miRNAs in stem cell aging and age-related disease

miRNAs in stem cell aging and age-related disease

Mechanisms of Ageing and Development 168 (2017) 20–29 Contents lists available at ScienceDirect Mechanisms of Ageing and Development journal homepag...

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Mechanisms of Ageing and Development 168 (2017) 20–29

Contents lists available at ScienceDirect

Mechanisms of Ageing and Development journal homepage: www.elsevier.com/locate/mechagedev

Review

miRNAs in stem cell aging and age-related disease Soon Won Choi a b

a,b,1

, Jin Young Lee

a,b,1

, Kyung-Sun Kang

a,b,⁎

T

Adult Stem Cell Research Center, College of Veterinary Medicine, Seoul National University, Seoul 08826, Republic of Korea Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University, Seoul 08826, Republic of Korea

A R T I C L E I N F O

A B S T R A C T

Keywords: Ageing Somatic stem cell MicroRNA Senescence SASP

MicroRNAs (miRNAs) are short, non-coding RNAs that regulate the expression of mRNA targets and play a part in the post-transcriptional silencing. To date, the prominent roles of miRNAs in stem cells have been investigated in a wide range of biological processes, including self-renewal, differentiation and proliferation. In this commentary, we first demonstrate the causes and mechanisms of somatic stem cell aging in a new aspect of miRNAs. The functions of stem cells decline with age in diverse tissues due to cellular damages and congenital disorders. The somatic stem cells exhibit type-specific phenotypes with cellular senescence during the aging process. We explore the specific miRNAs regulating stem cell aging and age-related diseases. The functional investigations of the miRNAs in somatic stem cells and degenerative diseases might facilitate the translation of knowledge into clinical practice for the regulation of stem cell aging and aging-related diseases.

1. Introduction Aging is an unavoidable biological process, which gradually increases risk factors for the onset of age-related diseases. Among the environmental and genetic risk factors, there are a substantial variety of different RNAs that are actively transcribed from the human genome apart from protein-coding mRNA transcripts. To date, short regulatory non-coding RNAs (ncRNAs) have been reported to be implicated in the onset of diseases related to aging. The smallest of the ncRNAs is the miRNA consisting of the single guide strand. The guide strand together with the RNA-induced silencing complex (RISC) directs target recognition to the untranslated region of target mRNAs. This binding event leads to the post-transcriptional silencing (Carthew and Sontheimer, 2009). Somatic stem cells, also referred to as adult stem cells, reside in diverse tissues of the body and provide organs with the abilities to grow and regenerate to maintain homeostasis during their lifetimes. However, the life-long persistence of stem cells in tissues leads to the accumulation of cellular damage, which ultimately may result in cellular senescence and death as well as in the loss of regenerative function. These changes contribute to the impaired response to injuries and diminished regenerative capacity in aged organisms. In this review, we discuss the aging phenotypes in the following stem cell populations: neural stem cells (NSCs), hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs) and muscle stem cells, which are also known as satellite cells of skeletal muscle. We also



1

explore the causes of stem cell aging including extrinsic stresses and hereditary diseases. The role of specific miRNAs in stem cell aging is reviewed in relation to the causes of the functional changes in somatic stem cells. The important roles of miRNAs in age-related diseases, such as age-associated cardiac degeneration and age-related macular degeneration (AMD) as well as cancers are reviewed. 2. The characteristics of stem cell aging 2.1. Stem cells and the organismal aging The life-long persistent stem cells in the body accumulate cellular damage, which may lead to the degeneration and loss of functions of organs. The persistent growth arrest and decreased self-renewal capacity of stem cells contribute to the impairment in tissue regeneration. For example, the decline of the self-renewal capacity of HSCs impairs both the innate and adaptive immune systems (Geiger et al., 2013). Similarly, the decrease of self-renewal of muscle satellite cells has deleterious effects on muscle regeneration (Blau et al., 2015). In addition to the impaired functions, senescent stem cells show distinctive cellular senescence phenotype. Cellular senescence refers to the state of irreversible growth arrest in response to various stress factors. In general, senescent cells exhibit distinguishing characteristics, including increased cell sizes, DNA damage markers and heterochromatin foci. Furthermore, senescent cells are reported to accumulate in aged tissues as indicated by the high senescence-associated β-galactosidase (SA-β-

Corresponding author at: Adult Stem Cell Research Center, College of Veterinary Medicine, Seoul National University, Seoul 08826, Republic of Korea. E-mail address: [email protected] (K.-S. Kang). These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.mad.2017.08.013 Received 29 November 2016; Received in revised form 21 July 2017; Accepted 21 August 2017 Available online 25 August 2017 0047-6374/ © 2017 Elsevier B.V. All rights reserved.

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gal) activity and increased expression of the senescence marker p16Ink4a (Krishnamurthy et al., 2004; Burd et al., 2013). Recently, emerging evidence suggests that senescent cells, particularly senescent fibroblasts induced by DNA damage, secrete pro-inflammatory factors referred to as senescence-associated secretory phenotype (SASP) (Kang, 2015; Herranz et al., 2015; Laberge et al., 2015; Freund et al., 2011). In general, SASP factors are induced at the mRNA levels and encompass a wide range of cytokines, chemokines, proteases and growth factors (Tchkonia et al., 2013; Freund et al., 2010). Senescent cells spread the stress response by SASP and change their microenvironment by interacting with neighboring cells. Senescent stem cells also secrete pro-inflammatory cytokines, which may induce senescence in neighboring cells. Jin et al. reported that the late-passage human MSCs secrete a member of SASP, namely MCP-1 (Jin et al., 2016). The knock-down of CCR-2, which is a receptor recognizing MCP1, improved the therapeutic efficacies of human MSCs in a murine allergic asthma model. Moreover, Insulin-like growth factor binding proteins 4 and 7 (IGFBP4 and IGFBP7) have been reported as secretory factors in senescent human MSCs (Severino et al., 2013). Since two SASPs, IGFBP4 and IGFBP7 were secreted, it induced cellular senescence in the early passage MSCs. The SASP of stem cells may ameliorate the functions of neighboring stem cells as well as exert deleterious effects in the mesenchymal niche. HSCs also exhibited decreased functions and blood output in response to the exposure to interleukin-1 (IL1), which is a key pro-inflammatory cytokine (Pietras et al., 2016). IL-1 treated HSCs exhibited an elevated myeloid output and suppressed lymphoid production that is observed in senescent HSCs. In addition, chronic exposure to IL-1 impaired the self-renewal of HSCs, which suggests that IL-1 may induce the senescence of HSCs. These results imply that secretory cytokines from the niche of stem cells may also induce senescence and impair the functions of residing stem cells in vivo. The senescent stem cells may contribute to the degeneration of organs and consequently organismal aging. The organismal aging is associated with decreased functions of organs and impaired maintenance of tissues. The declined regenerative capacity is accompanied by a dysfunction of stem cells, resulting in degenerative diseases. Furthermore, the ability to regenerate somatic tissues and stem cells is affected by the niche and the systemic milieu. Recent studies demonstrated the contribution of the systemic environment to the regeneration of tissues and stem cells in aging. Young blood infusion into old animals improves cognitive function and exercise capability (Baht et al., 2015; Sinha et al., 2014; Villeda et al., 2014). The circulating protein growth differentiation factor 11 (GDF11) in the young blood rejuvenates muscle stem cells and increased their functional capacities. Understanding the interactions of microenvironments and the specific stem cells is important to study aging and it may contribute to the regenerative therapies.

and T cells. The altered composition of the hematopoietic system may be responsible for the immunosenescence phenotype identified in elderly individuals (Henry et al., 2010). To address this, various factors and mechanisms have been investigated in HSC aging, including cell cycle-related genes and epigenetic changes (Chambers et al., 2007; Dumble et al., 2007; Miyamoto et al., 2007). Florian et al. reported that an age-dependent increase of RhoGTPase Cdc42 activity results in the decline of functional HSCs (Florian et al., 2012). Furthermore, comprehensive study using mouse HSCs revealed the relationship between the age-associated decline of HSCs and epigenetic changes (Beerman et al., 2013). The physiological aging led to the hyper-methylation of differentiation promoting genes, resulting in a functional decline in HSCs. 2.2.2. Mesenchymal stem cells MSCs have been commonly used in clinical trials because they have potent immunomodulatory properties and they are safe to use in therapeutic applications (Kim et al., 2013, 2015; Le Blanc et al., 2008; Reinders et al., 2013). For the application of MSCs as clinical therapeutics, the regulation of cellular senescence is important to protect from decline in their functions (Yu et al., 2014). BMI1 has been reported to improve therapeutic effects of hMSCs through the regulation of SASP and immune modulatory pathways (Jin et al., 2016; Lee et al., 2016). In addition to the therapeutic potentials, MSCs have significance for the investigations of the mechanisms of aging and progeroid. Interestingly, MSCs from progeria patients exhibit a wrinkled nuclear morphology and impaired differentiation potentials as well as phenotypes of premature senescence (Scaffidi and Misteli, 2008; Liu et al., 2011). Progeria is a premature aging disorder such as Hutchinson-Gilford Progeria Syndrome (HGPS) and Werner syndrome. The patients of the diseases recapitulate physiological aging at early age. Importantly, MSCs differentiated from HGPS patient-iPSC show accelerated aging phenotype. Because of these properties, mesenchymal stem cells have been used to elucidate the mechanism of physiological aging and premature aging syndrome (Liu et al., 2011; Zhang et al., 2011). 2.2.3. Muscle stem cells Muscle stem cells (MuSCs), also referred to as satellite cells, regenerate muscle tissues in response to damage signals. With age, satellite cells lose their self-renewal and repair abilities, which result in declined regeneration of muscle tissues (Blau et al., 2015; Bernet et al., 2014). Conboy et al. reported that notch signaling is a key factor in the muscle regenerative capability that declines with age (Conboy et al., 2003). Aged muscle exhibited an impaired response to injury as a result of a diminished activation of Notch signaling. In contrast, enforced activation of Notch improved the regenerative potential in aged muscle. Furthermore, similar to HSCs, senescent MuSCs have a biased differentiation potential that drives to a fibrogenic lineage rather than a myogenic lineage, because of altered Wnt and TGF-β signaling (Brack et al., 2007). In aged mice, the functional decline and biased differentiation potential of MuSCs lead to the loss of recovery from injuries.

2.2. Somatic stem cells with age 2.2.1. Hematopoietic stem cells Among somatic stem cells, HSCs are the most intensely investigated and well-characterized stem cells. Interestingly, the number of HSCs, defined by its surface markers, increases in old mice and humans (Dykstra et al., 2011; Kowalczyk et al., 2015). The alterations in proliferation and self-renewal potential were analyzed via clonal assays as well as single cell RNA-sequencing. These studies revealed that purified HSCs lose their self-renewal as demonstrated by serial transplantation assays in vivo and proliferation assays in vitro. The senescent HSCs directly affect the innate and acquired immune systems through the changes in the populations of immune cells. The exclusive property of HSC aging is the skewed differentiation into the myeloid lineage at the cost of the lymphoid lineage (Dykstra et al., 2011; Geiger and Rudolph, 2009). Young HSCs produce a balanced myeloid and lymphoid progenitor cells. However, aging causes increased differentiation of HSCs into myeloid progenitor cells and results in decreased production of B

2.2.4. Neural stem cells To date, individual senescence phenotypes have not been reported in NSCs. In vivo, the number of NSCs decreases with age, which suggests that the loss of NSCs may lead to age-related neurodegeneration (Maslov et al., 2004; Silva-Vargas et al., 2013). The neural progenitors of aged mice have a decreased self-renewal potential, which correlates with an increased expression of p16Ink4a (Molofsky et al., 2006). Hmga2, which is highly expressed in fetal development and tumors, has a critical role in fetal NSCs and it declines with age. Furthermore, the NSCs of Hmga2 knockout mice exhibit decreased proliferation with increased p16Ink4a and p19Arf expressions (Nishino et al., 2008). HMGA2 indirectly regulates p16Ink4a and p19Arf expressions through binding to JunB locus. In addition to Hmga2, Foxo3 is indispensable in the maintenance of NSC homeostasis (Renault et al., 2009). Knock-out 21

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caused the increased acetylation of H4K16 at the Hoxa9 promoter, resulting in the reduced tri-methylation of H3K27 and the up-regulation of Hoxa9. Moreover, the repressive marker, H3K27me3 increased with age in both HSCs and MuSCs (Sun et al., 2014; Liu et al., 2013). In HSCs, the increased pattern of H3K27me3 was consistent with the decline of the lymphoid differentiation potential in aged HSCs (Sun et al., 2014). Senescent MuSCs also exhibited altered patterns of H3K4me3 and H3K27me3 in myogenic genes (Liu et al., 2013). The age-dependent altered epigenetic markers revealed that the accumulation of epigenetic changes may result in a functional decline in stem cells. In MSCs, histone deacetylase inhibitors increased the acetylation of histones adjacent to the coding regions of miRNAs, which engage in MSC aging (Lee et al., 2011).

of Foxo3 decreased the self-renewal potential of NSCs in mice. Furthermore, NSCs isolated from Foxo3 knock-out mice exhibited impaired ability to generate neural lineages. These identified factors may provide important clues in age-associated neurodegeneration. 2.3. Causes of stem cell aging Though somatic stem cells exhibit different senescence phenotypes according to the lineages, they have several causes in common. The persistence of stem cells in vivo and in vitro makes them susceptible to the accumulation of cellular damage. The cellular damage encompass DNA damages, epigenetic alterations and metabolic changes. As mentioned above, progeria syndromes, which are rare congenital disorders, result in senescence specifically in MSCs. These causative factors interact with mechanisms of aging, thereby resulting in the previously described phenotypes.

2.3.3. Metabolism Quiescent somatic stem cells maintain a low-rate of metabolism to avoid cellular damage from ROS and ensure life-long tissue renewal abilities (Folmes et al., 2012). Nutrient sensing signalings, such as the mTOR, Akt and AMPK pathways, regulate the balance between quiescence and proliferation in stem cell aging (Jasper and Jones, 2010). Caloric restriction delays age-related pathologies and increases the lifespan of diverse species; thus, the underlying mechanism has been vigorously investigated in stem cells (Igarashi and Guarente, 2016; Cerletti et al., 2012). Caloric restriction is thought to enhance stem cell function and proliferation via mTOR signaling, IGF signaling, and AMPK signaling (Ito and Suda, 2014). Many somatic stem cells reside in hypoxic niche limiting ROS; thus, the effects of a hypoxic environment are also thought to have a role in stem cell aging (Takubo et al., 2010). Hypoxia inducible factors stabilized by hypoxia directly associate with the quiescence, proliferation and oxidative metabolism pathways of stem cells (Beegle et al., 2015; Tsai et al., 2011).

2.3.1. DNA damage The DNA damage accumulates with age in various organs and tissue-resident stem cell lineages. Moreover, an impaired DNA damage repair is one of the main causes of the stem cell aging. A DNA damage response is induced when single stranded DNA is exposed or DNA double-strand breaks are generated during the cell cycle or in response to the reactive oxygen species (ROS). The accumulation of DNA damage may alter the functions of genes through mutations or chromosomal rearrangements. Somatic stem cells are equipped with advantages in the cell cycle and metabolism; however, robust activation of the DNA damage response and the subsequent activation of tumor suppressor genes may lead to senescence or decline in functions. For example, physiological aging results in declined functional capacities of HSCs due to the accumulation of DNA damage (Rube et al., 2011). HSCs from the mice deficient in DNA damage repair also exhibited a loss of proliferative potential, diminished self-renewal and functional exhaustion (Rossi et al., 2007). In addition, culture of human MSCs in a low oxygen environment increased proliferation with decreased foci of 53BP1, a well-characterized DNA damage repair marker (Estrada et al., 2012). It would be possible to prevent the age-associated defects of stem cells by an increase in the DNA repair pathway.

2.3.4. Abnormal nuclear lamin proteins The premature aging disease Hutchinson-Gilford Progeria syndrome (HGPS) is caused by progerin, a mutant form of the nuclear membrane protein lamin A. HGPS is a unique ‘window’ to investigate the mechanisms of aging because the disease has in common with physiological aging, including an abnormal nuclear morphology and increased DNA damage (Scaffidi and Misteli, 2006). Besides, lamin A processing enzyme, ZMPSTE24, has a causative role in nuclear lamina defects and premature senescence. The depletion of ZMPSTE24, which is a metalloproteinase involved in prelamin A processing to produce mature lamin A, leads to nuclear architecture abnormalities, a shortened lifespan, and multiple aging-related phenotypes (Espada et al., 2008). Importantly, MSCs that express progerin or prelamin A exhibit a senescence phenotype with a loss of the differentiation potential (Scaffidi and Misteli, 2008; Espada et al., 2008; Yu et al., 2013a). MSCs differentiated from HGPS patient fibroblast-derived iPSCs also retain nuclear abnormalities, DNA damage and senescence with high expression of progerin (Zhang et al., 2011). In addition, modeling of Werner syndrome, another premature aging disorder, leads to an accelerated cellular senescence of MSCs with changes in the heterochromatin architecture (Zhang et al., 2015a).

2.3.2. Epigenetic alterations The term “epigenetic” refers to changes of the genome without a change in sequence (Brunet and Berger, 2014). In a broad sense, the term indicates the mode of genomic regulation that is not encoded in nucleotides (Goldberg et al., 2007; Benayoun et al., 2015). Specifically, “epigenetic” defines heritable alterations in the gene expression or cellular phenotype without changes in DNA sequence. Diverse epigenetic changes encompass modifications of histones, including methylation, acetylation, ubiquitination and changes of chromatin remodeling. Many histone modifications are involved in activation or suppression of genes and have regulatory roles in transcriptional initiation or elongation. In addition, the age-dependent altered expression of chromatin modifying enzymes could induce epigenetic changes in senescent stem cells. Changes of histone modifications and chromatin remodeling proteins in stem cell aging have been intensely investigated as well as age-dependent chromatin profilings. For example, polycomb group proteins, which are transcriptional suppressors, repress the INK4a locus to prevent senescence with the suppressive histone marker H3K27me3 (Martin et al., 2013; Jacobs et al., 1999). Interestingly, BMI1 has a significant role in the immunomodulatory functions as well as the regulation of senescence of hMSCs (Lee et al., 2016). BMI1 increased COX-2/PGE2 synthesis which is critical in immune suppressive properties by direct suppression of DUSP1 with repressive histone marks. BMI1 also has been reported to increase the self-renewal potential and maintain quiescence of human HSCs (Rizo et al., 2008). Another chromatin modifier, Sirt1 controls HSC homeostasis through epigenetic regulation of Hoxa 9 (Singh et al., 2013). Sirt1 deletion

3. miRNAs in stem cell aging Most miRNAs regulating senescence of stem cells have been reported in HSCs and MSCs. miRNAs control the senescence of stem cells by targeting genes involved in DNA damage, epigenetic changes, and metabolism as previously discussed. We will discuss reported miRNAs and their mechanisms in aging of stem cells in relation to their direct targets (Fig. 1, Table 1). 3.1. let-7 The let-7 miRNA family was one of the first members of miRNAs 22

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Fig. 1. Summary of human somatic stem cell types and age-related diseases. The specific miRNAs discussed in this review are indicated for proper cell type or disease.

newborn mice. These results imply that the role of HMGA2 in NSC aging is closely associated with the let-7b expression. Based on these findings, Yu et al. reported that the over-expression of anti-let-7b additionally to SOX2 increased the efficiency of direct reprogramming of human somatic cells into human induced NSCs (Yu et al., 2015). In hMSCs, histone deacetylase inhibitor induced senescence that miRNAs of the let-7 family were up-regulated during the senescence process, thereby decreasing HMGA2 expression (Lee et al., 2011). The diminished expression of HMGA2 resulted in the activation of the p16INK4a gene, which induced the senescence phenotype of human MSCs. Taken together, these data suggest that let-7 regulates senescence in both NSCs and MSCs via the suppression of HMGA2, which further increases p16INK4a.

identified to regulate stem cells. Expression of the evolutionarily conserved miRNA family is hardly detectable in the embryonic stages; however, it increases in mature tissue during development (Schulman et al., 2005). Similarly, let-7 is expressed at a low level in cancer, and enforced expression reduces the progression and cell proliferation of cancer cells (Lee and Dutta, 2007; Johnson et al., 2007). In NSCs, HMGA2, a target gene of let-7b, is highly expressed in fetal mice; however, the expression declines with age due to an increase of let-7b (Nishino et al., 2008). Furthermore, the over-expression of let-7b reduced the formation of neurospheres with decrease of the Hmga2 expression, and increased the p16Ink4a expression in NSCs. Interestingly, the over-expression of Hmga2 that lacks the let-7b binding sites significantly increased the neurospheres formation in old mice similar to 23

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Table 1 Senescence-associated miRNAs and their miRNA target(s). miRNA

Cell types

mRNA Targets

Mechanisms

References

Let-7

Neural stem cells Mesenchymal stem cells Neural stem cells

HMGA2

decreases self-renewal increases p16Ink4a expression

Lamin A/C

suppresses prelamin A expression

p85a, IGF-1, B-myb CNOT6 HDM4, p53

activates p53 pathway

miR-34a

Muscle stem cells Mesenchymal stem cells Mesenchymal stem cells

activates p53 pathway regulates ROS production

miR-212/132 miR-146 miR-195 miR-196 miR-141

Hematopoietic stem cells Hematopoietic stem cells Mesenchymal stem cells Mesenchymal stem cells Mesenchymal stem cells

miR-335 miR-431 miR-486-5p miR-543/miR-590-3p

Mesenchymal stem cells Muscle stem cells Mesenchymal stem cells Mesenchymal stem cells

FOXO3 TRAF6 Tert HOXB7 ZMPSTE24 CDC25A AP-1 SMAD4 SIRT1 AIMP3/p18

regulates FOXO3-dependent autophagy suppresses NF-kB and IL-6 expression shortens telomere length suppresses proliferation and differentiation increases prelamin A expression inhibits cell proliferation suppresses migration, differentiation and proliferation increases myogenic differentiation induces senescence SIRT1 dependently suppresses tumor suppressor AIMP3/p18

Maslov et al. (2004) and Lee and Dutta (2007) Folmes et al. (2012) Jung et al. (2012a) Hu et al. (2014) Ugalde et al. (2011) Boon et al. (2013) Zhang et al. (2015b) Mehta et al. (2015) Bhaumik et al. (2008) Okada et al. (2016) Candini et al. (2015) Burk et al. (2008) Qiu and Kassem (2014) Lee et al. (2015) Tome et al. (2014) Tome et al. (2014) Kim et al. (2012) Lee et al. (2014)

miR-9 miR-29

cancer biology because of its low expression level in cancer and its target, p53 (Rokhlin et al., 2008; Wiggins et al., 2010). Analysis of clinical samples implicates the functional significance of miR-34, in which miR-34a also represses HDM4, a negative regulator of p53 (Okada et al., 2014). In MSCs, the expression level of miR-34a was increased according to the passage number (Park et al., 2015). Furthermore, over-expression of miR-34a decreased cell cycle regulators, such as cell cycle-dependent kinases and cyclins, with the exception of p53 and p21. The treatment of miR-34 also reduced the differentiation efficiencies of human MSCs into osteogenesis and adipogenesis. Another study investigated by Zhang reported that miR-34a exhibited an increased expression level in hypoxia and serum depleted environment and over-expression of miR-34a induced senescence in MSCs of rats (Zhang et al., 2015b). The underlying mechanism of the functions of miR-34a is the Sirt1- and Foxo3a-dependent ROS production. miR-34a induced senescence of MSCs; however, the treatment of NAC, which is an ROS scavenger, alleviates the senescence process.

3.2. miR-9 miR-9 is a brain specific miRNA that plays a key role in neural development and neural cell behaviors. The differentiation of HGPS patient derived iPSC revealed that the lack of lamin A/C appeared in neurons due to the high expression of miR-9 (Nissan et al., 2012). The experiments using mi-Rs and anti-miRs in MSCs from HGPS-iPSC confirmed that functional effects of miR-9 on lamin A/C expressions. These results suggest that the physiological effects of miR-9 on the absence of neural degeneration in HGPS patients. Furthermore, progerin expressing knock-in mice exhibited extremely low levels of progerin and prelamin A in the brain as a result of the effects of miR-9 (Jung et al., 2012a). A luciferase reporter assay confirmed that miR-9 binds to the specific site of prelamin A 3′-UTR (untranslated region), which results in the depletion of prelamin A in neural tissues and NSCs. The knock-in mice did not exhibit a senescent phenotype in neural tissues because of the low expression level of prelamin A. These results imply that the expression of miR-9 in neurons leads to the protection of neural cells from mutant progerin induced premature senescence.

3.5. miR-212/132

3.3. miR-29

The miR-132 cluster is another miRNA that regulates the aging of HSCs (Mehta et al., 2015). The miR-212/132 cluster (Mirc19) was highly expressed particularly in long-term HSCs. The enriched miRNAs play a role in the maintenance of appropriate hematopoiesis by targeting the transcription factor FOXO3, a significant regulator of senescence. Furthermore, deletion of miR-132 in mice decreased the hematopoietic output and reconstitution potential with age. miR-132 leads to FOXO3-dependent alterations in the autophagy and viability of HSCs.

Early studies using miR-29 include the tumor suppressive mechanisms. miR-29 suppresses tumor growth by increasing p53 expression. miR-29 expression has been reported to increase in the muscles of old rodents (Hu et al., 2014). miR-29 impaired muscle progenitor cell proliferation in old mice by binding to the 3′-UTR of p85a, IGF-1, and Bmyb. In addition, miR-29 modulated the DNA damage response through binding of Ppm1d in Zmpste24 deficient mice (Ugalde et al., 2011). The expression of miR-29 was increased in Zmpste24 knock-out mice and old mice compared with normal young mice. The increase in the expression was dependent on p53 expression. In addition, senescent human MSCs exhibited an escalated expression of miR-29 (Wagner et al., 2008). CNOT6 was identified as the target gene of miR-29 and activated both the p53-p21 and p16-pRB pathways in human MSCs (Shang et al., 2016). These reports indicate that miR-29 may regulate senescence in MSCs and the muscle progenitor cell p53 pathway dependently.

3.6. miR-146 miR-146 has been reported as a negative regulator of cancer metastasis via the suppression of NF-kappa B (Bhaumik et al., 2008). In addition, increased NF-kappa B in senescent fibroblasts induced miR146a expression with the correlation of IL-1alpha (Bhaumik et al., 2009). Intriguingly, the depletion of miR-146a leads to a decline in the number and quality of HSCs (Zhao et al., 2013). The expression of miR146a decreased during the differentiation of HSCs, particularly in myeloid progenitor cells. Furthermore, a competitive repopulation assay indicated that miR-146a knock-out HSCs lost their ability to generate the entire hematopoiesis in vivo. miR-146 is involved in the dysregulated inflammatory hematopoiesis by targeting TRAF6, transcriptional factor NF-kB. These reports suggest that chronic inflammation could be a cause of the age-dependent decline of HSC function.

3.4. miR-34a Members of the miR-34 family also serve as important regulators of senescence through their roles in diverse pathways that include cell cycle, telomere shortening, and the DNA damage response (Boon et al., 2013; Sun et al., 2008). Specifically, miR-34 has been investigated in 24

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3.7. miR-195

implies their therapeutic potentials.

A recent study reported that the increased expression of miR-195 in human MSCs from old donors shortened the length of telomeres (Okada et al., 2016). Among the miRNAs that exhibited increased expression levels in old MSCs, inhibition of miR-195 significantly reduced the expression of SA-β-gal. miR-195 directly targeted the 3′-UTR of the Tert gene, and knock-down of the miRNA elongated the telomere length. In addition, the abrogation of miR-195 improved the therapeutic effects in cardiac repair as well as the proliferative ability of human MSCs. The transplantation of old MSCs, which have a decreased expression level of miR-195, exhibited an improved effect for cardiac repair. These results demonstrated that miR-195 regulates therapeutic functions via suppression of senescence in human MSCs.

3.11. miRNAs related to MSC aging In addition to the described miRNAs that are reported as regulators of senescence in cancer and stem cell biology, several miRNAs also have been reported in MSC aging. miR-335 is increased in human bone marrow-derived MSCs from old donors as well as in senescent MSCs induced by diverse stimuli (Tome et al., 2014). Forced expression of miR-335 induced a senescent phenotype in human MSCs and further disabled the chondrogenic differentiation capacity and immunomodulatory properties via the suppression of AP-1, which regulates the migration, differentiation and proliferation of cells. In human adipose-derived MSCs, miR-486-5p was increased in response to the replicative senescence, and over-expression of the miRNA induced premature senescence (Kim et al., 2012). miR-486-5p exerts the inhibitory effects by binding to the 3′-UTR of the SIRT1 gene, which is important in the aging of mammalian cells. miR-543 and miR-590-3p also have been reported to regulate the cellular senescence of human MSCs by the suppression of AIMP3/p18 pathway, which is a well-established tumor suppressive mechanism (Lee et al., 2014). The protein expression level of AIMP3/p18 increased in senescent MSCs, and the regulation of AIMP3/p18 controlled the cellular senescence and differentiation potential of MSCs. Among the predicted miRNAs that target AIMP3/p18, miR-543 and miR-590-3p significantly decreased the AIMP3/p18 expression. Furthermore, over-expression of miR-543/miR590-3p inhibits the senescence of late passage MSCs, and the inhibition of miR-543/miR-590-3p induced senescence via an increase of AIMP3/ p18. These miRNA dependent senescence mechanisms reported in MSCs might be applied for the cancer therapy and further investigation into other lineage stem cells.

3.8. miR-196 miR-196 is another miRNA up-regulated in senescent human MSCs from old donors. Compared with a pediatric group, MSCs obtained from an adult group exhibited an increased expression level of miR-196 and decreased expression of Ki-67. The silico analysis revealed that HOXB7 is a direct target of miR-196; moreover, the over-expression of HOXB7 increased the proliferation and osteogenic differentiation of human MSCs (Candini et al., 2015). The age-dependent dynamics of HOXB7 in human MSCs was also confirmed in skeletal tissues of old mice. The immunohistochemical studies exhibited a progressive decline of HOXB7 staining in the endosteal and periosteal areas. 3.9. miR-141-3p It has been reported that miR-200 family members (miR-200a, miR200b, miR-200c, miR-141 and miR-429) are regulators of EMT (epithelial to mesenchymal transition) and mediate the feedforward loop with ZEB1, which has critical effects on various malignant tumor progressions (Burk et al., 2008; Mateescu et al., 2011). Furthermore, miR200c inhibited the expansion of breast cancer cells and mammary duct formation from mammary stem cells via the direct suppression of BMI1, which is essential for the self-renewal of stem cells (Shimono et al., 2009). Interestingly, the miR-141-3p expression is increased during the senescence of human MSCs, thereby inducing prelamin A via the direct inhibition of ZMPSTE24, an enzyme that processes prelamin A into Lamin A (Yu et al., 2013b). The senescent human MSCs induced by replication and the treatments of HDAC inhibitors exhibited an abnormal nuclear morphology because of increased prelamin A. The expression of miR-141-3p is dependent on the histone acetylation at the promoter during replicative and HDAC-inhibitor-induced senescence. miR-141-3p also targets CDC25A and results in G1-phase cell cycle arrest and impaired osteogenic differentiation of human MSCs (Qiu and Kassem, 2014). The miR-141-3p significantly decreased the proliferation of human MSCs among the same precursor families. The downregulation of CDC25A, the direct target of miR-141-3p, also inhibited the cell proliferation of human MSCs.

4. miRNA in age-related disease It is clear that miRNAs play an important role in nearly all aspects of cellular functions as well as in disease initiation and progression (Ruan et al., 2009; Garzon et al., 2009). According to two studies regarding the rejuvenation of aged mice by parabiosis with young mice, the introduction of blood from young mice into old mice restored the regenerative capacity of skeletal muscle stem cells (Conboy et al., 2005) and improved cognitive function and synaptic plasticity (Villeda et al., 2014). In many studies, miRNAs have been reported to exist in plasma and serum (Freedman et al., 2016; Noren Hooten et al., 2013; Huang et al., 2013a). Furthermore, the expression of miRNAs and their mRNA targets altered with age in circulating peripheral blood mononuclear cells (Lai et al., 2014; Serna et al., 2012; ElSharawy et al., 2012; Noren Hooten et al., 2010). Current evidence supports the association and role of various miRNAs with age-related diseases. Recent research elucidating the exact role miRNAs may serve as potential candidates for therapeutic interventions. 4.1. miRNAs in cardiovascular diseases Age-associated cardiac degeneration is the major risk factor for cardiovascular disease (CVD), which is regulated by biochemical pathways and genetic processes (Zhang et al., 2012; Rippe et al., 2012; Dutta et al., 2012). CVD is characterized by a series of complex events, which consist of left ventricular hypertrophy, diastolic dysfunction, an increased risk of atrial fibrillation, valvular degeneration and fibrosis (Zhang et al., 2012; van Rooij et al., 2008; Ikeda et al., 2007). The main molecular hallmarks of cardiac aging in mammals are characterized as genomic instability, telomere attrition, epigenetic alterations, mitochondrial dysfunction, cellular senescence and stem cell exhaustion (Seeger et al., 2013; Lopez-Otin et al., 2013; Rodier and Campisi, 2011). During physiological aging, there is a gradual accumulation of senescent cells in cardiovascular tissues, the outcomes of which include

3.10. miR-431 Lee et al/ reported that miR-431 played a pivotal role in the myogenic ability of skeletal muscle with age (Lee et al., 2015). Transfection of miR-431 improved the myogenic differentiation of senescent myoblasts among miRNAs which were decreased with age. miR-431 directly targets SMAD4, which negatively regulates myogenic differentiation. The down-regulation of SMAD4 also promoted myogenesis in old myoblasts. In muscular tissue regeneration in vivo, the SMAD4 expression levels increased in response to the injury and remained elevated in old muscle. In contrast, miR-431 exhibited a lower expression in old muscles than young muscles. Interestingly, siSMAD4 and miR-431 treatments in old muscles enhanced the regenerative capability, which 25

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miR-23 and their inhibition suppresses postnatal retinal vascular development (Zhou et al., 2011). Another mouse model has demonstrated that pre-miR-31, -150, and -184 reduce ischemia-induced retinal neovascularization (NV AMD). In addition to NV AMD, increased oxidative stress with age causes RPE dysfunction, such as phagocytosis, which is crucial for the maintenance of photoreceptors. miR-184 is down-regulated in primary cultures of human RPE cells isolated from the eyes of AMD donors and in oxygen-induced retinopathy (OIR) mouse retina (Murad et al., 2014; Shen et al., 2008). Inhibition of miR-184 significantly reduced the phagocytosis efficiency in adult RPE cells (Murad et al., 2014). In contrast, the inhibition of miR-410 enhances phagocytosis in human adult stem cells (Choi et al., 2015). miR-17 is also up-regulated in OIRderived RPE cells and targets several pro-angiogenic genes (Doebele et al., 2010). However, miR-17 has also recently been shown to be antiapoptotic (Song et al., 2015). The miR-17-92 cluster is a negative regulator of angiogenesis, and miR-92a is up-regulated in RPE cells after oxidative stress as well as mouse retinae after OIR (Anand and Cheresh, 2011).

several age-related diseases (Rippe et al., 2012; Tsirpanlis, 2008). miRNAs play an important role in adult cardiovascular diseases by targeting key factors in the cardiovascular system, such as hypertrophic growth, a process defined as an increase in cardiomyocyte cell size without an increase in cell number (Topkara and Mann, 2011). This cardiomyocyte expansion leads to not only molecular changes, including α-MHC, β-MHC, ANF and SERCA2, but also morphological changes, including cardiac hypertrophy. Importantly, the reactivation of this specific set of key cardiac genes is regulated by miRNAs. In a mouse model of cardiac hypertrophy, miR-22 over-expression inhibits cell cycle progression by targeting ERRa, CDK6, SIRT1, HDAC4 and Sp1 (Jazbutyte et al., 2013; Huang et al., 2013b). miR-22 is sufficient to provoke dilated cardiomyopathy, a cardiac remodeling, in response to stresses by directly repressing SIRT1 and HDAC4. Furthermore, miR-22 and miR-1 are differentially expressed in Ca2+ transients and contractile work, which are regulated by PLN, RYR2, SERCA2a and α-MHC. In the aged heart, the α-MHC and Ncx1 genes, which are associated with the aging process, are up-regulated. Moreover, the ATPase pump Serca2a, which is reported to be a suppressor of senescence, is gradually down-regulated as the Ca2+ uptake into the sarcoplasmic reticulum (SR) decreases (Lafferty-Whyte et al., 2009). A change in the intracellular free Ca2+ is a common cause of cardiomyocyte death. Notably, miR-1 at the early stage of aging suppresses the aging process through marked down-regulation of α-MHC and Ncx1 for efficient Ca2+ uptake into the SR, whereas the over-expression of miR-22 impairs the Ca2+ transient and its reloading into the SR (Lafferty-Whyte et al., 2009; Takaya et al., 2009). miR-214 has also been identified as a central regulator of Ca2+ transients, which represses Ca2+ overload and cell death by directly targeting NCX1, BIM, CaMKIId, and Cyclophilin D (Liu et al., 2013). Moreover, miRNAs play a major role in cardiac fibrosis (CF). In general, miR-23, 29, 30, and 133 are down-regulated at the end stage of myocardium hypertrophy, whereas miR-21, 24, and 29 are key players of CF regulation in cellular senescence (Jazbutyte et al., 2013; Wang et al., 2012; Duisters et al., 2009). For example, these three key miRNAs are involved in CF proliferation and fibrosis. The miR-21 levels are selectively increased in fibroblasts of the failing heart and regulate the ERK-MAP kinase signaling pathway in cardiac fibroblasts (Thum et al., 2008). miR-24 belongs to the miR-23 cluster, and miR-23 is down regulated after myocardial injuries (Topkara and Mann, 2011). The over-expression of miR-24 reduces the conversion of CFs to myofibroblasts and attenuates the serum-mediated migration of CFs by repressing the TGF-β pathway (Wang et al., 2012). miR-29b regulates CF by targeting cytokines and growth factors, such as leukemia inhibitory factor (LIF) and insulin-like growth factor 1 (IGF-1), as well as a member of the pentraxin superfamily, pentraxin 3 (PTX-3) (Abonnenc et al., 2013). miR-29b also attenuated the cardiac fibroblast response to transforming growth factor-β.

4.3. miRNAs in cancer Cancer increases common cellular proliferation, thus bypassing an irreversible growth arrest. This cellular change is referred to as carcinogenesis. Cancer in elderly individuals is a complex disease, which involves various changes in gene expression that cause cancer development, including cell proliferation-mediated metastasis, invasion and angiogenesis. Many studies have discussed the importance of miRNAs in cancer biology by regulating the expression of their target mRNAs to enhance tumor growth, invasion, angiogenesis and immune evasion (Esquela-Kerscher and Slack, 2006). In addition, miRNA profiles in tumors may identify subtypes, potential survival rates and responses to specific treatments (Lu et al., 2005). For the classification of cancer by miRNAs, the WHO arranged leukemia subtypes with miRNAs (Wandt et al., 2010). Breast cancer and prostate cancer have been reported to have distinct miRNA expression patterns (Liu et al., 2012; Farazi et al., 2014). The miRNA analysis according to the investigated platform may provide accuracy with respect to the current diagnosis. Many examples have been reported; however, miRNAs as tumor suppressors are the most intensely investigated in tumor biology. The miR-15/16 cluster, let-7 paralogs and miR-34 family are the major tumor suppressor miRNAs. miR-15 and miR-16 are the first established miRNAs associated with cancer (Calin et al., 2002). The loss of miR-15/16 has been observed in prostate cancer and multiple myeloma (Ambs et al., 2008; Roccaro et al., 2009). miR-15/16 deteriorated the tumor-supporting ability of stromal cells in vitro and in vivo via the direct suppression of Fgf2, which enhances the survival, proliferation and migration of tumor cells (Musumeci et al., 2011). The let-7 family has been considered tumor suppressors because they have been reported to be down-regulated in human lung, breast and cervical cancers (Calin et al., 2004). RAS, an oncogene, was constitutively over-expressed in response to the loss of the let-7 family (Johnson et al., 2005). Several laboratories have reported that members of the miR-34 family directly target p53, and the up-regulation of the family induced apoptosis and cell-cycle arrest (He et al., 2007). In addition, miR-34a targets BCL2 and MYCN and causes the tumor suppressive phenotype in neuroblastoma (Bommer et al., 2007). Moreover, miR-34b suppresses cell proliferation via the down-regulation of Met in lymph nodes (Wang et al., 2013). Decreased Met expression increases the phosphorylation of p53, whereas p53 up-regulates miR-34b in feedback. The investigation of miRNAs in cancer biology may provide clues to cancer mechanisms and improve the molecular diagnostic strategy in human agerelated diseases.

4.2. miRNAs in age-related macular degeneration As a result of the time-dependent mechanisms of AMD, age undoubtedly is the strongest risk factor for the development of AMD. AMD is a complex disease of the central retina, which consists of highly specialized photoreceptors with a multifaceted support system. In AMD, the dysfunction of these photoreceptors and support system causes progressive and debilitating visual impairment and ultimately results in vision loss in the eyes. Pathological angiogenesis is strongly associated with AMD pathogenesis and progression. To date, many miRNAs, including miR-let-7a,b,c, miR-16, -20, -23a,b, -24, -103, -106a, -125a,b, -133a, -221, and -222, have been suggested to potentially target several angiogenic factors, such as VEGF, bFGF, HGF, and SCF (Poliseno et al., 2006). Of these miRNAs, miR-23 may play a key role of neovascular AMD. miR-23 is significantly down-regulated in macular RPE cells from AMD eyes (Lin et al., 2011). In an animal study, it has been reported that endothelial cells and vascularized tissues are highly enriched with 26

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Acknowledgments

5. miRNAs as a source of diagnostic markers for age-related diseases

This work was performed with the support of the “Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ01100201)”, Rural Development Administration, Republic of Korea, and was partially supported by the Research Institute for Veterinary Science, Seoul National University (SNU), Republic of Korea.

As reported in many miRNA studies in cancer prognosis (Jung et al., 2012b; Yuan et al., 2016; Mitchell et al., 2008), changes in miRNA expression and their mRNA targets with age within a cellular context have been investigated and play a key role in cellular senescence and the senescence-associated phenotype (Abdelmohsen and Gorospe, 2015). The development of next generation sequencing (NGS) technologies has led to the identification of different types of intracellular and extracellular RNAs (exRNAs) in circulation and other body fluids. In particular, miRNAs are very stable in patient serum, whole blood and tissues. These mature miRNAs in circulating systems are transported though the plasma to target cells (Vickers et al., 2011; Arroyo et al., 2011). Due to the relative ease of acquiring blood and serum from patients, most exRNA studies have been performed within circulation and have indicated that exRNAs may be used as biomarkers for diseases (Freedman et al., 2016; Weber et al., 2010). Recent studies have demonstrated that miRNAs in human blood and saliva or other body fluids may represent non-invasive, promising biomarkers for many diseases and disease states. For example, an NGS analysis of miRNAs in the cerebrospinal fluid (CSF) may provide potential clues to the diagnosis and response to the treatment of neurodegenerative diseases, such as Alzheimer’s and Parkinson’s diseases. Various miRNA-profiling studies have shown that predominant miRNA expression is associated with specific retinal disorders. A recent human study reported circulating miRNAs in serum as candidate biomarkers for AMD (Szemraj et al., 2015). The expression of miR-661 and miR-3121 in the serum of patients with dry AMD is increased. In contrast, miR-4258, miR-889 and let-7 in patients with wet form are up-regulated. Furthermore, miR-661, -889 and -3121 are highly expressed in graphic atrophy AMD compared with NV AMD. Furthermore, an NGS analysis of miRNAs in circulation demonstrated that heart- and muscle-specific circulating miRNAs, including miR-1, -133b, -208a,b and -499, increased up to 140-fold in advanced heart failure, which coincided with a similar increase in cardiac troponin I protein, the established marker for heart injury (Akat et al., 2014). Another study identified two miRNAs, miR-125b and miR-320b, that are down-regulated in myocardial infarction patients (Huang et al., 2014). These two miRNAs regulate many genes and pathways associated with cardiovascular diseases, such as TGF-β, apoptosis and cytokine signaling.

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6. Conclusion The functional analysis of miRNAs has provided deeper insights into how aging affects stem cells and degenerative diseases. The advances in the understanding of the roles of miRNAs in aging might offer new therapeutic modalities. The expression of miRNAs which are involved in the repair, regeneration and normal turnover of tissues could be altered in response to stimuli of inflammatory cytokines and oxidative species in age-related diseases. Moreover, mature miRNAs along with extracellular vesicles might be secreted and circulate the body. Thus, miRNAs may represent biomarkers for states of aging and diseases via altered expressions of endogenous and circulating non-coding RNAs. Undoubtedly, the continued investigation of miRNAs within senescent/ aged cells will shed light on their functional role in the context of aging and may unveil clues to potential roles in the context of extracellular environments.

Disclosures of potential conflicts of interest The authors have no conflicts of interest to disclose.

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