MiRNAs to senescence

2.1. Cellular senescence

How our cells are affected by aging? The answer might be that as our body turns aged, our cells turn senescent (Figure 11). Cellular senescence was formally describled more than 40 years ago as a limited proliferation ability of normal human cells in culture²⁹⁸. The irreversibility of the senescence arrest is tightly associated with the senescent cell’s special chromatin architecture, the so-called senescence-associated heterochromatin foci (SAHF). It constitutes a hallmark of senescent cells that contain several common markers of transcriptionally repressed heterochromatin and are hypothesized to silence genes important for cell proliferation, in particular those regulated by the E2F/RB1 repressor complex²⁹⁹. Despite an irreversible cell cycle, senescent cells actively secrete a spectrum of proinflammatory cytokines, chemokines, matrix metalloproteinases and growth factors, which is termed the senescence-associated secretory phenotype (SASP)³⁰⁰. SASP might be one of the manners that senescence employs to promote aging.

Nowadays, it has been demonstrated that senescent cells accumulate in various tissues and organs overtime demonstrated by accumulations of senescence markers such as senescence-associated β-galactosidase (SA-β-gal), p53 and cyclin-dependent kinase inhibitors p21 and p16. Senescence contributes to aging process. For example, Darren J.B. et al has shown that genetic or pharmacological removal of p^16Ink4a-positve cells (senescent cells)

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would induce extended lifespan associated with reduced pathological alterations in various organs such as adipose tissue, heart and kidney³⁰¹.

It has been demonstrated that various stimuli could induce cellular senescence. On the one hand, the telomeric DNA loss during each S phase would result in telomeres erosion, which then generates a persistent DNA damage reponse (DDR). DDR then could further reinforce the senescence-related growth arrest³⁰². On the other hand, senescence could also occur without DDR development under various culture stressors such as serum deprivation or oxidative stress, etc³⁰³.

Figure 11: Hallmarks of senescent cells.

Hallmarks of senescent cells include an essentially irreversible growth arrest; expression of SA-Bgal and p16;

robust secretion of SASP); nuclear foci contanining DDR proteins (DNA-SCARS/TIF) or heterochromatin (SAHF).

Adapted from³⁰⁴.

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Interestingly, miRNAs have emerged as key regulators in the cellular senescence associated gene programs. Disruption of global miRNAs biogenesis by silencing of DGCR8, Dicer or Drosha could lead to a senescence-like state in human and mouse primary fibroblasts demonstrated by flatted morphology, increased SA-β-gal activity or formation of SAHF^305,306. So, how miRNAs could affect senescence? In the following part, we are going to focus on the effects of miRNAs on the two major pathways involved in senescence.

2.2. miRNAs in p53/RB tumor-supressor pathways

Proliferative arrest, one of hallmarks of senescent cells, is induced by both endogenous and external influences that ultimately converge on either or both of the p16^INK4a/ RB and p14^ARF/ p53 pathways. The binding of p16 to the cyclin-dependent kinase 4-6/ cyclin D complex inhibits the phosphorylation of pRb family proteins and causes a G1 cell cycle arrest (Figure 12).

 miRNAs upstream of p53/ RB pathways

In senescence, let-7/agronaute2 (AGO2) signaling induced implementation of silent-state chromatin modification at target promoters. Inhibition of the let-7/AGO2 effector

Figure 12 : The p53 and RB sequestering MDM2, which facilitates the degradation and inactivation of p53.

In the RB pathway, stress signals indude INK4A, the product of the CDKN2A locus. INK4A inhibits CDKs that phosphorylate, and therefore inactivate, RB during the G1 phase of the cell cycle. Reviewed in ¹.

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complex perturbed the timely execution of senescence³⁰⁷, suggesting that some miRNAs could potentially affect senescence via modifying the promoters of p16/p53 and thereby their expressions. Similarly, in human mammary epithelial cells, senescence-associated miRNAs (SA-miRs) such as mir-26b, mir-181a function in concert to repress expressions of polycomb group proteins, embryonic ectoderm development or enhancer of zeste homologue 2, thereby activating expression of p16 and senescence³⁰⁸. Likewise, amongst the human cancer-associated mir-17-92 cluster, mir-17/mir-20a seed family are necessary and sufficient for conferring resistance to oncogene-induced senescence, by directly targeting p21^WAF1309.

Senescence in the type II alveolar epithelial cells (AECs) contributes to the development of idiopathic pulmonary fibrosis (IPE). The upregulation of SA-miRs including mir-34 family and mir-20a, mir-29c and let-7f might participate in the induction of senescence in AECs, with its ectopic overexpression inducing senescence markers: p16, p53 and SA-β-gal³¹⁰. In addition, induction of mir-29 and mir-30 is necessary for the RB pathway driven-senescence through targeting and inhibition of the 3’UTR of B-Myb³¹¹, a transcription factor that negatively regulates senescence³¹². Moreover, mir-29 overexpression induced by Wnt-3a in muscle progenitor cells (MPC) during aging, induces senescence via targeting the 3’ UTR of p85α, IGF and B-Myb, suppressing the translation of these mediators of myoblast proliferation. Finally, electroporation of mir-29 into muscles of young mice recapitulates aging-induced responses³¹³.

Amongst these SA-miRs in human keratinocytes (NHKs), ectopic overexpression of mir-137 or mir-668 induced senescence in rapidly proliferating NHKs, demonstrated with increased SA-β gal activity and expression of p16 and p53³¹⁴.

P16 could improve and stabilize the levels of p53 through negatively regulated p53 suppressor MDM2 oncoprotein, which was mainly mediated by mir-141 and mir-146-5p dependent negative regulation of MDM2³¹⁵.

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 miRNAs downstream of p53/RB pathways

On the other hand, some miRNAs are identified as down stream effectors of senescence marker genes. Recent evidence indicated that p16 is a modulator of gene expression. It has been demonstrated that p16 could contribute to senescence through modulating miRNAs expression. For instance, through the formation of the p16-CDK4-Sp1 heterocomplex which binds to the Sp1 consensus-binding motifs present in the promoters of mir-141 and mir-146b-5p, p16 enables the transcription of mir-141 and mir-146b-5p, which mediate p16’s response to ultraviolet (UV) induced DNA damage³¹⁶.

2.3. miRNAs in DNA damage pathways

The most significant phenotype associated with mammalian cellular aging is telomere shortening which induces disruption of chromosome integrity by DNA damage and apoptosis³¹⁷. Telomerase lengthens telomeres in DNA strands by adding DNA sequence repeats (TTAGGG in all vertebrates) to the 3’ end of DNA strands in the telomere regions, which are found at the ends of eukaryotic chromosomes, thereby preventing senescent cells from postmitotic condition and apoptosis³¹⁸. However, aging is linked with reduced telomerase activity which reduces the regenerative capacity of proliferative organs and increases incidences of diseases onset and development³¹⁹. Interestingly, mir-195 was overexpressed in old mesenchymal stem cells (OMSCs) in mice, which targeted telomerase reverse transcriptase. Abrogation of mir-195 in OMSCs resulted in increased length of telomere in the nucleus, markedly improving their proliferative abilities. Moreover, transplantation of mir-195-knockout OMSCs reduced infarction size and improved heart function in mice³²⁰.

MiRNAs are also involved in the regulation of cellular reactive oxygen species (ROS) level which is major contributor to DDR. Surprisingly, in human HEK293 cells, induction of DDR by exposure to H2O2 resulted in phosphorylation of dicer (the miRNA producing enzyme) and subsequent nuclear recruitment of dicer to promote DNA repairment mediated by repair factors MDC1 and 53BP1³²¹, indicating that some miRNAs might be involved in this process.

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Specifically, mir-31, a novel target of histone deacetylase inhibitor (HDACi) in breast cancer cells, induces cellular senescence via repression of the polycomb group (PcG) protein BMI1. Mir-31 overexpression leads to γH2AX foci formation largely due to induction of mitochondrial ROS³²². Although in kidney the molecular basis for aging is not well understood, it was reported that mir-335 and mir-34a were increased in aged rat kindney, corresponding with down regulation of their targets superoxide dismutase 2 (SOD2) and thioredoxin reductase 2 (Txnrd2), respectively, which are critical antioxidants located in mitochondria. In vitro experiments demonstrated that overexpression of these two miRNAs leaded to senescence of young mesangial cells via suppression of SOD2 and Txnrd2 associated with a concomitant increase in ROS. Conversely, inhibition of these two actors in aged mesangial cells inhibited senescence via upregulation of SOD2 and Txnrd2 with decreased ROS³²³.