Long noncoding RNAs(lncRNAs) and the molecular hallmarks of aging.

During aging, progressive deleterious changes increase the risk of disease and death. Prominent molecular hallmarks of aging are genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, cellular senescence, stem cell exhaustion, and altered intercellular communication. Long noncoding RNAs (lncRNAs) play important roles in a wide range of biological processes, including age-related diseases like cancer, cardiovascular pathologies, and neurodegenerative disorders. Evidence is emerging that lncRNAs influence the molecular processes that underlie age-associated phenotypes. Here, we review our current understanding of lncRNAs that control the development of aging traits.


INTRODUCTION
Aging is associated with a progressive deterioration in the function of cells, tissues, and organs. This decline in function eventually leads to age-related declines in physiologic function, such as loss of muscle mass, declining blood flow, impaired immune recognition, decreased ability to utilize energy, diminished cognitive function, and reduced ability to respond to stress stimuli. These age-related declines culminate in the onset of age-associated diseases like sarcopenia, cardiovascular disease, cancer, obesity, diabetes, neurodegenerative diseases, and many other pathologies. Recently, Lopez-Otín et al [1] cataloged the molecular processes that decline with advancing age and underlie the phenotypes of aging. They include epigenetic changes, loss of telomere function, declining protein homeostasis, increased cellular senescence, depletion of the stem cell pool, and altered intercellular communication.
The phenotypic changes that characterize the aging process are governed by specific alterations in the pools of expressed proteins. Therefore, there is heightened interest in understanding the mechanisms that drive age-Review associated gene expression programs. These processes are mainly directed by proteins that bind DNA and RNA, as well as by a variety of noncoding (nc) RNA, both short ncRNAs (mainly microRNAs) and long (lncRNAs). Together, this vast and heterogeneous group of factors affects aging by controlling gene expression transcriptionally and post-transcriptionally in myriad different ways. The influence of transcription factors, RNA-binding proteins, and microRNAs in ageassociated processes has been reviewed elsewhere [2][3][4]. Some lncRNAs have been studied for decades, but the broad range of expressed lncRNAs and their impact on protein expression programs has only come into view in recent years. LncRNAs modulate gene expression patterns at all levels: transcriptional, posttranscriptional, and post-translational [5][6][7]. Through their impact on the type and abundance of proteins, lncRNAs affect key cellular processes such as proliferation, differentiation, quiescence, senescence, the cellular response to stress and immune agents, and many others cellular functions relevant to the biology of aging. In this review, we describe and discuss the emerging impact of lncRNAs on these processes and their possible implications in senescence, aging and age-related pathologies.
LncRNAs. LncRNAs vary widely in size, genomic localization, and biogenesis. They can be expressed from intergenic regions (lincRNAs), from the opposite strand of mRNAs (antisense lncRNAs), from vestigial genes that lost their coding potential (pseudogeneencoded lncRNAs), from introns of annotated genes (long intronic ncRNAs), or from the promoter regions of coding mRNAs (promoter-associated lncRNAs); they can also be generated by the splicing machinery (circular RNAs) [8].
LncRNAs can be classified according to their molecular mechanism of action. Some nuclear lncRNAs can regulate gene expression epigenetically by recruiting chromatin-modification factors to activate or inactivate different loci [9,10]. LncRNAs can also serve as transcriptional regulators by assembling transcriptional activators and repressors to modulate transcription initiation by RNA polymerase II [10,11]. Other lncRNAs can function in nuclear compartmentalization and help maintain nuclear structures such as nuclear speckles, paraspeckles, and interchromatin granules [11].
In addition, lncRNAs can regulate gene expression post-transcriptionally by base-pairing with mRNAs to modulate their translation and/or stability [12][13][14] and by interfering with RNA-binding proteins to influence splicing and translation [14][15][16]. Competing endogenous RNAs (ceRNAs) and circular RNAs are stable lncRNAs that accumulate in large numbers and modulate gene expression in different ways, including as decoys or sponges for microRNAs [17,18]. Finally, some lncRNAs have been shown to control protein turnover by facilitating ubiquitination [7] (Figure 1, Table 1).

LncRNAs that modulate telomere length
Telomeres are structures that protect the ends of chromosomes against damage. Cellular senescence is generally associated with a gradual shortening of telomere length [19]. In order for DNA replication to take place on the telomeres, a specific polymerase, telomerase reverse transcriptase (TERT), is required to extend telomeres to preserve their lengths [1]. The length of telomeres is regulated by the telomerase ribonucleoprotein complex that contains the protein TERT and the lncRNA TERC (telomerase RNA component), as well as by the telomeric repeatcontaining RNA lncRNA TERRA.
TERC. The essential RNA component of the telomerase enzyme complex, lncRNA TERC, has been directly implicated in the maintenance of telomere length and thus the prevention of premature senescence and aging. In support of this function, TERC-deficient mice displayed short telomeres, chromosomal instability, and premature aging [20]. TERC serves as a template for the synthesis of telomeric repeats and acts as a scaffold that brings the protein subunits of telomerase together with other accessory proteins associated with the complex. Additionally, TERC was shown to have a catalytic function in the process of adding telomere repeats [21].

TERRA.
While TERC promotes and maintains telomere length, the lncRNA TERRA suppresses telomere elongation. TERRA transcripts are transcribed from telomeres by RNA polymerase II, an unexpected discovery, since telomeres were long believed to be transcriptionally silent, and have variable lengths ranging between 100 and >9000 nt in mammals [22]. The suppression of telomeric RNA elongation is linked to the presence of numerous copies of the telomere UUAGGG repeat in the TERRA transcript, spanning ~200 nt [23], which renders TERRA a high-affinity ligand (and hence a competitive inhibitor) for TERT [24].
Abnormal expression of TERRA may contribute to premature senescence and aging.
For instance, mutation in the gene DNA methyltransferase 3B (DNMT3B) leads to hypomethylation of the telomeric region and elevated levels of TERRA. These changes www.impactaging.com result in the syndrome ICF (immunodeficiency, centromeric instability, and facial dysmorphism), in which fibroblasts exhibit premature senescence linked to the suppression of telomere elongation [25]. TERRA is also involved in the removal of 3'G overhangs of uncapped telomeres during DNA damage-induced senescence [23]. Although the loss of telomeres is believed to accelerate senescence, telomerase-deficient cells can avoid senescence by a unique mechanism that requires TERRA during telomere recombination [26]. Early in S phase, the levels of TERRA decline, rising again at the end of S phase and thereby displacing hnRNPA1 from the telomeres and recruiting the singlestranded DNA-binding protein POT1 (protection of telomeres 1). Thus, while TERRA suppresses telomere elongation, it can also protect telomere ends [27]. Histone methylation in stem cells Associated with ES cell pluripotency [133] www.impactaging.com

LncRNAs associated with epigenetic alterations in aging and senescence
Epigenetic changes modulate gene expression in a variety of processes that characterize age-associated pathology and physiology [28,29]. In this section, we review the involvement of lncRNAs in epigenetic changes including DNA methylation, histone modification, and heterochromatin formation.
www.impactaging.com expression allows gene expression of genes on the Kcnq locus relevant to age-associated diseases like type 2 diabetes and cancer [43]. One of the genes within this locus is the cyclin-dependent kinase (cdk) inhibitor and tumor suppressor CDKN1C, encoding P57 KIP2 [44]. Thus, Kcnq1ot1 can affect aging and senescence through its impact upon the cell cycle.
Airn. The lncRNA Airn is transcribed in antisense orientation to the maternally expressed Igf2r gene and controls Igf2r mRNA expression by silencing its transcription in cis. Interestingly, it is not the Airn lncRNA per se, but rather Airn transcription what drives methylation changes and Igf2r silencing [45]. Airn is likely involved in aging and senescence through its effect on IGF2R, since senescent cells show enhanced IGF2R expression compared to proliferating cells and IGF2R is implicated in longevity [46,47]; however, a role for Airn in these age-related processes has not been studied directly.

ecCEBP.
The lncRNA ecCEBP was also found to recruit DNMT1 in order to regulate local DNA methylation and silence the CEBP gene [48]. The encoded protein, CAAT enhancer-binding protein (C/EBP), influences cell cycle progression by interacting with and inhibiting kinases cdk2 and cdk4, triggering growth arrest [49]. The levels of several C/EBP family members, which are key regulators of adipogenesis, decline with age [50]. Recently, age-associated changes in C/EBP proteins were shown to cause severe liver injury and accelerated liver proliferation after treatment with CCl 4 (carbon tetrachloride) [51], while C/EBPγ suppressed senescence and inflammatory gene expression by heterodimerizing with C/EBPβ [52]. Together, these findings indicate that the lncRNA ecCEBP regulates C/EBP expression and highlight its putative role in the age-and senescence-dependent changes in C/EBP abundance.
pRNA. The lncRNA pRNA regulates transcription of ribosomal RNA (rRNA) by interacting with DNA at the target site of the transcription factor TTF1; the resulting DNA-RNA triplex is specifically recognized by the DNA methyltransferase DNMT3b [53]. Accelerated rDNA methylation was observed in senescent Werner syndrome fibroblasts [54], although the possible involvement of pRNA in this process has not been examined. rRNA levels are selectively reduced with brain aging and this reduced ribosomal activity may contribute to Alzheimer disease (AD) [55, 56], although pRNA has not yet been implicated in the age-related changes in rRNA.

PAPAS.
The heterogeneous population of lncRNAs termed PAPAS (promoter and pre-rRNA antisense) is derived from rDNA gene transcription in the antisense orientation [57]. During quiescence, PAPAS is upregulated to guide the H4K20 methyltrasnferase Suv4-20h2 to nucleolar chromatin, leading to increased trimethylation of H4K20 and chromatin compaction at rDNA loci [58].

PTENpg1-AS.
The lncRNA PTENpg1-AS, which represses expression of the tumor-suppressor gene phosphatase and tensin homolog (PTEN), exists in two isoforms, α and β. PTENpg1-ASα represses PTEN expression by lowering its transcription via its interaction with DNMT3a and enhancer of Zeste on the PTEN promoter, while PTENpg1-ASβ regulates PTEN mRNA stability [59]. Given the role of PTEN in aging, senescence, and age-related diseases such as cancer, we anticipate a direct function for PTENpg1-AS in these processes [60-64].
TARID. The lncRNA TARID (TCF21 antisense RNA inducing demethylation) has the ability to trigger expression of the tumor suppressor protein TCF21 (transcritpion factor 21) by inducing promoter demethylation. During this process, TARID binds to GADD45A (Growth arrest and DNA-damage inducible alpha), a regulator of DNA demethylation, and recruits it to the TCF21 promoter. Interestingly, TARID is expressed in the antisense orientation to the TCF21 gene [65].
In this section, we examine lncRNAs implicated in histone modifications, particularly methylation, that play a role in processes such as cell cycle progression, inflammation, and senescence.
Kcnq1ot1. The levels of the potassium channel protein KvLQT1 (encoded by the gene KCNQ1) decline with aging and age-related cardiovascular disease [73,74]. The 91-kb long lncRNA Kcnq1ot1 influences histone modifications by recruiting the histone methyltransferases G9a and polycomb repressor complex (PRC2) to increase H3K9 and H3K27 trimethylation and repress transcription of the Kcnq1 locus [75].
NeST. The lncRNA NeST (nettoie Salmonella pas Theiler's) binds to the H3K4 methyltransferase component WDR5 (WD repeat domain 5) and recruits it www.impactaging.com to the IFNγ locus. Given that lncRNA NeST is involved in inflammation during microbial infection [76] and that IFNγ methylation increases with advancing age, NeST could contribute to the inflammatory response and infection in elderly.

ANRIL.
The lncRNA ANRIL is transcribed from the same locus as INK4b/ARF/INK4a but in the opposite direction. ANRIL is involved in cell cycle regulation at least in part by recruiting CBX7 (Chromobox 7), a protein component of the of polycomb repressor complex 1 (PRC1), to the locus, increasing H3K27 methylation and thereby repressing INK4a transcription. Interestingly, CBX7 variants with point mutations that disrupt binding to RNA or methylated H3K27 repress genes in the INK4a locus, impairing cellular senescence [77].

ANRASSF1.
The promoter of gene RASSF1A, which encodes the Ras association domain-containing protein 1, was found to be increasingly hypermethylated with advancing age [78]. The lncRNA ANRASSF1 forms an RNA/DNA hybrid at the RASSF1A transcriptional start site and recruits the chromatin-modifying PRC2 complex to the RASSF1A promoter.

Heterochromatin formation
Global disruption of heterochromatin is a hallmark of senescence and aging. In senescent cells, dramatic remodeling of the chromatin is associated with the formation of senescence-associated heterochromatin foci (SAHF) [83,84]. The loss of heterochromatin with aging is believed to underlie various cellular processes associated with aging [85,86].
Heterochromatin formation was found to promote longevity in Drosophila and the function of heterochromatin binding protein 1 (HP1a) correlates with life span in flies [87]. In addition, chromosomal stability is directly related to heterochromatin formation and maintenance.
Epigenetic features including H3K9 trimethylation, H4K20 trimethylation, and HP1a binding are observed at constitutive heterochromatin in chromosomal DNA repeat regions such as subtelomeric and pericentromeric regions [88].
BORDERLINE. This lncRNA was found to prevent spreading of the HP1 protein SWI6 and H3K9 methylation beyond the pericentromeric region in yeast. Interestingly, BORDERLINE is processed into short RNAs (brdrRNAs) by Dicer, a type-III RNase implicated in aging and senescence [90][91][92]. Accordingly, Dicer might modulate BORDERLINE levels, function, and processing to brdrRNAs in agerelevant processes.

lncRNAs and proteostasis
Proteostasis (protein homeostasis) encompasses several biological processes that govern protein biogenesis, folding, trafficking, activity, interactions, degradation, and elimination. Disruption of proteostasis can lead to age-related diseases such as Alzheimer's, Parkinson's, and Huntington's diseases [93,94]. Major proteolytic systems, including autophagy and the ubiquitinproteasome pathway, decline with age, indicating that proteostasis is a common feature of aging [1]. In this section, we review the lncRNAs associated with proteostasis, including autophagy, and protein synthesis, trafficking, assembly, and degradation.

LncRNAs in autophagy
Changes in autophagy are another hallmark of aging [1]. Disruption of autophagy induces changes in mammalian tissue that resemble those associated with aging. Inhibition of autophagy also compromises the aging-inhibitory effects of interventions such as caloric restriction and sirtuin 1 activation [95]. Although their interdependence is not clearly understood, autophagy and senescence are integral processes of the cellular response to stress agents [96].
HULC, MEG3, and 7SL. These lncRNAs modulate autophagy in different ways. Overexpression of HULC in SGC7901 human gastric cancer cells promoted cell proliferation and invasion, and inhibited apoptosis by inducing autophagy [97]. In contrast, the levels of MEG3 and 7SL correlated inversely with the level of the autophagy marker LC3-II in vivo [14,98,99]. The underlying mechanisms through which these lncRNAs influence autophagy are not well understood, but they may involve p53, since 7SL can regulate p53 mRNA translation and MEG3 can regulate p53 directly via RNA-protein association or indirectly by lowering MDM2 expression levels [100,101]. In sum, the agingwww.impactaging.com and senescence-associated process of autophagy is robustly affected by lncRNAs.

LncRNAs in protein trafficking
Some lncRNAs influence homeostasis by modulating the traffic of proteins and hence the abundance of a protein in a particular subcellular compartment. By recruiting transcription factors to the nucleus and specifically to certain DNA regions, lncRNAs can modulate transcription. Similarly, by recruiting certain RNAbinding proteins into ribonucleoprotein complexes, lncRNAs can elicit other gene regulatory functions. Although the complete spectrum of mechanisms through which lncRNAs affect protein trafficking and their impact on aging and senescence are largely unknown, a few examples are beginning to emerge.
GAS5. This lncRNA induces growth arrest by acting as a decoy for the transcription factor glucocorticoid receptor (GR) and thus inhibits GR-mediated gene expression [102]. Specifically, GAS5 binds GR in the cytoplasm and prevents its mobilization to the nucleus. Interestingly, the level of GR protein decreased in the nucleus but not the cytosol of aged Long-Evans rats with cognitive impairment [103], although it is not yet known if GAS5 is directly involved in the aberrant GR distribution in this animal model of aging.

PANDA.
The lncRNA PANDA is induced by p53 upon DNA damage. It binds the transcription factor NF-YA and interferes with its transcriptional activity, lowering the expression of apoptotic genes. Interestingly, NF-YA interaction with p53 impacts upon cell cycle regulation and senescence [104,105]. These findings indicate that PANDA could be involved in DNA damage-induced senescence through NF-YA and p53.
Gadd7. In Chinese hamster ovary (CHO) cells, Gadd7 was induced by DNA damage and oxidative stress and controlled the G1/S checkpoint and cell growth [106]. Gadd7 associated directly with TDP-43 and interfered with the binding of TAR DNA-binding protein (TDP-43) to Cdk6 mRNA, leading to Cdk6 mRNA destabilization, abnormal cell cycle progression, and possibly senescence [107,108]. It is not known if Gadd7 interferes with other TDP-43 targets or if it influences other RNA-binding proteins involved in senescence or aging.
7SL. This highly abundant lncRNA interacts with the TP53 mRNA and suppresses p53 translation. The RNA-binding protein HuR can displace 7SL and enhances p53 translation. According to this competitive interaction, silencing 7SL increased HuR binding to TP53 mRNA and promoted p53 translation, in turn enhancing cell cycle arrest and senescence [14].

lncRNAs associated with protein synthesis and degradation
Protein synthesis and degradation are two critical processes that determine the steady-state abundance of proteins in cells. Protein synthesis is controlled by the rates of mRNA translation, while the ubiquitin proteasome pathway is the major driver for protein degradation. Some lncRNAs modulate protein levels indirectly by influencing the available pool of microRNAs and thereby affecting mRNA turnover and translation; for instance, lncRNAs such as linc-MD1 and lincRNA-RoR (below) can act as decoys for miRNAs that usually suppress the translation or stability of other mRNAs [17,109]. However, some lncRNAs interact directly with mRNAs to enhance or suppress their translation or with proteins to modulate their half-lives: AS Uchl1. AS Uchl1 enhances translation of UCHL1 (ubiquitin carboxy-terminal hydrolase L1) through an embedded SINE (short interspersed nuclear element) B2 repeat present in AS Uchl1 [110]. UCHL1 is involved in brain development and age-related neurodegenerative pathologies such as Parkinson's disease [111]. Overexpression of UCHL1 was found to induce senescence, likely due to increased production of p14ARF, p53, p27KIP1 and decreased MDM2 levels [112]. Accordingly, AS Uchl1 lncRNA may influence both senescence and neurodegeneration.
HOTAIR. The levels of HOTAIR, a lncRNA upregulated in senescence, are reduced by the senescence-repressor HuR, an RNA-binding protein, via degradation of HOTAIR in a microRNA-dependent manner. HOTAIR was shown to serve as a scaffold to promote the ubiquitination and subsequent degradation of Ataxin-1 and Snurportin-1 [7]. These findings suggested that senescence-associated lncRNAs can function as platforms to facilitate protein ubiquitination and degradation to elicit cellular senescence.

LncRNAs and complex assembly
The assembly of protein complexes is widely required for the modulation of gene expression patterns. Several lncRNAs function in complexes with proteins, RNA, and DNA, as explained above. For instance, the lncRNA TERC is essential for the telomere complex formation that maintains telomere length [21, 117,118], H19 interacts with MBD1 to form a ribonucleoprotein complex that recruits histone lysine methyltransferases to suppress gene expression [36], and ecCEBP forms a complex with DNMT1 to regulate DNA methylation and silencing of the CEBP gene [48]. The lncRNA pRNA interacts with DNA at a site which is specifically recognized by the DNMT3b to regulate rRNA transcription [53], PTENpg1-AS lncRNA interacts with DNMT3a forming a ribonucleoprotein complex to regulate PTEN transcription [59], and lncRNAs Kcnq1ot1, ANRIL, ANRASSF1, and PINT form complexes with histones or histone modifiers to regulate gene expression as explained above. Other examples include lncRNA-protein complexes NcRNA CCND1 -TLS, which are recruited to the promoter to suppress transcription of the CCND1 gene following DNA damage [119], and THRIL-hnRNPL, which is recruited to the tumor necrosis factor (TNF)α promoter site to regulate TNFα expression [120].
These complexes broadly regulate gene expression programs relevant to the control of cell cycle progression and inflammation, two processes central to senescence and aging.

LncRNAs and Stem cells
Stem cells are progenitor cells for the development of specific cell types in different tissues. The ability of stem cells to regenerate tissues (also known as 'pluripotency') declines with age. Like adult cells, stem cells are also exposed to stimuli that promote aging or senescence such as DNA damage and cell cycle inhibition. Disruption of stem cell gene expression programs is deleterious for various tissues and promotes age-related physiologic declines like immunosenescence (diminished production of adaptive immune cells) and reduction in muscle mass. They also underlie many pathologies, including cancer [1]. Pluripotency in human and mouse embryonic stem cells (ESCs) is tightly regulated and involves transcription factors (TFs) such as Oct4, Sox2 and Nanog along with several other coregulators [121]. These TFs transcribe genes that encode proteins involved in the maintenance of pluripotency and suppress the transcription of genes that encode proteins necessary for developing specific cell types [122]. Examples of lncRNAs that affect stem cell homeostasis are emerging, as discussed below.

Cross-regulation between lncRNAs and stem cell transcription factors
LncRNAs can regulate certain stem-cell transcription factors, which reciprocally can regulate the expression of lncRNAs. The abundance of some conserved lncRNAs is transcriptionally regulated by Oct4 and Nanog in mouse ESCs: AK028326 is activated by Oct4, while AK141205 is repressed by Nanog. Interestingly, knockdown or overexpression of these lncRNAs altered Oct4 and Nanog mRNA levels and subsequently cellular lineage-specific gene expression and pluripotency, suggesting a feedback regulatory loop [123]. In mouse ESCs, several lncRNAs involved in stem cell gene regulation were identified that bound chromatin-regulatory proteins and coordinated gene expression programs [124].

ES1, ES2, ES3.
The levels of the transcription factor REST are elevated in ESCs and decline as ESCs convert to neuronal SCs [125]. REST is involved in neurodegeneration, neurogenesis, and neuronal pathologies like ischemia, epilepsy, and Huntington's disease [126,127]. REST has been suggested to regulate the expression of lncRNAs which could be involved in neurodegeneration and cancer [128]. A more detailed study of human lncRNAs that promote pluripotency and neuronal differentiation indicated that lncRNAs ES1, ES2, and ES3 are exclusively expressed in human ESC or iPSC and also regulated by Nanog and/or Oct4 [129]. Interestingly, depletion of these lncRNAs was proposed to inhibit neurogenesis through binding to the epigenetic regulators REST and SUZ12 [129].

linc-RoR.
Recently, another report indicated that the expression of 28 lncRNAs was higher in iPSCs than in ESCs, suggesting that they might be required for the development of iPSCs. One of these lncRNAs, linc-RoR, was found to regulate the reprograming of hESCs. Silencing of linc-RoR inhibited the formation of iPSC colonies while its overexpression favored reprograming, prompting the hypothesis that linc-RoR might act as an endogenous miRNA sponge to regulate Oct4, Nanog, and Sox2 in hESCs [130]. As linc-RoR levels decrease rapidly under differentiation conditions, it was proposed to be a self-renewal and pluripotency marker for hESCs [109,131]. Linc-RoR was also found to lower the levels of the tumor suppressor and cell cycle regulator p53 under DNA damage conditions; similar to UCA1 (below), linc-RoR interacts with p-hnRNP I, which is required for p53 mRNA translation [132]. These findings indicate that linc-RoR is a potential stem cell regulator and modulator of cell growth and survival. The influence of linc-RoR on p53 expression connects www.impactaging.com linc-RoR to the control of cell cycle progression and cellular senescence.

Histone methylation regulates stem cell lncRNAs
Profiling of mouse ES cells differentiated into embryoid bodies identified 174 differentially expressed lncRNAs, many of which were associated with developmental genes (e. g., Dlx1, Dlx4, Gata6, Ecsit), including the pluripotency-associated lncRNAs Evx1as and Hoxb5/6as. They are derived from homeotic loci and associated with trimethylated H3K4 histones and the histone methyltransferase MLL1 [133]. Further studies suggested that lncRNA transcription in mouse ESCs is regulated by promoter methylation at CpG similar to protein-coding genes.
For example, loss of the repressive H3K27me3 upon differentiation transcriptionally activated many lncRNAs, suggesting that ESC-specific lncRNAs are regulated via mechanisms similar to those that control protein-coding gene expression to influence stem cell differentiation and maintain pluripotency [134].

Cell cycle-associated lncRNAs
Environmental stresses, telomere dysfunction, oxidative stress, and DNA damage negatively influence normal cell cycle progression causing cells to withdraw from cell cycle, halt replication and enter senescence. Thus, as tissues and organs age, terminally arrested senescent cells accumulate [135][136][137]. Accordingly, DNA damage, a suggested primary cause of cellular senescence and organismal aging, increases with advancing age and elevates cell cycle inhibitors (e.g., p53 and p21) that promote senescence [138]. Recent insight into lncRNAs involved in cell cycle regulation and senescence were mentioned earlier in this review. Here, we discuss cell cycle-related lncRNAs that may influence senescence and organismal aging.

MALAT1.
Several lines of evidence suggest that MALAT1 represses senescence. For example, depletion of MALAT1 in CaSki human cervical cancer cells induced G1 arrest and reduced cell growth, cell proliferation, and tumor size [139], and depletion of MALAT1 in the breast cancer cell line MB231 decreased cell proliferation and triggered G1 arrest [140]. Similarly, silencing MALAT1 in proliferating WI-38 cells enhanced senescence and induced G1/S arrest, two sets of effects that were linked to p53 actions [34,141]. Together, these studies indicate that MALAT1 might be essential for cell proliferation. Unexpectedly, however MALAT1-knockout mice lacked an obvious phenotype or histological abnormalities, indicating that MALAT1 is not essential for development [142], although its impact may be revealed under specific pathological or environmental conditions. ANRIL. As mentioned above, the lncRNA ANRIL is expressed from the p15/CDKN2B/CDKN2A/ARF gene cluster [143]. Downregulation of ANRIL significantly enhanced the levels of the cdk inhibitor (cdki) p15 and moderately increased the levels of cdki p16. Genomewide analysis indicated that ANRIL levels decline in senescent WI-38 cells, and its silencing increased the abundance of cell cycle inhibitors and induced senescence [34,144]. While these findings indicate that ANRIL is required for cell cycle progression and suppression of senescence, further studies are warranted to understand the roles of ANRIL more broadly in aging.
The CCND1-associated lncRNA NcRNA CCND1 modulates the expression of the cell cycle regulator cyclin D1 (CCND1), which is required for the activity of cdks, particularly Cdk2 and cdk4, to progress through the G1/S phase transition [145].
Upon exposure to DNA-damaging agents, the NcRNA CCND1 binds the RNA-binding protein TLS to form a ribonucleoprotein complex that is recruited to the CCND1 promoter and inhibits transcription [119]. Since CCND1 is a major regulator of the cell cycle progression, NcRNA CCND1 will likely be implicated in aging-relevant processes such as cancer.
SRA. SRA is a co-activator of steroid receptors and other transcription factors such as the myogenic regulatory transcription factor MyoD and the major regulator of adipogenesis PPARγ [146,147]. Overexpression of SRA promotes the differentiation of mesenchymal precursor cells into adipocytes; conversely, SRA knockdown inhibits preadipocyte differentiation. SRA decreases the expression of cdkis p21 and p27 and it increases phosphorylation of Cdk1 [146,147]. Since SRA is expressed in a wide range of tissues [148], similar regulatory mechanisms may occur in a variety of tissues that impact upon cell cycle progression, senescence, and adipogenesis.

HEIH.
The lncRNA HEIH is highly expressed in human Hepatitis B virus-related hepatocellular carcinoma (HCC). It suppresses the expression of cdkis p16, p21, p27, and p57 and hence facilitates tumor cell growth [149]. Future studies are warranted to study the role of HEIH lncRNA in aging.

HULC.
The lncRNA HULC is highly expressed in hepatocytes, HCC, and hepatic colorectal carcinomas [150,151]. Hepatitis B virus X protein induces CREBwww.impactaging.com mediated HULC expression, which subsequently promotes hepatoma cell proliferation by lowering the expression of the cdki p18 [152,153]. Thus, HULC may play a role in cell cycle progression and cellular senescence.
Gadd7. As mentioned above, Gadd7 was upregulated by DNA damage [106]. Gadd7 decreases cell growth by binding TDP-43 and preventing its interaction with Cdk6 mRNA, leading to Cdk6 mRNA decay [107]. As CDK6 affects cell cycle and senescence [108,154], it is expected to influence mammalian aging. UCA1. UCA1 is highly expressed in bladder transitional cell carcinoma [155]. Recently, UCA1 was found to promote breast cancer cell growth by lowering the expression of the tumor suppressor and cdki p27. Silencing UCA1 enhanced hnRNP I abundance in the cytoplasm, facilitating p27 mRNA translation and triggering G1 cell cycle arrest [156]. These findings suggest that UCA1 can affect cellular senescence and carcinogenesis by modulating p27 levels [157].

H19.
As mentioned above, H19 is an epigenetic regulatory RNA that affects cell growth and proliferation. In breast cancer cells, H19 knockdown decreased cell growth and formation of colonies in culture [162]. Hypoxia elevated H19 abundance in HHC cells and H19 knockdown significantly reduced tumor growth after recovery following hypoxia. Additionally, H19 overexpression enhanced tumor growth in Hep3B cells [163]. These findings indicate that the high levels of H19 in cancer cells led to enhanced growth and proliferation, as well as to the avoidance of cellular senescence.

MEG3.
The lncRNA MEG3 is repressed in many human cancer cell lines due to gene deletion or to DNA hypermethylation.
Forced expression of MEG3 inhibited the growth of human cancer cell lines such as HeLa, MCF-7, and H4, suggesting that MEG3 may act as a tumor suppressor [168], while downregulation of MEG3 enhanced autophagy, increased cell proliferation and inhibited cell death [99]. A major mechanism through which MEG3 regulates cell proliferation is by lowering the levels MDM2, a repressor of p53 [169]. MEG3 expression also induces apoptosis and growth arrest in cervical cancer cells [170]. Global analysis on transcriptional networks and cellular senescence in human mammary fibroblasts suggested that the NR4A3 (nuclear receptor subfamily 4, group A, member3) is upregulated in senescent cells and affects cell growth via MEG3 [171], further linking MEG3 to cellular senescence and carcinogenesis.

7SL.
Unlike MEG3, 7SL lncRNA is highly expressed in cancer cells [14,172]. 7SL silencing was found to promote cell cycle arrest and senescence in HeLa cells. These effects were mediated, at least in part, through binding to and suppressing TP53 mRNA translation by competing with the RNA-binding protein HuR, as explained above [14].

LncRNAs and intercellular communications
Factors associated with aging in one tissue can be secreted, transported, and received by other tissues. These intercellular communication events involve the sharing of factors such as inflammatory cytokines and extracellular vesicles, which are associated with senescence, aging-associated declines, and diseases of advancing age [1,173,174].

Inflammation-associated lncRNAs
Senescent cells actively secrete pro-inflammatory cytokines, a phenomenon known as the senescenceassociated secretory phenotype (SASP) [1,175]. Given that senescent cells accumulate in older tissues, they are believed to be directly responsible for the low-grade proinflammatory state that characterizes aging.
The emerging role of lncRNAs in inflammation is discussed here.
17A. The lncRNA 17A is upregulated in cerebral tissues derived from Alzheimer disease patients as well as in response to inflammatory stimuli such as IL-1α. Interestingly, 17A is encoded within the G-proteincoupled receptor 51 (GPR51) gene and 17A overexpression enhanced secretion of Aβ and regulated GABA B alternative splicing and signaling [176]. In light of the fact that GPR function is associated with age-associated declines [177], 17A provides an interesting link between GPRs and age-associated neurodegeneration.
Lethe. In mouse embryonic fibroblasts (MEFs), 54 lncRNAs were found to be regulated following www.impactaging.com treatment with TNFα, a pro-inflammatory cytokine associated with inflammation, aging, age-related diseases, and cellular senescence [178,179]. Among these lncRNAs, Lethe was found to be particularly important for the pro-inflammatory state of aging tissues by providing an key negative feedback loop: binding of Lethe to the NF-κB subunit RelA inhibited DNA binding and reduced the production of inflammatory proteins [178].
THRIL. TNFα was also found to be induced by lncRNA THRIL (TNFα-and hnRNPL-related immunoregulatory lincRNA). This lncRNA interacts with hnRNP L, forming a ribonucleoprotein complex that binds the TNFα promoter and regulates its expression in THP1 macrophages [120]. Together, lncRNAs lethe and THRIL are involved in inflammation in a TNFα-dependent manner.

Lnc-IL7R.
Recently, Lnc-IL7R was identified as a regulator of the lipopolysaccharide (LPS)-induced inflammatory response. Depletion of Lnc-IL7R reduces trimethylation of histone H3 at lysine 27 (H3K27me3) causing a reduction in the levels of inflammatory mediators including E-selectin, VCAM-1, IL-6 and IL-8 [180]. Given that pro-inflammatory cytokines are secreted by senescent cells and contribute to the aging phenotype [181,182], Lnc-IL7R might be involved in sense-cence or aging by regulating these inflammatory factors.
Other lncRNAs. Toll-like receptors (TLRs) are vital in the response of macrophages to pathological stimuli. In normal aging, defects in TLR signaling enhanced inflammation [183]. Recently, inflammatory lncRNA signatures were characterized by RNA sequencing in healthy individuals treated with low-dose LPS, which activates TLR4 signaling. The study revealed tissuespecific regulation of distinct lncRNAs in the inflammatory physiology and pathology of the cardiovascular system [184]. However, the impact of these lncRNAs on age-related processes has not been investigated directly.
The TGF-β/Smad3 pathway is involved in inducing and resolving the inflammatory response [185]. Highthroughput RNA analysis was used to identify Smad3dependent lncRNAs related to renal inflammation. The analysis identified numerous lncRNAs altered in Smad3 knockout mice [186]. As this pathway is impaired with age and Smad3 regulates senescence phenotype [187,188], this network of proteins and lncRNAs differentially expressed may play a role in agingassociated inflammation ('inflammaging').

LncRNAs and extracellular signaling through exosome
Like inflammatory cytokines, RNAs can serve as extracellular signaling molecules.
RNAs can be transferred between cells through gap junctions or via extracellular vesicles such as exosomes and microvesicles [189]. Due to their presence in all body fluids, exosomes are believed to participate in many biological processes, including those that directly affect aging [173]. All RNAs, including mRNAs, dsRNAs, and miRNAs, can be transported among cells [190,191].
The examples below highlight emerging functions of lncRNAs in intercellular communication in age-relevant processes, particularly inflammation and growth inhibition.
TUC339. Among the lncRNAs found in extracellular vesicles from hepatocellular carcinoma cells, the lncRNA TUC339 modulates cell cycle progression, tumor growth, and adhesion [192,193]. Since TUC339 can be transported to distant tissues via extracellular vesicles, it can serve as a messenger to modulate the cell division cycle in distant tissues.
Tie-1as. Also involved in cell-cell communication, the lncRNA tie-1as interacts with and represses the mRNA encoding for tyrosine kinase-containing immunoglobulin and epidermal growth factor homology domain-1 (Tie-1), causing defective endothelial cell contact junctions [194]. Although endothelial function declines with age, the impact of Tie-1as on aging has not been examined directly.

Linc-ROR.
This lncRNA is highly enriched in extracellular vesicles derived from hepatocellular carcinoma cells. TGFβ regulates the levels of Linc-ROR in the vesicles and incubation of vesicles with tumor cells reduces chemotherapy-induced cell death in recipient cells. Suppression of Linc-ROR leads to increased chemotherapy-induced apoptosis and cytotoxicity, suggesting that this lncRNA is a modulator of chemoresistence in hepatocellular carcinoma cells [195].

Other lncRNAs.
Recently, several lncRNAs that are typically present in low abundance in cells were found to be selectively enriched in secreted exosomes, including MALAT1, HOTAIR, LincRNA-p21, GAS5, TUG1 and CCND1-ncRNA. Some of these lncRNAs are discussed above, as they influence cell cycle regulation, cellular senescence, and other processes. Treatment with the DNA damaging agent bleomycin elevated the concentration of LincRNA-p21 and ncRNA-CCND1 in exosomes [196]. Since damaged DNA accuwww.impactaging.com mulates with advancing age, the accumulation of these lncRNAs in exosomes may serve as a marker for aging in tissues.

Concluding remarks and perspectives
Aging traits are governed through changes in subsets of expressed proteins. LncRNAs can modulate protein expression patterns by controlling gene transcription, mRNA stability, and protein abundance. Through this influence, lncRNAs modulate key molecular events underlying the aging process, including those discussed here -the control of telomere length, epigenetic gene expression, proteostasis, stem cell function, intercellular communication, cell proliferation and cellular senescence.
The examples discussed in this review underscore the growing recognition that lncRNAs critically affect both the physiologic decline that occurs with aging and the pathologies associated with advancing age. Accordingly, there is mounting interest in the diagnostic, prognostic, and therapeutic value of lncRNAs. The facile detection of lncRNAs in body fluids (e.g. exosomes) and the ease of design of molecules to increase or decrease lncRNA levels make them particularly attractive clinical targets.
However, the potential usefulness of lncRNAs in aging dysfunction and disease cannot be fully realized at present. First, we must have a more comprehensive understanding of age-associated lncRNAs, their spatiotemporal pattern of expression, the molecules with which they interact (proteins, DNA, and RNA), and the impact of altering their abundance upon cell function. Second, we need to develop suitable animal models in which we can study age-associated lncRNAs, assess their function in tissues, organs, and systems, and evaluate how they influence the aging process. By advancing in these areas of knowledge, we expect to gain a deeper molecular understanding of aging and develop more effective interventions to ameliorate the losses of advancing age.