhibits re-epithelization 
 HaCaT cell line [73TGF-β1-induced miR-21 expression increased keratinocyte migration 
 Excisional wound model (mouse) [84Elevated expression in migrating epithelial cells following skin injury 
 HaCaT cell line [85TGF-β signalling induced miR-21 expression; miR-21 blocks inhibition of growth by TGF-β1 
130a Healthy skin punch biopsy (human) [72Inhibits re-epithelization 
31 Keratinocytes (human) [74Expression induced by TGF-β2 
 Skin punch biopsy (human) [74Expression gradually increased from day 1 until day 7 
203 HEKn cells [75Overexpression attenuated cell proliferation and migratory capacity 
 Skin punch biopsy (mouse) [75Expression found in suprabasal epidermal layers; minimal expression in migrating keratinocytes 
221/222 Endothelial cells (human vascular) [80Transfection reduces c-Kit, thereby reducing angiogenesis 
200b Dermal microvascular endothelial cells (human) [83De-repression of angiogenic transcription factor ETS-1, and thus increased angiogenesis, following hypoxia-induced miR-200b down-regulation 
29a Thermal wound injury model (rat)/BJ fibroblast cell line (human) [87Augmented expression concurrently with attenuated COL1α2 and VEGF-A (confirmed as targets of miR-29a in vitro
 Excisional and thermal wound models (mouse) [88Expression unaltered in excisional wound model, but down-regulated in thermal wound model 
 NRK-52E cell line [893′-UTR of numerous collagen genes targeted by miR-29 family 
 SSc fibroblasts/skin sections (human) [90Down-regulation in SSc fibroblasts and skin sections; overexpression of miR-29a caused a decrease in type I and type III collagen mRNA, with the opposite effects observed upon miR-29a knockdown 
 Dermal fibroblasts (human) [91Transfection of miR-29a decreased collagen and TIMP-1; TAB-1, a regulator of TIMP-1, found to be a novel target of miR-29a 
29b Excisional and thermal wound models (mouse) [88Down-regulation in both models; transcription inhibited by TGF-β1, enhancing collagen 1 production 
145 Hypertrophic scar tissue (human) [92α-SMA and miR-145 elevated 
 Dermal fibroblasts (human) [92Expression augmented by TGF-β1, causing a decreasing in KLF4, thereby de-repressing α-SMA 
129-5p SSc patient skin and serum samples [93IL-17A transfection attenuated miR-129-5p expression; down-regulation of IL-17A signalling by TGF-β1 releases inhibitory effect of miR-129-5p on collagen type 1 
miR Cell/Model Role 
132 Keratinocytes (human) [71Augments proliferation and attenuate chemoattractive ability 
21 Skin punch biopsy (human/rat) [72Inhibits re-epithelization 
 HaCaT cell line [73TGF-β1-induced miR-21 expression increased keratinocyte migration 
 Excisional wound model (mouse) [84Elevated expression in migrating epithelial cells following skin injury 
 HaCaT cell line [85TGF-β signalling induced miR-21 expression; miR-21 blocks inhibition of growth by TGF-β1 
130a Healthy skin punch biopsy (human) [72Inhibits re-epithelization 
31 Keratinocytes (human) [74Expression induced by TGF-β2 
 Skin punch biopsy (human) [74Expression gradually increased from day 1 until day 7 
203 HEKn cells [75Overexpression attenuated cell proliferation and migratory capacity 
 Skin punch biopsy (mouse) [75Expression found in suprabasal epidermal layers; minimal expression in migrating keratinocytes 
221/222 Endothelial cells (human vascular) [80Transfection reduces c-Kit, thereby reducing angiogenesis 
200b Dermal microvascular endothelial cells (human) [83De-repression of angiogenic transcription factor ETS-1, and thus increased angiogenesis, following hypoxia-induced miR-200b down-regulation 
29a Thermal wound injury model (rat)/BJ fibroblast cell line (human) [87Augmented expression concurrently with attenuated COL1α2 and VEGF-A (confirmed as targets of miR-29a in vitro
 Excisional and thermal wound models (mouse) [88Expression unaltered in excisional wound model, but down-regulated in thermal wound model 
 NRK-52E cell line [893′-UTR of numerous collagen genes targeted by miR-29 family 
 SSc fibroblasts/skin sections (human) [90Down-regulation in SSc fibroblasts and skin sections; overexpression of miR-29a caused a decrease in type I and type III collagen mRNA, with the opposite effects observed upon miR-29a knockdown 
 Dermal fibroblasts (human) [91Transfection of miR-29a decreased collagen and TIMP-1; TAB-1, a regulator of TIMP-1, found to be a novel target of miR-29a 
29b Excisional and thermal wound models (mouse) [88Down-regulation in both models; transcription inhibited by TGF-β1, enhancing collagen 1 production 
145 Hypertrophic scar tissue (human) [92α-SMA and miR-145 elevated 
 Dermal fibroblasts (human) [92Expression augmented by TGF-β1, causing a decreasing in KLF4, thereby de-repressing α-SMA 
129-5p SSc patient skin and serum samples [93IL-17A transfection attenuated miR-129-5p expression; down-regulation of IL-17A signalling by TGF-β1 releases inhibitory effect of miR-129-5p on collagen type 1 

miR-145 appears to regulate a similar cascade; in vivo, miR-145 levels and α-smooth muscle actin (α-SMA) were significantly augmented in hypertrophic skin tissue compared with controls, while TGF-β1-induced elevation of miR-145 attenuated expression of KLF4, thereby de-repressing α-SMA in skin myofibroblasts [92]. Thus, aberrant miR-145 appears to contribute to scarring due to α-SMA’s contribution to permanent tissue contracture. Furthermore, inhibition of miR-145 attenuated not only COL1α1 expression, but also TGF-β1 secretion and migration.

A reduction in miR-129-5p also appears to possess a significant regulatory role in aberrant fibrotic processes associated with SSc pathology; in vitro, IL-17RA siRNA transfection significantly reduced expression of the anti-fibrotic miR-129-5p. Moreover, TGF-β1-induced down-regulation of IL-17A signalling due to attenuation of the receptor IL-17RA expression in SSc fibroblasts releases the inhibitory effect of miR-129-5p on type 1 collagen, thereby promoting fibrosis [93].

A paucity of literature appears to exist concerning miR-mediated longer term remodelling of the skin, whereby type III collagen is replaced by type I collagen and re-aligned. This area, therefore, warrants further investigation so that the molecular mechanisms that govern wound healing are fully characterized (Figure 3).

Schematic represention of miRs involved in collagen regulation and scleroderma pathogenesis

Figure 3
Schematic represention of miRs involved in collagen regulation and scleroderma pathogenesis

Col., collagen; ITGB3, integrin β 3; MMP-1, matrix metalloproteinase-1 [88,90,91,9399].

Figure 3
Schematic represention of miRs involved in collagen regulation and scleroderma pathogenesis

Col., collagen; ITGB3, integrin β 3; MMP-1, matrix metalloproteinase-1 [88,90,91,9399].

Foetal wound healing

Although not as common as tissue injuries in adults, foetal skin possesses the capacity to heal without scarring due to its own unique molecular response to injury. Microarray analysis has revealed a plethora of differentially expressed genes between mid-gestational and post-natal dermal wounds, concomitantly with the expected full re-epithelialization and lack of scarring in the former, and dense scar tissue present in the latter mice [100]. Most importantly, a number of these differentially regulated genes were involved in growth factor signalling and cell proliferation. Further support for the significance of growth factors in the striking phenotypic differences between early-mid foetal skin and late foetal or post-natal skin has been reported. Unwounded human adult and foetal skin possess distinct TGF-β expression profiles, with lower expression of all three isoforms found in foetal skin, in addition to differential ratios compared with adult skin [101]. Given the role of TGF-β in the regulation of extracellular matrix deposition, it was unsurprising that deletion of TBF-βRII significantly attenuated dermal scar formation and enhanced epidermal proliferation in post-natal fibroblasts [102]. The dermis of TBF-βRII knockout mice also exhibited a decrease in collagen deposition together with augmented keratinocyte proliferation, and thus, re-epithelialization [86].

In addition to growth factors, aberrant collagen composition and organization also contribute to the scarring phenotype which is not characteristic of non-scarring foetal skin. Mid-gestational rats exhibited a much higher collagen type III:collagen type I ratio than adult rats following surgical dermal injury [103]. In corroboration, Goldberg et al. [104] discovered that COL1α1 expression was attenuated following injury during mid-gestation only, while COL1α2 and COL1α3 were significantly lower in the late-gestational group, in both wounded and normal conditions. The pattern of collagen deposition in the mid-gestational mice was characteristic of the surrounding unwounded skin with no visual evidence of scarring, whereas irregular collagen deposition in the late-gestational mice was observed, suggesting scar tissue formation. In vivo, undamaged human foetal skin also exhibited a greater type III:type I ratio than adolescent, adult and elderly skin [105].

A comparison of mid-gestational (non-scarring phenotype) and late-gestational (scarring phenotype) mouse skin revealed a number of differentially expressed miRs and predicted targets between the two time periods and associated phenotypes. In particular, expression of miR-29b, -29c and 338-3p were altered 24, 20 and 19-fold respectively [106]. Importantly, bioinformatic analysis revealed that the differentially expressed miRs were also shown to putatively target a number of signalling pathways, including TGF-β. Thus, it could be postulated that miRs may be significant regulators of the foetal non-scarring phenotype due to modulation of TGF-β signalling, thereby fine tuning the response. The role of the miR-29 family in the regulation of collagen expression has already been discussed within this review, and may present a further molecular mechanism by which differential miR expression contributes to scarless healing.

MicroRNA therapeutics

In addition to the essential role of miRs in normal development of the skin and regeneration from injury, they also play a pivotal role in the pathogenesis of a vast number of diseases, including conditions of the skin such as vitiligo, psoriasis, SSc, dermatomyositis and melanomas. The detection of miRs in serum or plasma, which resist degradation due to containment within extracellular vesicles, is emerging as a non-invasive diagnostic marker for major diseases like cancer [107] and cardiovascular disease [108], in addition to RA [109], SSc [110], and as a biomarker of the severity of inflammation in children with AD [111].

In terms of treatment of specific conditions, recent research has demonstrated that miRs can manipulated via administration of miR mimics which are chemically synthesized, double-stranded RNAs which mimic endogenous miRNAs, while antagomiRs are chemically engineered oligonucleotides which inhibit miRNAs. There is potential for both to be used therapeutically for tissue regeneration or fibrotic diseases. As highlighted by Christopher et al. [112], however, a number of steps are required before a miR could potentially be used as an effective therapy, namely, profiling of the miR associated with a specific disease state, in vitro studies to validate the miR using loss/gain of function, in vivo studies to investigate pharmacokinetics, followed by clinical trials if all other stages are successful. Other important considerations are specificity of binding to the target miR, resistance to degradation and the method of in vivo delivery.

Chemical modification of the miRs is required to enhanced stability and stop the breakdown by copious endogenous nucleases, in addition to improving affinity of the antagomiR to the cognate miR. Such examples are locked nucleic acids (LNAs), whereby the ribose of the RNA nucleotide is chemically modified by the addition of a 2′-O, 4′-C-methylene bridge [113]. Alternatively, tiny LNAs which are 8-mer LNA anti-miRs which specifically target the miR seed region, can be used [114]. Chemical substitution of the 2′-hydroxyl group, to 2′-O-methyl or 2′-O-methoxyethyl, for example, also occur [115].

The issue of specificity is being addressed via a number of methods; sponge miRs, for example, contain several complementary binding sites to the specific miR of interest, whereas miR erasers utilize two copies of the exact miR complementary antisense sequence [116]. Long non coding RNAs (lncRNA) are longer forms of RNA arbitrarily defined as 200 nt and over, and often act as sponges to sequester faulty miR expression. Indeed, the X-linked lncRNA H19 is aberrantly expressed in keloids [117]. Targeting of the miR to the correct tissue could also be employed by conjugating the chemically synthesized miR to a monoclonal antibody which identifies that specific tissue antigen, thereby ‘hitting the target’ and causing the miR to bind to its cognate mRNA. This could also be PEGylated to increase stability.

Viral vectors have been utilized in order to deliver miR mimics or inhibitors. For example, adeno-associated virus (AAV) is a popular gene delivery system; however, a consistent issue which has inhibited the progression of some AAV therapies to human clinical trials is the induction of low grade immune activation [118]. Lenti-viral vectors for delivery suffer from the same immunogenicity issues. miR mimics for the tumour suppressors miR-34a and let-7 have been successfully delivered in a complex with neutral lipid emulsion in mice [119].

A number of miR therapies are currently in pre-clinical trials, with a small number having already met the requirements to advance to clinical studies. Although no miR therapies designed specifically for skin disorders are at this stage to the authors’ knowledge, MiRagen Therapeutics currently have an ongoing phase 1 clinical trial for MRG-201, an miR-29b mimic which is designed to attenuate collagen expression, as this is a true target of miR-29. A number of studies have also utilized miR mimics and antagomiRs in vitro, and could potentially inform future studies which may progress to clinical trials.

Loss/gain of function studies are essential in order to validate particular miRs which may be future therapeutic targets with regard to skin disorders and/or healing. miR mimics and inhibitors have shown that miR-200b expression was up- and down-regulate respectively, in human microvascular endothelial cells, with a subsequent attenuation in tube formation and wound closure following mimic administration, with contrasting effects observed following addition of the 200b inhibitor [83]. In a similar investigation, the addition of an miR-29b mimic to primary human dermal fibroblasts elicited a significant augmentation of miR-29b expression with a concomitant attenuation of HSP47 and COL1α1 expression. As expected, contrasting outcomes were observed following administration of an inhibitor [88]. These miRs could, therefore, be future targets for therapeutic intervention. Furthermore, due to the data presented above by Gras and colleagues [92], they concluded that miR-145 may also be a promising target for future therapeutic intervention.

With specific regard to the utilization of antagomiRs, Krützfeldt et al. [120] have demonstrated that intravenous injection of antagomiRs against miR-16, -122, -192 and -194, resulted in a subsequent attenuation of the miR levels in a number of major tissues, including the skin. Subcutaneous injection of an miR-203 antagomiR attenuated miR-203 expression, but importantly, resulted in a greater number of proliferative cells in the dorsal epidermis of neonatal mice, demonstrating a functional effect [23]. Similarly, in a mouse excisional wound model, injection of an antagomiR to miR-155 attenuated expression as expected, in addition to phagocytic cell migration, pro-inflammatory cytokine secretion, and COL1α1, COL2α1 and α-SMA expression. Functionally, this elicited an overall positive effect as demonstrated by better aligned and thinner collagen fibres upon wound healing [121]. Most recently, topical epicutaneous administration of the miR-155 antagomiR elicited a reduction in collagen deposition and dermal thickening in bleomycin-induced fibrotic mice [122]. Of greatest interest, however, was that due to the topical delivery method, miR-155 was down-regulated only in the skin, and not liver, bone marrow or blood cells.

Direct injection of an miR-21 antagomiR to the dermis surrounding a would site did indeed attenuate expression of miR-21; however, impaired collagen deposition and delayed wound healing were observed which the authors did not expect, and therefore, miR-21 may not be an effective therapeutic target for wound healing [84].

Another issue is that targeting one specific miR might not be sufficient to elicit a significant clinical effect due to large redundancy among miRs; a reduction in one miR may have negligible effects on the protein output due to one or more miRs compensating for this. Thus, it may take multi-miR targeting approaches to repress a specific pathway.

Whilst haematopoietic stem cell transplantation (HSCT) isn’t an miR-based therapy per se, we hypothesize that miRs could be reset to non-aberrant levels thereby attenuating IL-6, TNF-R and CD3+ cell numbers, which was indeed observed in the dermis of the patient presented in Figure 4 [123]. Concomitantly, restoration of the skin structure also occurred, which would be expected given the role of IL-6 in promoting fibrosis [124]. It must be noted that no current data exist to support or refute this postulation however.

Haematoxylin and eosin (H.E.) and T cell (CD3) immunohistochemistry staining of an SSc patient’s dermis, before and after HSCT.

Figure 4
Haematoxylin and eosin (H.E.) and T cell (CD3) immunohistochemistry staining of an SSc patient’s dermis, before and after HSCT.

There is a major reduction in T cells and collagen post HSCT.

Figure 4
Haematoxylin and eosin (H.E.) and T cell (CD3) immunohistochemistry staining of an SSc patient’s dermis, before and after HSCT.

There is a major reduction in T cells and collagen post HSCT.

Concluding remarks and future directions

Although originally referred to as ‘junk DNA’ with little to no biological function, it has become increasingly apparent that non-protein coding regions of the genome can still exert a multitude of diverse effects and are no longer deemed insignificant. Non-coding RNAs are one such example which mediate a vast number of functions via post-transcriptional gene regulation, in the case of miRs [125].

The skin and the underlying molecular mechanisms by which it develops, is maintained, and regenerated, while complex, has been reasonably well characterized at this point. Of interest however, is how these mechanisms can be manipulated in order to elicit salubrious outcomes in the context of wound healing or skin disorders such as scleroderma.

Persistent perpetuation of dysregulated miRs in skin disorders suggest that they are key players in disease progression, and may also, therefore, be promising biomarkers and/or therapeutic targets. Data, thus far, have highlighted a variety of potential therapeutic targets in vitro and using murine models; however, as discussed, a large number of stages and considerations are required prior to a specific miR becoming eligible for clinical trial. Further research should continue to identify and validate miRs associated with aberrant epidermal development and homoeostasis associated with disease in the hope that efficient miR-based therapies may be established. It may be possible in the future with the advent of viral gene delivery to alter the expression of miRs with the use of viral vectors providing immune activation is avoided. In particular, SSc may be a tractable target for miR viral gene delivery due to the wealth of knowledge of miRs in this condition.

Competing Interests

The authors declare that there are no competing interests associated with the manuscript.

Abbreviations

     
  • α-SMA

    α-smooth muscle actin

  •  
  • AAV

    adeno-associated virus

  •  
  • AD

    atopic dermatitis

  •  
  • BMP

    bone morphogenetic protein

  •  
  • BMP4

    bone morphogenic protein 4

  •  
  • COL1α2

    collagen type 1 α 2

  •  
  • EGF

    epidermal growth factor

  •  
  • HSCT

    haematopoietic stem cell transplantation

  •  
  • HSP47

    heat shock protein 47

  •  
  • iASPP

    inhibitor of apoptosis stimulating protein of p53

  •  
  • LNA

    locked nucleic acid

  •  
  • lncRNA

    long non coding RNA

  •  
  • MCP-1

    monocyte chemoattractant protein 1

  •  
  • M-CSFR

    macrophage colony stimulating factor receptor

  •  
  • miR

    microRNA

  •  
  • MMP-1

    matrix metalloproteinase-1

  •  
  • NFI-A

    nuclear factor I type A

  •  
  • pAKT

    phosphorylated protein kinase B

  •  
  • PDGF

    platelet-derived growth factor

  •  
  • PKC

    protein kinase C

  •  
  • pre-miR

    precursor miRs

  •  
  • RA

    rheumatoid arthritis

  •  
  • RISC

    RNA-induced silencing complex

  •  
  • SSc

    systemic sclerosis

  •  
  • VSMC

    vascular smooth muscle cell

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