Mature sequences of miRNAs from miR-30, miR-200, and miR-181 families.
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.
- oxidative stress
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 . 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 .
Skin is also constantly exposed to environmental insults such as ionizing or UV radiations, chemicals, or climatic variations . 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 . 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 . 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 . 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 . 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 . More precisely, mitochondria play a pleiotropic role in pigmentation by modulating the melanin production through interacting with melanosomes . Moreover, increased oxidative stress linked to mitochondrial dysfunction is observed in aged melanocytes and hair follicle epithelium .
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  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 . 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 from multiple tissues, including epidermis, revealed a clonal heterogeneity of gene expression level among a defined cell population [15, 16, 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 (
2. The miR-30 family in the control of the autophagic flux
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) . 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 . 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] and, more recently, in other several types of normal cells such as cardiomyocytes [26, 27], endothelial cells [28, 29, 30], thymocytes , lens epithelial cells , or hepatic stellate cells .
Autophagy is well-conserved catabolic process across phyla that directs the degradation of either bulk or selective cellular components. Molecular mechanisms governing autophagy in mammals has been extensively reviewed . Briefly, the process is regulated by a core machinery involving a step-by-step interaction of multiple molecular partners called autophagy-related (ATG) proteins. The initiation step is under the control of the protein kinase ULK1, which phosphorylates Beclin-1 (BECN1) on S14, thus boosting the activity of the VPS34-P150 complexes that induce the nucleation of the autophagophore. Subsequent phagophore extension requires first the intervention of ATG5-ATG12-ATG16L complexes. The closure of the autophagosome relies then on the activity of the ATG4-ATG3-ATG7 complexes that convert the inactive microtubule-associated protein LC3-I into the active LC3-II form by conjugation with phosphatidylethanolamine. Finally, LC3-II allows the autophagosomes to fuse with lysosomes to form autolysosomes where all contents are enzymatically digested (Figure 1).
Plethora of microRNAs have been shown to modulate the different proteins involved at each step of the autophagic process [35, 36, 37]. Although the six members of the miR-30 family have distinct mature sequences, the seed sequence is perfectly conserved (Table 1). Thus, it is not surprising that other members of the miR-30 family have been involved as well in the negative control of autophagy, such as miR-30b in vascular smooth muscle cell , miR-30c in neurons , miR-30d in astrocytes , and miR-30e in cardiomyocytes . The different members of the miR-30 family have been primarily associated to the targeting of
The functional link between these miRNAs and the mechanisms of skin aging is not clarified yet; however, the decline in effectiveness of autophagy is clearly one of the hallmarks of aging . This has been observed in diverse organisms from nematodes to rats, including human cells, and this is accompanied in part by a downregulation of
In fibroblasts, the defect in the autophagic flux was identified at the final degradation step of the autophagolysosome and was correlated with weakened turnover of dermal extracellular proteins, possibly leading to a collapse of the dermis structure and skin fragility . In keratinocytes, the autophagic process, especially the nucleophagy, plays a key role in the control of the terminal differentiation [63, 64]. In addition, it has been demonstrated that
The importance of the multiple autophagy pathways in skin function has been recently reviewed elsewhere . The causative link between miR-30 members’ induction, autophagy reduction, and tissue aging still needs to be demonstrated especially in skin for miR-30a. Toward this aim, we already showed that a reduced epidermal differentiation is correlated with an abnormal barrier function in an organotypic skin models prepared with keratinocytes artificially overexpressing miR-30a . Finally, the impairment of the various modes of autophagy (aggrephagy, lipophagy and mitophagy) leads to the accumulation of protein aggregates, to the aberrant handling of lipid droplets causing changes in lipid metabolism, and to the accumulation of dysfunctional mitochondria responsible for ROS production, which are all some features of aged tissue (Figure 2), including skin . Interestingly, the upregulation of miR-30b and miR-30d (clustered at the same locus) significantly increases by about twofold in retinal epithelial cells treated with a sublethal dose of H2O2, a potent inducer of ROS . All of these converging data from the literature strongly suggest that the negative regulation of the autophagic pathway by miR-30 could be an important mechanism contributing to tissue aging.
3. The miR-200 family in the control of the oxidative balance
Oxidative stress results from an imbalance between the production of free radicals and their molecular scavengers aiming at the restoration of the redox equilibrium in the cell. At moderate levels, ROS induce biochemical modifications of lipids, proteins, and DNA and thus take part in the signaling cascades controlling cellular processes such as differentiation or trafficking of intracellular vesicles. However, a sustained excessive concentration of ROS will disturb the cellular homeostasis and eventually induces cell death or senescence. Every day, parts of the skin are directly exposed to solar radiations that induce the production of ROS. Since keratinocytes are exposed to both UV-A and UV-B, they display a powerful antioxidant system and an efficient DNA repair machinery compared to dermal fibroblasts.
Since the epidermis turnover takes about a month, even a little decline in this ROS scavenging mechanism will contrariwise strongly affect keratinocytes homeostasis and leads to a progressive accumulation of senescent cells that no longer participate in the regenerative process. Indeed, the steady-state ROS levels were found to be 2.6-fold higher in primary human keratinocytes from old donors (60–82 years) compared to young ones (2–45 years) . This was correlated with a particular increase in 8-hydroxy-2′-deoxyguanosine (8-OH-dG) residues, a highly mutagenic DNA lesion leading to the transversion of GC to TA upon replication by a DNA polymerase [72, 73].
Consistently, a previous report has shown that about 3% of proliferating human keratinocytes contain these oxidized guanines versus 19% in senescent human keratinocytes . The 8-oxoguanine DNA glycosylase (OGG1) is a key enzyme coordinating the removal of 8-OH-dG lesions by catalyzing the first step of the repair process (Figure 1), and its expression level is significantly decreased with aging in keratinocytes .
To note, we have not observed a differential expression of miR-200a-3p in our own miRNA sequencing between keratinocytes from young (3–6 years), adult (20–40 years), and elderly (60–71 years) human skin samples . This difference may come from the differential segregation of young and old keratinocytes in the two studies and/or from the anatomical location of the biopsies as sun-exposed or photo-protected areas would present a different pattern of expression. Nevertheless, the miR-200 family is composed of five members, including miR-141, miR-200a, miR-200b, miR-200c, and miR-429 (Table 1), and we effectively detected a significant twofold increase in miR-200c-5p levels in both adult and aged keratinocytes as compared to the young cells from photo-protected skin biopsies . The functional significance of the elevated level of expression of the so-called passenger strand still needs to be depicted. Recently, a study on miR-122 has opened up a new perspective in the miRNA field as it showed that the passenger strand is not always an innocent bystander but could also cooperates with the guide strand to achieve the same function through different mechanisms . In agreement with our miRNome analysis in aged keratinocytes, miR-200c-3p was increased by about threefold in aged skin fibroblasts (65–80 years) as compared to young ones (4–6 years), isolated from skin biopsies protected from sun exposition . Thus, overexpression of miR-200 family members cannot be solely interrelated to photo-aging but also to intrinsic chronological aging.
Interestingly, the existence of a crosstalk between the miR-200 family and oxidative stress has been investigated in other physiological contexts. Oxidative stress induced by H2O2 treatment in normal liver cells or normal endothelial cells triggers progressive overexpression of all of the five members, with discrepancy among them [77, 78]. Even if the five members of the miR-200 family are divided into two clusters, namely miR-200a/miR-200b/miR-429 on chromosome 1 and miR-200c/miR-141 on chromosome 12, it has been reported that the promoters of both miR-200 clusters comprise TP53-binding sites and that all of the three transcription factors TP53/TP63/TP73 are able to activate the transcription of the miR-200 family members [77, 78, 79, 80]. MiR-200c-3p has been shown to target
Another consequence of miR-200c-induced
Moreover, miR-200a-3p is regulating the ROS-stress response signaling by targeting the MAP kinase p38 alpha (
4. The miR-181 family in the control of mitochondria homeostasis
In our microarray approach aiming at identifying modulated miRNA with epidermis aging, we found out that three members of the miR-181 family were significantly upregulated, namely miR-181-a (fold change 1.61), miR-181-b (fold change 1.54), and miR-181-d (fold change 2.40). Two of which were previously associated with keratinocytes replicative senescence: miR-181a (fold change 1.30) and miR-181b (fold change 1.38) and with human skin aging as well, although this latter result was not statistically significant in this particular study . Additionally, miR-181a was also found to be tightly related to human dermal fibroblasts senescence , making it a consistent miRNA associated with skin aging. The miR-181a and miR-181b are two intronic clustered miRNAs existing in double copies on chromosome 1 (miR-181a-1 and miR-181b-1) and on chromosome 9 (miR-181a-2 and miR-181b-2), whereas the miR-181c and miR-181d constitute a third cluster on chromosome 19 (Table 1).
Multiple targets have been identified for the miR-181, including SIRT1, a key regulator of cell survival in the context of oxidative stress. The essential crosstalk between oxidative stress and SIRT1 has been fully reviewed elsewhere . As discussed previously, SIRT1 deacetylates the FOXO transcription factors and subsequently stimulates the expression of antioxidants. In addition, the SIRT1-FOXO axis is also involved in autophagy induction. SIRT1 promotes the activation of FOXO transcription factors that positively regulate the expression of several autophagy-related genes such as
The disruption of the mitochondrial function is also retrieved when miR-181 are overexpressed, independently of
Remarkably, consistent enriched expression of miR-181 is found in mitochondria across different cell models even though these miRNAs are not encoded in the mitochondrial genome but come from the nucleus [104, 105]. The precise subcellular localization of particular miRNAs at the mitochondria led to the classification of miRNAs such as “mitomiRs,” a group of approximately 60 members . The miR-181a is one of the most consistent—if not the most consistent—mitomiR. Since mitochondria play a key role in the aging process, it is reasonable to assume that mitomiRs disrupt gene regulatory networks eventually contributing to tissue decline with aging. Very recently, a seminal study has demonstrated that miR-181a/b is controlling a group of elemental genes for mitochondrial biogenesis and function . NRF1, a master regulator of mitochondrial biogenesis; the cytochrome
In order to keep up with redox equilibrium, the cell has to maintain a perfectly tuned balance between mitochondrial biogenesis and its recycling. The elimination of defective mitochondria is mediated by the BCL2-related outer membrane protein BNIP3L/NIX, which contains a conserved LC3-binding motif and acts as a receptor for addressing damaged mitochondria to autophagosomes, which then deliver the organelle to lysosomes for degradation and recycling. A recent
Accumulating compromised mitochondria will ultimately lead to the failure of the respiratory chain to produce ATP and will alongside generate even more ROS affecting in cascade the global cell homeostasis. A positive feedback loop may exist between miR-181 and oxidative stress since a recent study has shown that H2O2 was able to boost the expression of miR-181a, probably through the activation of the NF-κB signaling . Altogether, these data show that miR-181 overexpression observed with skin aging would exert a deleterious effect by simultaneously preventing mitochondrial turnover and overactivation of cell death through apoptosis (Figure 2). However, it is to be noted that
5. Circulating miRNAs in the spreading of the aging message
MiRNAs have been found in all biological fluids such as blood, saliva, urine, or breast milk. These circulating miRNAs are very stable as they are protected from RNAse degradation thanks to a packaging into extracellular vesicles (EVs) made of a lipid bilayer [112, 113] or through complexing with carrier proteins [114, 115]. Indeed, an important part of circulating miRNAs is bound to the argonaute (AGO) proteins and is released in the extracellular compartment after cell death . As AGO proteins are very stable in the presence of both RNAses and proteases, conjugated miRNAs are secured . EVs are classically divided into three different groups: (1) apoptotic bodies, with a diameter comprised between 1 and 5 μm, (2) microvesicles, formed by direct budding of the plasma membrane and ranging in size from 0.1 to 1 μm, and (3) exosomes, the smaller EVs derived from an endosomal origin with a diameter ranging between 30 and 150 nm (Figure 1). They represent a new class of paracrine factors mediating cell-to-cell communication . They transfer a complex signal to more or less distant recipient cells through their composite cargo, including proteins, mRNAs, lipids, noncoding RNAs and particularly miRNAs, thus modulating their behavior [112, 118, 119]. Interestingly, miRNAs enclosed in exosomes do not necessarily reflect their relative abundance in the parent cell, indicating that the exosomal miRNA loading occurs through a selective sorting [120, 121]. This allows a controlled released of particular messages to the recipient cells depending on the biological context. The intercellular communication via circulating miRNAs is likely to be involved in aging and age-related diseases. It was recently observed that human senescent dermal fibroblasts, which progressively accumulate in aging tissues , release more exosomes than proliferating cells . In accordance, unpublished data from our group suggest that aged keratinocytes also secrete more exosomes as compared to young keratinocytes.
It is now well admitted that exosomes from senescent cells and their miRNA cargo are part of the senescent-associated secretory phenotype (SASP) . Indeed, most of the miRNAs contained in exosomes are predicted to silence proapoptotic pathways and so could be involved in the propagation of senescent cells in tissue, thus greatly contributing to the aging process . In parallel with an increased secretion of exosomes, two recent studies have demonstrated that immune cells are more capable of exosome uptake by internalization in older people [124, 125]. Even if the molecular mechanisms behind these observations are not understood yet, the decreased clearance of senescent cells by the innate immune system is clearly another factor enforcing tissue aging .
Specific circulating miRNAs have already been associated with different age-related diseases. For example, a concordance has been noticed between miR-29-3p increase in exosomes released by bone marrow mesenchymal stem cells and aging . This miR-29-3p increase leads to insulin resistance in adipocytes, myocytes, and hepatocytes by downregulation of SIRT1 protein level. Insulin resistance is often developing in elderly type 2 diabetes patients. This suggests a significant role of exosomal miRNAs in aging-associated insulin resistance and represents a new therapeutic target. Additionally, circulating miR-34a in plasma is also increased during aging. This increase is even more important in age-related hearing loss patients and has been correlated with a decrease of diverse miR-34a target expression (
In skin, few things are known concerning implication of circulating miRNAs in aging. However, several studies have demonstrated age-related changes of circulating miRNAs expression level in biological fluids [128, 132], including a decrease of miR-181a, miR-200c, and miR-30b in serum of older individuals [133, 134], one representative from each miRNA family that we focused on here. The observation that these miRNAs are increased in aged human primary keratinocytes compared to young cells  but decreased in the serum of elderly people well illustrates the fact that the proportion of miRNAs released by cells does not necessarily reflect the variations in parent cells. In another pathological context, the acute myocardial infarction, miR-30a, is enriched in exosome from patient serum .
Taken together, all of these emerging data exemplify the theory that circulating miRNAs and particularly miRNA exosome cargo play a crucial role in cell-to-cell communication. They are implicated in multiple physiological and pathological mechanisms, including aging and age-related diseases. As they are present in all the biological fluids, and more interestingly in blood, they could be used as biomarkers for various human diseases that limit lifespan. Moreover, the capability of exosomes to transfer information and affect the behavior of distant cells is very interesting for the development of new therapies. In skin context, the exosomes of the different cell types and their roles in skin homeostasis are not really described for the moment. It could be also interesting to consider if some miRNAs implicated in the complementary aging processes, as the ones we described here, are present in exosomes derived from skin cells and how they could affect some gene regulatory networks in recipient cells.
Many miRNAs have been already described in the skin to be involved in either keratinocytes or fibroblasts senescence . However, cellular senescence is only one parameter contributing to tissue aging. In this chapter, we described how miRNAs could drive tissue decline with aging, by regulating complex gene regulatory networks with a special focus on autophagy, oxidative stress, and mitochondria homeostasis. Indeed, since one miRNA is targeting multiple effectors at the same time, a dynamic buffering of inter-related pathways will ultimately tip the balance toward a cell fate or another. Here, we tend to demonstrate that some miRNAs are consistently found to be indirectly or directly associated to diverse mechanisms of aging, namely the members of the miR-30, miR-200 and miR-181 family. Interestingly, the three cellular processes detailed in here are closely nested. Autophagy is crucial to remove havocs due to oxidative stress, including damaged mitochondria. Mitochondria are also a direct source of ROS, so maintaining the homeostasis of this particular organelle ensures a good oxidative equilibrium. One particular finding when we gather different studies from the literature is the fact that the three miRNA families described here, miR-30, miR-200, and miR-181, are all upregulated following an oxidative stress. Since all of them could also worsen this oxidative stress by acting on different pathways, it is not clear what is the cause and what is the consequence so far. Finally, miRNAs are clearly exported out of the parent cell and could possibly diffuse into the whole body. According to all of the emerging data exposed in this chapter, it appears quite clear that circulating miRNAs have a central role in the propagation of functional defects associated to tissue aging. A central fundamental question still remains to date: What is driving in a first place the modulation of miRNAs expression with aging?
Conflict of interest
The authors declare no conflict of interest.