MicroRNAs in the Functional Defects of Skin Aging

*Fabien P. Chevalier, Julie Rorteau and Jérôme Lamartine*

#### **Abstract**

Humankind has always been intrigued by death, as illustrated by the eternal quest for the fountain of youth. Aging is a relentless biological process slowly progressing as life cycle proceeds. Indeed, aging traduces an accumulation of physiological changes over time that render organisms more likely to die. Thus, despite our mastery of advanced technologies and robust medical knowledge, defining the molecular basis of aging to control lifespan is still currently one of the greatest challenges in biology. In mammals, the skin is the ultimate multitasker vital organ, protecting organisms from the world they live in. As a preferential interface with the environment, the skin is reflecting the internal physiological balances. The maintenance of these balances, called homeostasis, depends on the concurrent assimilation of diversified signals at the cellular level. MicroRNAs (miRNAs) are noncoding RNAs that regulate gene expression by mRNAs degradation or translational repression. Their relatively recent discovery in 2000 provided new insights into the understanding of the gene regulatory networks. In this chapter, we focused on the role of three miRNA families, namely miR-30, miR-200, and miR-181, playing a key role in the progression of the skin aging process, with particular input in mechanistic considerations related to autophagy, oxidative stress, and mitochondrial homeostasis.

**Keywords:** skin, microRNA, epidermis, keratinocyte, fibroblast, aging, autophagy, oxidative stress, mitochondria, miR-30, miR-200, miR-181

#### **1. Introduction**

Skin, the largest vital organ in the body, is made of three distinct layers from the top to the depth: the epidermis, which is a fine layer of epithelial keratinized cells called keratinocytes; the dermis consisting of fibroblasts in an intracellular matrix with various additional structures such as hear follicles, sweat glands, nerve endings, and capillaries; and the profound subcutaneous tissue called hypodermis. As a physical barrier between the body and the environment, the skin is affected by both intrinsic and extrinsic aging. Intrinsic or chronological aging is a natural continuous dynamic process that normally begins in the mid-1920s. During this inexorable process, the skin undergoes a physiological deterioration characterized by skin atrophy, increased physical and immunological vulnerability, with a reduced capacity of tissue repair in case of wounding. More precisely, intrinsic aging is leading to a 10–50% thinning of the epidermis, the flattening of the dermal-epidermal junction, an atrophy of the dermis with disorganization of the collagen and elastic fibers, a reduction of the microvasculature, and a loose of adipose tissue [1]. The

thinning of the epidermis and the reduction of the skin regeneration capacities are mainly linked to the dysfunctions of the epidermal stem cell compartment, which progressively lose its capacity to generate progenitor cells that are able to ensure the physiological renewal of the epidermis or to sustain wound repair [2].

Skin is also constantly exposed to environmental insults such as ionizing or UV radiations, chemicals, or climatic variations [3]. UV exposure is the main player in extrinsic skin aging and leads to phenotypic changes named photoaging. The photo-aged skin is characterized by a thickening of the epidermis with abnormal keratinocytes differentiation, an accumulation of abnormal elastic tissues (called solar elastosis) with a disorganization and degradation of the collagen fibers in the dermis, an abnormal pigmentation, and an activation of the immune response [4]. Skin is affected by both UV-A and UV-B radiations. UV-B rays are mainly limited to the superficial epidermal part of the skin and directly induce DNA lesions such as cyclobutane dimers and 6–4 photoproducts in exposed cells, leading to keratinocytes senescence, apoptosis, or carcinogenesis [5]. UV-A rays penetrate deeper into the dermis and induce DNA, protein, and lipid damages through the generation of reactive oxygen species (ROS), which in turn activate MAP-kinase p38, JNK, and ERK pathways with induction of the AP1 transcription factor resulting in the expression of the MMP1, -3, and -9 responsible for extracellular matrix degradation [6]. ROS also oxidate cellular components including proteins, lipids, DNA, and RNA, with altered metabolism and further damages.

ROS are also produced during the chronological aging process mainly through mitochondrial activity of the electron transport chain and this is the basis of the free radical theory of aging [7]. It states that mutations acquired in mitochondrial DNA (mtDNA) during life can disrupt metabolisms in the mitochondria and increase ROS. In the skin, mtDNA mutations accumulate with age and UV stress can accelerate this damage. Mitochondrial dysfunctions specifically contribute to skin aging phenotype especially through abnormal pigmentation and hair graying and loss [8]. More precisely, mitochondria play a pleiotropic role in pigmentation by modulating the melanin production through interacting with melanosomes [9]. Moreover, increased oxidative stress linked to mitochondrial dysfunction is observed in aged melanocytes and hair follicle epithelium [10].

Elimination of damaged mitochondria seems then to be a safety mechanism preserving cellular function, tissue homeostasis and organismal soundness. Selective mitochondrial autophagy, named mitophagy, has been described to ensure this function. Autophagy is a cellular quality control mechanism preliminary nonselective playing an essential role in cells for bulk proteins and organelle recycling. Mitophagy modulates the turnover of mitochondria under equilibrium conditions and adjusts the number of mitochondria according to the cellular needs. Increasing evidence suggest that impairment of mitophagy is involved in aging and age-related diseases [11] but the genetics and epigenetics mechanisms modulating mitophagy during aging remain to be better understood.

Multiple epigenetic changes are considered as reliable hallmarks of tissue aging such as modification of DNA methylation motifs, histone post-translational modifications, and modulation of noncoding RNA expression [12]. The latter is an emerging scientific domain in which an incredibly expanding number of studies have been published over the last decade, highlighting day after day the key roles of long noncoding RNAs, circular noncoding RNAs, and microRNAs (miRNAs) in the control of physiologic balances. For example, the stem cell function is governed by numerous factors such as growth factors, cellular metabolism, mediators of inflammation, extracellular matrix, interaction with niche cells, and so on. It has been clearly described that the imbalance between stem cells renewal and commitment can give rise to deleterious effects leading to pathologies or accelerated aging [13, 14]. Recent single-cell analyses

**51**

*MicroRNAs in the Functional Defects of Skin Aging DOI: http://dx.doi.org/10.5772/intechopen.89689*

RNAs, which cooperate with each other and are co-regulated.

from multiple tissues, including epidermis, revealed a clonal heterogeneity of gene expression level among a defined cell population [15–17], thus reflecting a distinct fluctuating transcriptome in individual cells which governs cell fate. Thus, cell fate decisions rely on the integration of dynamic regulatory networks of gene expression and these fluctuating transcriptomes are likely to be under the control of noncoding

Isolated human primary keratinocytes are a valuable model for studying epidermal aging as they retain features of the tissue they are extracted from. Recently, we took advantage of this model to identify miRNAs modulated with chronological aging through a genome-wide expression analysis of cell extracted from skin biopsies of healthy infants (3–6 years old), young adults (20–40 years old), and aged adults (60–71 years old). This microarray screening allowed us to identify 60 miRNAs significantly modulated (*P* < 0.05, fold change >1.5) between at least two of the three sample groups analyzed [18]. Most of them were differentially expressed between the youngest group and the two adult groups. Considering that physiological aging starts as early as 20 years old and that our cell samples were prepared from photo-protected skin areas, one can speculate that constant modulation of miRNAs expression as soon as 20 years could constitute an epigenetic signature of intrinsic chronological aging. Thus, according to our miRNome analysis during aging, this signature would be constituted by the overexpression of miR-181d-5p, miR-1972, miR-200c-5p, miR-30a-3p, miR-30a-5p, miR-30c-2-3p, miR-30c-5p, miR-365a-5p, miR-4298, miR-6812-5p, and miR-6831-5p and the underexpression of miR-4443. Among these miRNAs of the epidermis aging signature, no mechanistic studies are referenced in the literature for miR-1972, miR-4298, miR-6812-5p, and miR-6831-5p, thus limiting any biological interpretation. Furthermore, if few studies are published to date for miR-365 and miR-4443, they mostly come from cancerology studies, as it is traditionally the case for miRNA studies. Considering the debate on the relevance of the cancer cell lines as reliable mechanistic models [19–21], we decided here to focus on data obtained from pathological and physiological models excluding the cancer field, as much as we can. Consequently, we will focus here our attention on miRNA members from three different families. Interestingly enough, the miR-30 family is highly represented in this signature, and a recurrent biological pathway targeted by miR-30 is autophagy. In addition, many members of the miR-200 family have been associated to oxidative damage. Finally, the miR-181 family is progressively enriched with overexpression of additional members (miR-181a-2-3p and miR-181b-5p) with elderly. Several published studies converge toward a control of mitochondrial homeostasis by miR-181. As described earlier, the autophagic flux, the response to oxidative stress and the maintenance of functional mitochondria are all affected with skin aging and thus

constitute cellular processes of particular interest regulated by miRNAs.

The miR-30 family is composed of six members (miR-30a, miR-30b, miR-30c-1, miR-30c-2, miR-30d, and miR-30e) transcribed from three clusters of two genes located on human chromosome 1 (miR-30c and miR-30e), chromosome 6 (miR-30a and miR-30c-2), and chromosome 8 (miR-30b and miR-30d) [22]. Each gene is able to produce two mature miRNA sequences, the 3p and 5p strands (**Table 1**), with various abundances. We specifically observed an induction of miR-30a-3p, miR-30a-5p, miR-30c-2-3p, and miR-30c-5p in aged human skin [18]. In our miRNome analysis, the miR-30a is the most overexpressed miRNA with aging, with a 4- to 6-fold increase depending on the mature strand. For a long time, mir-30a-5p has been associated with the regulation of autophagy in various cancer cells [24, 25]

**2. The miR-30 family in the control of the autophagic flux**

#### *MicroRNAs in the Functional Defects of Skin Aging DOI: http://dx.doi.org/10.5772/intechopen.89689*

*Non-Coding RNAs*

thinning of the epidermis and the reduction of the skin regeneration capacities are mainly linked to the dysfunctions of the epidermal stem cell compartment, which progressively lose its capacity to generate progenitor cells that are able to ensure the

Skin is also constantly exposed to environmental insults such as ionizing or UV radiations, chemicals, or climatic variations [3]. UV exposure is the main player in extrinsic skin aging and leads to phenotypic changes named photoaging. The photo-aged skin is characterized by a thickening of the epidermis with abnormal keratinocytes differentiation, an accumulation of abnormal elastic tissues (called solar elastosis) with a disorganization and degradation of the collagen fibers in the dermis, an abnormal pigmentation, and an activation of the immune response [4]. Skin is affected by both UV-A and UV-B radiations. UV-B rays are mainly limited to the superficial epidermal part of the skin and directly induce DNA lesions such as cyclobutane dimers and 6–4 photoproducts in exposed cells, leading to keratinocytes senescence, apoptosis, or carcinogenesis [5]. UV-A rays penetrate deeper into the dermis and induce DNA, protein, and lipid damages through the generation of reactive oxygen species (ROS), which in turn activate MAP-kinase p38, JNK, and ERK pathways with induction of the AP1 transcription factor resulting in the expression of the MMP1, -3, and -9 responsible for extracellular matrix degradation [6]. ROS also oxidate cellular components including proteins, lipids, DNA, and

ROS are also produced during the chronological aging process mainly through mitochondrial activity of the electron transport chain and this is the basis of the free radical theory of aging [7]. It states that mutations acquired in mitochondrial DNA (mtDNA) during life can disrupt metabolisms in the mitochondria and increase ROS. In the skin, mtDNA mutations accumulate with age and UV stress can accelerate this damage. Mitochondrial dysfunctions specifically contribute to skin aging phenotype especially through abnormal pigmentation and hair graying and loss [8]. More precisely, mitochondria play a pleiotropic role in pigmentation by modulating the melanin production through interacting with melanosomes [9]. Moreover, increased oxidative stress linked to mitochondrial dysfunction is

Elimination of damaged mitochondria seems then to be a safety mechanism preserving cellular function, tissue homeostasis and organismal soundness. Selective mitochondrial autophagy, named mitophagy, has been described to ensure this function. Autophagy is a cellular quality control mechanism preliminary nonselective playing an essential role in cells for bulk proteins and organelle recycling. Mitophagy modulates the turnover of mitochondria under equilibrium conditions and adjusts the number of mitochondria according to the cellular needs. Increasing evidence suggest that impairment of mitophagy is involved in aging and age-related diseases [11] but the genetics and epigenetics mechanisms modulating mitophagy

Multiple epigenetic changes are considered as reliable hallmarks of tissue aging such as modification of DNA methylation motifs, histone post-translational modifications, and modulation of noncoding RNA expression [12]. The latter is an emerging scientific domain in which an incredibly expanding number of studies have been published over the last decade, highlighting day after day the key roles of long noncoding RNAs, circular noncoding RNAs, and microRNAs (miRNAs) in the control of physiologic balances. For example, the stem cell function is governed by numerous factors such as growth factors, cellular metabolism, mediators of inflammation, extracellular matrix, interaction with niche cells, and so on. It has been clearly described that the imbalance between stem cells renewal and commitment can give rise to deleterious effects leading to pathologies or accelerated aging [13, 14]. Recent single-cell analyses

physiological renewal of the epidermis or to sustain wound repair [2].

RNA, with altered metabolism and further damages.

observed in aged melanocytes and hair follicle epithelium [10].

during aging remain to be better understood.

**50**

from multiple tissues, including epidermis, revealed a clonal heterogeneity of gene expression level among a defined cell population [15–17], thus reflecting a distinct fluctuating transcriptome in individual cells which governs cell fate. Thus, cell fate decisions rely on the integration of dynamic regulatory networks of gene expression and these fluctuating transcriptomes are likely to be under the control of noncoding RNAs, which cooperate with each other and are co-regulated.

Isolated human primary keratinocytes are a valuable model for studying epidermal aging as they retain features of the tissue they are extracted from. Recently, we took advantage of this model to identify miRNAs modulated with chronological aging through a genome-wide expression analysis of cell extracted from skin biopsies of healthy infants (3–6 years old), young adults (20–40 years old), and aged adults (60–71 years old). This microarray screening allowed us to identify 60 miRNAs significantly modulated (*P* < 0.05, fold change >1.5) between at least two of the three sample groups analyzed [18]. Most of them were differentially expressed between the youngest group and the two adult groups. Considering that physiological aging starts as early as 20 years old and that our cell samples were prepared from photo-protected skin areas, one can speculate that constant modulation of miRNAs expression as soon as 20 years could constitute an epigenetic signature of intrinsic chronological aging. Thus, according to our miRNome analysis during aging, this signature would be constituted by the overexpression of miR-181d-5p, miR-1972, miR-200c-5p, miR-30a-3p, miR-30a-5p, miR-30c-2-3p, miR-30c-5p, miR-365a-5p, miR-4298, miR-6812-5p, and miR-6831-5p and the underexpression of miR-4443. Among these miRNAs of the epidermis aging signature, no mechanistic studies are referenced in the literature for miR-1972, miR-4298, miR-6812-5p, and miR-6831-5p, thus limiting any biological interpretation. Furthermore, if few studies are published to date for miR-365 and miR-4443, they mostly come from cancerology studies, as it is traditionally the case for miRNA studies. Considering the debate on the relevance of the cancer cell lines as reliable mechanistic models [19–21], we decided here to focus on data obtained from pathological and physiological models excluding the cancer field, as much as we can. Consequently, we will focus here our attention on miRNA members from three different families. Interestingly enough, the miR-30 family is highly represented in this signature, and a recurrent biological pathway targeted by miR-30 is autophagy. In addition, many members of the miR-200 family have been associated to oxidative damage. Finally, the miR-181 family is progressively enriched with overexpression of additional members (miR-181a-2-3p and miR-181b-5p) with elderly. Several published studies converge toward a control of mitochondrial homeostasis by miR-181. As described earlier, the autophagic flux, the response to oxidative stress and the maintenance of functional mitochondria are all affected with skin aging and thus constitute cellular processes of particular interest regulated by miRNAs.
