The role of epigenetics and non-coding RNAs in autophagy: A newperspective for thorough understanding
Shahrzad Talebiana,c,1, Hossein Daghagha,d,1, Bahman Yousefic, Yusuf Ȍzkule, Khandan Ilkhania,b,Farhad Seiff, Mohammad Reza Alivanda,b,c,*a Department of Medical Genetics, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iranb Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, IrancMolecular Medicine Research Center, Tabriz University of Medical Sciences, Tabriz, IrandAging Institute, Tabriz University of Medical Sciences, Tabriz, Irane Department of Medical Genetics, Faculty of Medicine, Erciyes University, Kayseri, TurkeyfDepartment of Immunology & Allergy, Academic Center for Education, Culture, and Research, Tehran, Iran
Autophagy is a major self-degradative intracellular process required for the maintenance of homeostasis andpromotion of survival in response to starvation. It plays critical roles in a large variety of physiological andpathological processes. On the other hand, aberrant regulation of autophagy can lead to various cancers andneurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, and Crohn’s disease. Emergingevidence strongly supports that epigenetic signatures, related non-coding RNA profiles, and their cross-talkingare significantly associated with the control of autophagic responses. Therefore, it may be helpful and promisingto manage autophagic processes by finding valuable markers and therapeutic approaches. Although there is agreat deal of information on the components of autophagy in the cytoplasm, the molecular basis of the epigeneticregulation of autophagy has not been completely elucidated. In this review, we highlight recent research onepigenetic changes through the expression of autophagy-related genes (ATGs), which regulate autophagy, DNAmethylation, histone modifications as well as non-coding RNAs, including long non-coding RNAs (lncRNAs),microRNAs (miRNAs) and their relationship with human diseases, that play key roles in causing autophagy-related diseases.
1. Introduction
Autophagy is a cytoplasmic catabolic process, highly conservedduring evolution. It takes place at basal levels under normal conditionsof cells and its levels increase by a diversity of cellular stresses (Lapierreet al., 2015; Shaid et al., 2013; Zaffagnini and Martens, 2016). Pre-viously, it was thought that autophagy is a non-selective system, butnowadays it has been demonstrated that autophagy can also selectivelydestroy cargos (Zaffagnini and Martens, 2016). Autophagy, based onthe cellular context, is divided into three forms, including micro-autophagy, chaperone-mediated autophagy, and macroautophagy(Cuervo, 2004). In this review, we focus on macroautophagy because ofwide-range targeting of cells and plurality of studies. Autophagy iscontrolled through almost all the possible regulatory mechanisms intranscriptional, post-transcriptional, and epigenetic levels. Based on
previous studies, an aberration of especial epigenetic signatures inchromatin structure could play crucial roles in the regulatory processesand to be applicable in governing in this regard (Feng et al., 2015;Loscalzo and Handy, 2014).
According to recent studies, epigenetic dysregulation and subse-quently inappropriate autophagy represent a critical factor in the pa-thogenesis of related human diseases, including neurodegenerative andage-related diseases, especially various cancers, Alzheimer’s disease,Parkinson’s disease and Crohn’s disease (Baek and Kim, 2017; Choiet al., 2013). Improper autophagy in a variety of cancers such as lung,breast, gastric and colorectal cancers, is related to the aberrant functionof epigenetic factors, including DNA methylation-related enzymes,histone-modifying enzymes and non-coding RNAs (H-JR et al., 2016a;Yang and Liang, 2015). Therefore, there exists a mutual interactionbetween epigenetic and autophagy processes that could be helpful in
https://doi.org/10.1016/j.mad.2020.111309Received 10 December 2019; Received in revised form 22 May 2020; Accepted 28 June 2020
⁎ Corresponding author at: Department of Medical Genetics, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran.E-mail addresses: [email protected], [email protected] (M.R. Alivand).
1 These authors contributed equally to this work.
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the pathogenesis of the related diseases.Considering the vital importance of autophagy in biological pro-
cesses, such as regulation of cell metabolism and homeostasis, con-trolling of cell growth and aging, and balancing of cell retention anddeath, it is of pivotal importance to carry out a thorough study of au-tophagy regulatory processes, particularly to clarify the epigenetic as-pects of the regulation of autophagy (Choi et al., 2013; Madeo et al.,2010; Ryter et al., 2013). Hence, here we review the current researchesand advances in epigenetic modifications that influence the expressionof autophagy-related genes and their potential contribution to autop-hagy-associated diseases. Furthermore, we express the role of non-coding RNAs such as microRNA (miRNA) and long non-coding RNA(lncRNA) as further complementary epigenetic phenomena in autop-hagy.
2. Autophagy and its related factors
Autophagy activates the relevant catalytic pathways used in cellmetabolism and homeostasis, thereby controlling and balancing cellsurvival and death. Also, it participates in innate and adaptive immuneresponses, turnover of mitochondria, peroxisomes, endoplasmic re-ticulum, and other organelles. Generally, the autophagy process issummarized in the following steps: induction, autophagosome forma-tion through elongation of an isolated membrane called cisterna, cel-lular debris sequestration by autophagosome elongation, autolysosomeformation as a result of autophagosome-lysosome fusion, digestion ofsequestered cargos in the lysosome, and maintenance of cellularhomeostasis (Zaffagnini and Martens, 2016; Hassen et al., 2017).During autophagy, several conserved autophagy-related genes (ATG)and their proteins participate sequentially in each step as briefly de-scribed here (Fig. 1). Nowadays, according to genetic studies, > 40ATG genes have been reported in fungi, most of which have homologsin higher eukaryotes (Delorme-Axford and Klionsky, 2018). In this re-gard, studies on yeast have greatly contributed to a comprehensive
understanding of molecular mechanisms of the autophagy machineryrelated to signaling, including induction of autophagy through mTORsignaling, inactivation or integration of autophagy activation signalsvia upstream nutrient-sensing kinases such as PKA (protein kinase A)and Snf1 (Sucrose Non-Fermenting 1)/AMPK (AMP-activated proteinkinase) and the nucleation and expansion in autophagosome formationthrough the phosphatidylinositol 3-kinase (PtdIns3K) nucleation com-plex and ubiquitin-like conjugation systems in yeast (Cebollero andReggiori, 2009; Noda and Tor, 1998). Also, there exists an intracellularregion namely PAS (phagophore assembly site) for the formation ofautophagosomes; most ATG proteins/complexes are recruited into theorganization of PAS in a hierarchical order (Pyo et al., 2012). TheATG17-ATG31-ATG29 ternary subcomplex and ATG13, ATG1 andATG9, together with ATG18 and ATG2, congregate at the PAS, re-spectively (Feng et al., 2014). The phagophore, temporary surroundsthe specific cargo or bulk cytoplasm and mature autophagosome areformed following the expansion and closure of the phagophore (Turcoand Martens, 2016). The aforementioned events need two systems ofubiquitin-like (Ubl) conjugation, the ATG12 Ubl system, and ATG8 Ub1system (Grumati and Dikic, 2017). The ATG12 Ubl system consists ofATG7, ATG5, ATG12, ATG10, and ATG16 and contributes to the for-mation of ATG12-ATG5-ATG16 heterotrimeric complex, which mightact as an E3-like enzyme for the ATG8 conjugation system (Hanadaet al., 2007). In addition, ATG7, an E1 ubiquitin-like enzyme which isthe activator of ATG12, and also ATG10, an E2 ubiquitin-like enzymeare required for the sequential conjugation of ATG12 to ATG5 at thephagophore (Nakatogawa et al., 2009). Finally, ATG12-ATG5-ATG16complex is formed by further conjugation of a multimeric protein,ATG16 (ATG16 L in mammals) with ATG5-ATG12 (Kaur and Debnath,2015). In mammals, the ATG12-ATG5-ATG16 L complex is recruited tothe phagophore, and with the expansion of this isolation membrane, thelocalization becomes limited to the outer surface. Upon maturation ofautophagosome or immediately before that, the complex separates fromthe autophagosome (Noda et al., 2009). ATG8 Ubl system, the second
Fig. 1. Autophagy process. Mainly, after inhibition of mTOR because of various cellular stresses, the autophagy process begins with recruitment of the initiationcomplex, which consists of Atg1, Atg17, Atg29, Atg31 and Atg13 in yeast and ULK1/ ULK2, Atg13, Atg101 and FIP200 in mammalians. Subsequently, the matureautophagosome is formed by the nucleation, expansion and finally closure of the isolation membrane through the cooperation of Atg12 and Atg8 Ubl systems. Thelast step is the conversion of autophagosome to autolysosomes through autophagosome-lysosome\late endosome\vacoule fusion, and finally the cargo degradation.
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ubiquitin-like system, includes ATG7, ATG3, ATG8, and ATG4 (Fenget al., 2014). ATG8 is a highly conserved Ub-like protein and central tothe formation of autophagosome and phagophore membrane extensionand closure (Xie et al., 2008). In higher eukaryotes, there are severalcategorized mammalian ATG8 orthologs into the LC3/GABARAP pro-tein family (Lee and Lee, 2016). In order to function correctly in au-tophagy flux, ATG8 must be conjugated to PE (phosphatidylethanola-mine) lipid; ATG-PE conjugation has a critical function in thesubsequent dynamic rearrangement of membranes (Nair et al., 2012).For this purpose, the first ATG4 mediates proteolytic processing of ar-ginine residue in the C-terminus of nascent ATG8 to expose a preservedglycine residue; then this residue of glycine gets conjugated to phos-phatidylethanolamine by the sequential actions of ATG7 (E1-like) andATG3 (E2- like), and non-lipidated ATG8 conjugates to form the ATG8-PE, finally (Ichimura et al., 2000). Similar to the yeast LC3, the mam-malian homolog of ATG8 is also converted into PE to form LC3-II andthen it is assembled to the membrane of phagophore and autophago-some (Ravikumar et al., 2010). The following steps are autophagosomeouter membrane fusion with the vacuole, resulting in the final de-gradation of the cargo (Delorme-Axford et al., 2015).
Despite the existence of the autophagy in such human cell lines,various studies on animal models concern the capacity of autophagythat is reduced through down-regulation of autophagy-related genes(such as ATG2, ATG8a, ATG5, ATG6, ATG7, and ATG8) and lysosomal-associated membrane protein type 2a (LAMP2A) during aging of animalmodels, including C. elegans, D. melanogaster, mouse, and rate. Furtherevidence in specific tissues and cells will be needed for better under-standing of the status of autophagy in the aging of each tissue of animalmodels and humans.
3. Epigenetics and its mechanisms
The term “Epigenetics” was coined in the early 1940s by ConradWaddington, who described epigenetics as “the branch of biology whichstudies the causal interactions between genes and their products whichbring the phenotype into being.’’ Today, generally epigenetics is de-fined as consistent and heritable changes in gene expression with nounderlying alterations in the primary DNA sequence (Godfrey et al.,2007; Goldberg et al., 2007; Handy et al., 2011).
In general, there is strong evidence that many of the epigeneticcharacteristics are inherited through germ cell lines and that the ex-pression of some genes or their inheritance can be highly dependent onthe parent's gender through which they have been inherited (Allegrucciet al., 2005). On the other hand, a number of studies have suggestedthat some environmental signals, such as temperature variations, che-micals, nutrition and other stress factors, can also have a major effecton gene expression phenotype, leading to some acquired epigeneticchanges. Recently, a model has been proposed in which, in addition tothe vital role of evolutionary epigenetic changes inherited in thechromatin structure, the acquired epigenetic changes can also be in-heritable with a contribution to the course of evolution and speciation(Feil and Fraga, 2012; Jablonka and Lamb, 2015).
Epigenetic mechanisms play important roles in many aspects ofbiological diversity, developmental biology, and cellular processes suchas the catabolic process of autophagy. A key aspect of epigenetics is thestability of epigenetic marks and their adaptability in the face of evo-lutionary changes and environmental requirements. These abilities re-sult from the activity of specific enzyme groups named “epigeneticwriters”, “epigenetic erasers”, and “epigenetic readers”, which con-tribute to the insertion and deletion as well as the identification andreading of epigenetic marks, respectively (Treviño et al., 2015).
Based on previous studies, two main epigenetic mechanisms arehistone modifications and DNA methylation. In this regard, non-codingRNAs (nc-RNAs) also contribute to some epigenetic signatures in cel-lular processes that should be mentioned (Yang and Liang, 2015;Kondo, 2009; Xu et al., 2017).
Epigenetic histone modifications are PTM (post-translational mod-ifications) that mostly take place along the histone N-terminal tails,including phosphorylation, methylation, acetylation, sumoylation, andubiquitination (Sadakierska-Chudy and Filip, 2015; Yen et al., 2016).These modifications, chiefly on the arginine and lysine residues inhistone tails, are vital for the epigenetic regulation of gene expression(Smith and Denu, 2009).
Acetylation and methylation of histones are among histone mod-ifications that play an important role in the epigenetic regulation ofnumerous cellular events. Histone methylation levels are accuratelyregulated due to the activity of histone methyltransferases (HMTs) andhistone demethylases (HDMs) families, respectively classified in thehistone writers and erasers groups (Hyun et al., 2017; Rice and Allis,2001). Similarly, levels of histone acetylation are also regulatedthrough the coordinated function of the histone acetyltransferases(HATs) family as writers, and histone deacetylases (HDACs) as erasers(Turner, 2000). Moreover, some effector molecules, termed readers,have been identified, interacting with modified residues of histones,and determining the functional Consequences of Histone Modifications(Yun et al., 2011).
Histone ubiquitination has been less studied in autophagy comparedto histone acetylation and methylation. Ubiquitination marks generallyemerge due to the activity of specific enzymes, ubiquitin-activatingenzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin-proteinligase (E3), and binding of ubiquitin small protein to lysine residues inhistones, which are capable of both activation and inhibition of tran-scription (Weake and Workman, 2008).
Another important epigenetic signature is DNA methylation con-ducted by individually or interaction with other histone modificationsin the epigenetic regulation of gene expression and histone modifica-tions can also interact with methylated DNA. In other words, changes ingene expression and chromatin structure in the methylated regions ofDNA can occur as a result of DNA methyl-CpG-binding domain proteins(MBDs) collaboration and histone-modifying enzymes (Hashimotoet al., 2010; Vaissière et al., 2008).
DNA methylation mainly occurs in CpG islands where GC sequencesare more abundant than other genomic regions. As a whole, the humanhaploid genome contains approximately 30 million CpG dinucleotides(Fouse et al., 2010). Adjacent to CpG islands (up to 2 kb), there are alsoareas called CpG shores that are associated with the greatest methyla-tion differences called (differentiated methylated region) DMR betweenvarious normal tissues and also normal and cancerous samples (Irizarryet al., 2009).
DNA methyltransferase enzymes (DNMTs) catalyze a methyl grouptransferred from S-adenosyl- L-methionine (SAM) to the C-5 position ofcytosine in the CpG dinucleotides (Valente et al., 2014). Types ofknown DNMTs include DNMT1, DNMT2, DNMT3a, DNMT3b, andDNMT3L (Robertson and Jones, 2000). DNMT1 is the main enzyme inmaintaining the methylation pattern after DNA replication. The en-zymes DNMT3a, DNMT3b, and DNMT3L contribute to de novo DNAmethylation process. Also, DNMT2 is the smallest member of this familythat, unlike other members, seems to be mostly an RNA methyl-transferase and can be involved in the methylation of tRNA molecules(Zare et al., 2018).
Depending on their location, 5-methylcytosines (5mCs) can differ-ently affect gene expression. For example, hypermethylation of DNA inlocated CpGs in the promoter or enhancer region can suppress tran-scription and in the gene body can either increase or decrease tran-scription (Kumar et al., 2018).
In addition to the CpGs, DNA methylation also occurs in non-CpGsites (CpH; H = C, A, or T). Although non-CpG methylation is not yetfully understood in mammals, recent studies have shown that DNAmethylation in non-CpG regions is more specific to certain cells, in-cluding neurons and stem cells (Jang et al., 2017).
On the other side of the DNMTs enzymes, the ten-eleven translo-cation (TET) enzyme family is active as 'erasers'. The family consists of
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conserved dioxygenases that ultimately remove the methyl group withstep-by-step oxidation and conversion of 5mC to 5-hydro-xymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carbox-ycytosine (5caC), thus considered as key elements in the reversibility ofDNA methylation process (Rasmussen and Helin, 2016).
In addition to histone modifications and DNA methylation, mole-cules of noncoding RNAs (ncRNAs) can, directly and indirectly, influ-ence the epigenetic regulation of gene expression. ncRNAs play a cri-tical role in many cellular processes such as autophagy. According tothe number of nucleotides, non-coding RNAs have been categorized aslong non-coding RNAs (> 200 bp) and also small non-coding RNAs(< 200 bp), including microRNAs (miRNAs), small nucleolar RNAs(snoRNAs), short interfering RNA (siRNA) and piwi-interacting RNA(piRNA) (Esteller, 2011). Studies on the role of ncRNAs in the epige-netic regulation of autophagy have mostly focused on the two largefamilies of these molecules, miRNAs and lncRNAs. miRNAs are small(20 − 24 nucleotides) single-stranded RNAs, attaching to com-plementary sequences on target gene transcript, regulate gene expres-sion post-transcriptionally by suppression of mRNA translation or itsdestabilization (Costa, 2010; Knowling and Morris, 2011).
The lncRNAs are generally 1000 to 10,000 nucleotides in length,and some are up to 100,000 nucleotides in length. Moreover, lncRNAshave various functions in the epigenetic regulation of gene expression.For instance, some lncRNAs could activate transcription factors (TFs),through interactions with them and induction of allosteric changes. Onthe other hand, some of these molecules bind to TFs and inhibit tran-scription. Furthermore, lncRNAs can also evoke chromatin-modifyingenzymes to target specific regions in DNA molecule or, act as a mole-cular scaffold and contribute to the formation of ribonucleoproteincomplexes (Nobili et al., 2016).
Various epigenetic modifying enzymes are known to regulate in-tracellular processes like autophagy. In the following sections, we willdiscuss the epigenetic mechanisms and modifying enzymes that have
been identified so far in epigenetic regulation of autophagy.
4. Histone modifications and histone-modifying enzymes inepigenetic regulation of autophagy
Studies on the epigenetic regulation of autophagy process haveidentified a variety of histone marks that can regulate autophagy inthree levels, including induction or sustaining of autophagy, inhibitionof autophagy and regulation of autophagy function in the life or deathof the cell (Baek and Kim, 2017).
4.1. Histone methylation
The Protein lysine methyltransferases (PKMTs) and Protein argininemethyltransferases (PRMTs), two subfamilies of HMTs family, mostlyimplement histone modifications through the reversible transfer ofmethyl groups on lysine and arginine residues, which lead to the acti-vation or silencing of the target gene, respectively (Carr et al., 2015).The defects in these enzyme families cause the development of manyhuman diseases, especially neurodegenerative diseases and variouscancers, because of their critical regulatory function (Kudithipudi andJeltsch, 2014; Poulard et al., 2016). H3K9me2, H3K27me3, andH3R17me2 are known as histone methyl marks that are methylated bysome PKMTs and PRTMs family members and play a critical role in theepigenetic regulation of autophagy (Kudithipudi and Jeltsch, 2014;Ropero and Esteller, 2007).
H3K9 dimethylation is performed by G9a methyltransferase, alsoknown as EHMT2, a member of the PKMTs family (Tachibana et al.,2002). Due to its fundamental role in many biological processes,especially starvation tolerance, G9a widely expresses in somatic cells(An et al., 2017). G9a, mainly along with its homolog enzyme, G9a-likeprotein (GLP), in a heteromeric complex catalyzes the mono- and di-methylation of H3K9 (Tachibana et al., 2005). Based on previous
Fig. 2. A. EZH2 and G9a suppress autophagy by trimethylation of H3K27 and dimethylation of H3K9, respectively, under normal conditions. B. CARM1-mediateddimethylation of H3R17 contributes to autuphagy sustained under starvation conditions. C. The balancing effect of hMOF-SIRT1 that can regulate H4K16 acetylationcontributes to autophagic-mediated life-and-death decision. Deubiquitination of H2Bub1 is carried out by USP44, contributing to autophagy activation. H3K4 de-methylation is catalyzed by KDM5 and KDM1A and H4K20 is methylated by SUV420 and SET8 and both these histone marks can be involved in autophagy induction.
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studies, G9a performs its role through being linked to the promoter ofATG genes, such as WIPI1, LC3B, and DOR, and this makes these genesbe silenced and autophagy-suppressed during different stages undernormal conditions. Under starvation conditions, G9a is dissociated fromthe promoter of these genes, resulting in H3K9me2 decreasing andH3K9ac increasing, and ultimately to make autophagy (Kudithipudiand Jeltsch, 2014; de Narvajas et al., 2013; Ren et al., 2015). Somestudies on several human cancers have indicated that G9a inhibitioninduces autophagy and thus reduces cell proliferation, colonization,and tumorigenesis in a variety of human malignancies (Ren et al., 2015;Chen et al., 2010, 2015a; Ding et al., 2013; Dong et al., 2012; Hua et al.,2014; Huang et al., 2010; Ke et al., 2014). A study on HNSCC (head andneck squamous cell carcinoma) also has indicated that G9a inhibitionresulted in the upregulation of the ERK-specific phosphatase, dualspecificity of phosphatase-4 (DUSP4), and the inactivation of DUSP4-dependent ERK pathway. Subsequently, cellular death is mediated byautophagy in HNSCC (Li et al., 2014) (Fig. 2).
Although such pieces of evidence suggest that G9a as an epigeneticautophagy suppressor should be inhibited for the induction of autop-hagy. A recent study on drosophila reported that the G9a deletion orsuppression also reduced the expression of ATG8a/LC3 gene and sup-pression of autophagy under starvation conditions. It was further statedthat the positive role of G9a in tumorgenicity might be attributed to itsfunction to acquire starvation tolerance in cells. Finally, researchershave suggested that autophagy epigenetic regulatory function of G9amight vary depending on the cell or tissue (An et al., 2017; Ren et al.,2015).
H3R17 dimethylation is mediated by CARM1 (coactivator-asso-ciated arginine methyltransferase 1), a methyltransferase from PRMTsfamily, and is associated with autophagy induction and sustainingunder starvation conditions (H-JR et al., 2016a). Studies have suggestedthat SKP2-SCE ubiquitin ligase under nutrient-rich conditions degradesCARM1, and AMPK-SKP2-CARM1 pathway is activated under nutrient-poor conditions. AMPK (AMP-activated protein kinase) is a criticalelement in cellular responses to low energy levels. If glucose starvationpersists to sustain autophagy, AMPK accumulates in the nucleus andphosphorylates and directly regulates FoxO3a (Forkhead box O3a) as aFoxO family member of known transcription factors, which regulatesmajor cell survival processes through its target genes controlling (Greeret al., 2007). Although the FoxO3a, mostly recognized as a transcrip-tional activator, can also have function as a transcriptional suppressorand repress SKP2, an F-box protein of the SCF E3 ubiquitin ligasecomplex, leading to CARM1 stabilization (H-JR et al., 2016b; Wang andLi, 2010; Yang et al., 2014). As CARM1 is a transcription factor EB(TFEB) co-activator, an increase in CARM1 levels leads to increasedH3R17me2 levels and transcriptional induction of lysosomal- and au-tophagy-activating genes. TFEB is a master regulator for the activationof lysosomal biogenesis and autophagy genes (H-JR et al., 2016b;Settembre et al., 2011). A recent research on aged heart showed thatimpaired nuclear AMPK-FoxO3 activity and thus non-suppressed SKP2-E3 ubiquitin ligase lead to CARM1 instability; thus having a negativeimpact on nucleus TFEB-CARM1 and autophagy flux (Li et al., 2017a).
H3K27 trimethylation is catalyzed by EZH2 (Enhancer of ZesteHomolog 2), the catalytic subunit of PRC2 (polycomb repressive com-plex 2), and associated with transcription suppression (Schwartz andPirrotta, 2007). Studies have proposed that EZH2 inhibits autophagythrough activating the mTOR(The mammalian target of rapamycin), asignaling pathway under normal conditions. The mTOR pathway playsan effective role in autophagy regulation, and EZH2 suppresses thepromotor of negative regulatory genes (e.g., GPI, TSC2, RGS16,FKBP11, RHOA, and DEPTOR) on this pathway. In addition, MTA2(metastasis-associated 1 family, member 2), a component of the NuRDcomplex, is required for the EZH2-mediated gene repression of thesegenes. The EZH2 knockdown results in the accumulation of LC3B-II andthe formation of autophagosome (Wei et al., 2015).
Moreover, recently the intricate relationship between miRNAs
related to autophagy regulation and histone-modifying enzymes, suchas EZH2 has become a subject of increasing interest. Studies have re-ported that miR-124 targeting the EZH2–STAT3 signaling pathway andinducing autophagy-related apoptosis, plays an important tumor-sup-pressing role in CCA (cholangiocarcinoma). Mir-124 over expressionresulted in down-regulation of EZH2 and STAT3 (signal transducer andactivator of transcription 3) and up-regulation of some autophagy-in-ducing factors, including Beclin-1 and LC3-II. Accordingly, miR-124directly targets EZH2 by which it triggers the autophagic flux in CCAcells (Ma et al., 2018). Similarly, a recent study on endometrial carci-noma has reported that miR-101–3p inhibits the EZH2 expression viabinding to its 3′-UTR region, and increases autophagy and autophagy-related genes (Wang and Liu, 2018). To sum up, EZH2 regulates au-tophagy via its histone-modifying activity while, in turn, EZH2 ex-pression itself is modified by miRNAs.
H3K4me3 and H4K20me3 are the other autophagy epigenetic reg-ulators, which are known as histone ‘activating’ and ‘suppressing’modifications, respectively (Howe et al., 2017; Yu et al., 2008). Studieshave demonstrated that both of these histone marks are connected toH4K16ac as another important epigenetic autophagy regulator. Thetrimethylation of lysine 4 on histone H3 (H3K4me3) is performed bySET1 and MLL (mixed-lineage leukemia) family methyltransferases(Duncan et al., 2015). Genome-wide analyses have also indicated thatH3K4me3, together with H4K16ac, exists inside the units of single-nucleosomes of human cells (Füllgrabe et al., 2014a). Investigations ona variety of cell lines from yeasts to mammals have documented thatautophagy results in both a decrease in H3K4me3 levels and H4K16deacetylation. Such a mentioned decrease is probably caused by theformation of a negative feedback regulatory loop to inhibit the over-stimulation of autophagy and cell death. In other words, this decreasingof mentioned signatures is associated with a general transcriptionalinhibition process to save energy under nutritional deprivation (Duncanet al., 2015; Füllgrabe et al., 2013). Recently, the WNT/β-catenin sig-naling pathway has been shown to be involved in the negative reg-ulation of autophagy by transcription suppression of SQSTM1/P62autophagy adaptor gene. During autophagy, WNT is degraded, and thusthe expression of the SQSTM1 gene is enhance, consequently autophagyis induced (Petherick et al., 2013). Furthermore, WNT promotes MLL toenhance the H3K4me3 levels in promoters; hence, the WNT destructionduring autophagy causes a global decrease in H3K4me3 levels (Sierraet al., 2006; Wend et al., 2013). Tang et al. (2018) demonstrated thathistone deacetylase 6 inhibitors stimulated autophagy through in-creasing expression of the autophagy‐associated genes ATG7 andBECN1 to improve the function of acute kidney diseases (Fig. 2).
The H4K20 methylation is catalyzed by a series of KHMTs, includingSETD8 and SUV420 (H1/H2). SETD8 mono-methylates H4K20 and, inturn, H4K20me1 play the substrate role for the SUV420 enzyme toperform H4K20me2 in the same position and residue (Füllgrabe et al.,2014a). H4K20me3 is mainly present in constitutive heterochromatinregions, and is remarkably influenced by a well-known autophagy in-ducer, serum starvation (Kapoor-Vazirani et al., 2011). The number ofcells with high H4K20me3 levels is enhanced under starvation condi-tions (Kourmouli et al., 2004). As mentioned above, like H3K4me3,H4K20me3 is connected to H4K16ac, and the last H4K16ac deacety-lation leads to the accumulation of H4K20me3 and determines theH4K20me3 level over the genome (Serrano et al., 2013). Moreover,these two histone marks regulate RNA polymerase II (RNAP II) pausingon the opposite side; RNAP II pausing is a transcriptional regulatorymechanism in about 20− 30 % of the genome (Füllgrabe et al., 2014a).This mechanism is inhibited in the presence of H4K16ac and is per-formed in the presence of H4K20me3 (Füllgrabe et al., 2014a; Kapoor-Vazirani et al., 2011). Accordingly, both an increase in H4K20me3 anda decrease in H4K16ac during autophagy prevent the expression of asubgroup of genes controlled by RNAP II pausing mechanism (Kapoor-Vazirani et al., 2011). In other words, SUV420H2-mediated H4K20trimethylation reinforces RNAP II pausing through inhibiting the
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hMOF-mediated H4K16 acetylation (Figs. 2 and 3,Table 3).
4.2. Histone acetylation
Some studies have recently highlighted the significant role of acet-ylation in autophagy regulating. Some research investigations have alsobeen conducted on the gain/loss of function mutations in HATs andHDACs and the use of HDACs inhibitors in order to further clarify therole of the acetylation in the epigenetic regulation of autophagy(Bánréti et al., 2013). The acetylation of lysine residues on histones isperformed by lysine acetyltransferases (KATs), mainly leading to tran-scriptional activation. The role of some KAT family members (e.g.,KAT3A, KAT3B, KAT5, and KAT8) and different HDAC family members(e.g., Sirtuin 1–3 (Sirt1–3), and HDAC (1–7)) in autophagy regulationare now established (Bánréti et al., 2013).
Regarding some research studies, the H4K16ac changes could havean effect on Atgs transcriptional regulation and trigger a regulatoryfeedback loop, which plays a vital role in the survival or death decisionsabout autophagy induction. A molecular histone is shifted by balancingthe effects of KAT8/hMOF/MYST1 and SIRT1 on H4K16 acetylationand regulating the autophagy outcome (Füllgrabe et al., 2014a, 2013).Rapamycin/starvation-induced autophagy is associated with hMOF(human ortholog of Drosophila males absent in the first) down-reg-ulation and H4K16ac reduction. In addition, although SIRT1 can induceautophagy in the cytoplasm through direct de-acetylation of key au-tophagy proteins such as LC3/ATG8, ATG5, and ATG7, SIRT1 seems toact as a factor limiting the autophagy in the nucleus and H4K16 dea-cetylation by SIRT1 would downregulate ATGs involved in differentautophagy steps (Füllgrabe et al., 2014b). The suppression of H4K16acdeacetylation through SIRT1 inhibition or hMOF overexpressionthrough autophagy might disturb the regulatory feedback loop andpromote autophagic flux, ultimately leading to cell death. In otherwords, H4K16 acetylation status, determined by SIRT1 and hMOF, isdecisive in cell death/survival specified by autophagy (Füllgrabe et al.,2013; Huang et al., 2015) (Fig. 3,Table 3).
4.3. Histone ubiquitination
Ubiquitination is an extensive PTM that positively or negativelyregulate various autophagy steps in both selective and non-selectivepathways (Grumati and Dikic, 2017). Similar to other histone marks,the role of histone ubiquitination in autophagy regulation is alsoclaimed in some experiments. The literature indicates that H2Bub1conducts as a vital switch between epigenetic pathways and autophagy(Wang et al., 2016). Histone H2B lysine 120 mono-ubiquitination ismainly carried out by RAD6-RNF20 ubiquitination machinery. Understarvation conditions, H2Bub1 is reduced and autophagy is activated;however, there is no change in RAD6-RNF20 level, and the H2Bub1
level is set by DNMT3-USP44 (DNA methyltransferase 3-UbiquitinSpecific Protease 44) axis in the starved cells. Under basal conditions,USP44 deubiquitinase levels are kept low by two DNA methyl-transferases (DNMTs), DNMT3A, and DNMT3B, which are recognizedas the required de novo methyltransferases to establish embryonic site-specific DNA methylation patterns. Under poor nutrient conditions,DNMT3a and DNMT3b are degraded by the pathway of ubiquitin-pro-teasome pathway; hence, the USP44 expression is activated, USP44deubiquitinates H2Bub1, and the H2Bub1 level is decreased (Chenet al., 2016; Okano et al., 1999). Furthermore, autophagy, in turn,modulates DNA repair by regulating histone ubiquitination. Pieces ofevidence suggest that P62/SQSTM1, the ubiquitin and LC3-bindingprotein, accumulates in cells with disturbed autophagy, and suppressesthe recruitment of some factors for the double-stranded break (DSB)sites such as RAD51, BRCA1 and RAP80 through inhibiting nuclearRNF168, a crucial E3 ligase for histone H2A ubiquitination and DSBrepair. The effectiveness of the DSB repair is then decreased, resultingin enhanced cell deaths (Wang et al., 2016) (Fig. 2,Table 3).
5. Role of DNA methylation in autophagy regulations
DNA methylation is a further process of epigenetic regulation inautophagy, which can perform its role through MBD proteins or usingchromatin remodeling proteins such as HDACs with a physical weak-ening of transcription factors binding (Klose and Bird, 2006). Aberra-tion of DNA methylation is mainly carried out at three levels, namelyloss of imprinting (LOI), hypomethylation and hypermethylation (Wonget al., 2007). According to various studies, hypermethylation of DNAhas a more pronounced role in autophagy regulation compared withother mentioned mechanisms. Hence, in this section we focus on hy-permethylation of genes that are directly or indirectly linked to theautophagy regulation process. DNA methylation can affect specifichistone modification patterns, and vice versa (Espada et al., 2004;Estève et al., 2006). In contrast, another study reported no decrease inH3K9me2 modification level in the knock-out of DNMT1 or DNMT3a,DNMT3b, and DNMT1 triple-knockout in mouse ES cells. In general, theinteractions between these two regulatory epigenetic processes arecomplex (Tsumura et al., 2006) (Fig. 4).
Several studies have clarified the fundamental role of DNMTs inepigenetic reprogramming of autophagy, and documents suggest thataberrant methylation of genes associated with autophagy and conse-quently changes in the expression of certain proteins, has a close re-lationship with various types of human complex diseases fromAlzheimer's disease to cancers (Khalil et al., 2016).
Surveying the level of expression and epigenetic modifications ofATG5 and LC3B (a member of the ATG8/LC3 family), two essentialgenes for the initiation and elongation stages of autophagy, respec-tively, as well as the expression level of DNMT1, DNMT2, and different
Fig. 3. The balancing effect of hMOF-SIRT1, which can reg-ulate H4K16 acetylation, contributing to autophagic-mediatedlife-and-death decision. During autophagy, H4K16ac is re-duced by the down-regulation of hMOF, triggering a negativeregulatory loop to prevent overexpression of the key Atg genesand thereby over‐activation of autophagy, which might causecell death. Inhibition of H4K16ac deacetylation by hMOF up-regulation or SIRT1 down-regulation upon induction of au-tophagy can disrupt the regulatory feedback loop and promoteautophagic flux, ultimately leading to cell death.
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DNMT3 isoforms in childhood acute lymphatic leukemia (ALL) patients,have revealed that in these patients the expression level of DNMT1 washigher compared with healthy subjects, leading to promoter hy-permethylation of ATG5 and LC3B genes. Consequently, there was asignificant reduction in the expression level of these genes (Hassenet al., 2017). Interestingly, investigation of these genes expression inthe macrophages of old mice showed that the promoters of ATG5 andLC3B genes are hypermethylated in these cells due to a significant in-crease in the levels of DNA methyltransferase2 (DNMT2), a conservedmethyltransferase, the precise roles of which are controversial but it ismostly known as tRNA-aspartic acid methyltransferase 1 (Trdmt1)(Shanmugam et al., 2015). DNMT2 inhibition could restore the ex-pression of these two genes to normal in vivo and in vitro levels, and itis suggested that DNMT2 mediates methylation of a distinct DNA motifin these old cells and several studies showed that it is an RNA me-thyltransferase involved in the methylation of tRNA molecules com-pared with DNMT1 and DNMT3a (Khalil et al., 2016).
It is also suggested that in ESCC (esophageal squamous cell carci-noma), LC3Av1, another member of the LC3 gene family, is co-localizedwith LC3B in the same autophagosomes during the induction of au-tophagy, and aberrant methylation of LC3Av1 can be associated withdecreased autophagy in the ESCC cell line and also carcinogenesis (Baiet al., 2012). In breast cancer cells, the expression of the GABARAPgene family (GABARAP, GABARAPL1, and GABARAPL2) is regulatedwith epigenetic modifications, and the down-regulation of the GABA-RAPL1 autophagy gene, in particular, occurs through two epigeneticprocesses of DNA methylation and histone deacetylation, which can bethe cause of autophagy reduction in these cells (Boyer-Guittaut et al.,2014). Studies on colorectal and gastric cancer cells on the Klotho (KL)gene, which was initially characterized as an aging suppressor gene,and also investigations on the PCDH17 gene in gastric cancer cells haveshown that both of these genes are tumor suppressor genes which im-pose their effect through induction of autophagy, and the promoters ofthese genes in almost all these cancerous cells are methylated as aspecific tumor event and they can be used as an epigenetic biomarkerfor these tumors (Hu et al., 2013a; Kuro-o et al., 1997; Xie et al., 2013).Increased levels of PCDH17, along with an increased number of
autophagic vacuoles and upregulated ATG12, ATG5, and LC3BII au-tophagic proteins, induce autophagy, although the exact relationshipbetween this gene and autophagy should be further studied (Hu et al.,2013a). Aberrant methylation of the BECLIN-1(BECN1(the mammalianortholog of Atg6)) gene, an autophagy-related gene with tumor sup-pressor function, has been observed in breast cancer (Khalil et al.,2016). Beclin-1 is a key component of the autophagy-inducing lipid-kinase complex PI(3)KC3(PI(3) kinase class III, and according to stu-dies, hypermethylation of this gene's promoter in the CpG islands canbe a mechanism of autophagy inhibition and tumor induction in thiscancer (Li et al., 2010; Liang et al., 2006) (Table 2).
In addition to DNA methylation, DNA demethylation can play acritical role in the epigenetic control of autophagy. The members of ten-eleven translocation (TET) family enzymes catalyze the stepwise oxi-dation of 5mC (5-methylcytosine) to 5 hmC (5-methylcytosine), leadingto DNA demethylation (Hu et al., 2013b). Three TET paralogs havebeen identified, TET1, TET2, and TET3, which demonstrate dissimilarpatterns of tissue-specific expression (Cimmino et al., 2011). Amongthem the performance of TET1 is more evident in relation to autophagy.It has been suggested that TET1, which acts as a tumor suppressor andCpG demethylase in tumor cells, can exert its functions by regulation ofautophagy at early stages (Fu et al., 2017). Furthermore, it has beendemonstrated that TET1 can regulate the levels of autophagy-relatedgenes, including DNA damage-regulated autophagy modulator protein1 (DRAM1) and ATG13 in tumor cells (Fu et al., 2018a). Also studies onatherosclerosis have indicated that TET2 can control autophagy andautophagic flux by altering the methylation of BECN1 promoter invascular endothelial cells (Peng et al., 2016). In general, further studiesare required to clarify the exact role of TET family enzymes in autop-hagy.
6. Role of non-coding RNA in autophagy regulations
In contrast to histone and chromatin alterations, a gene regulationepigenetic mechanism that is called RNA interference happens post-transcriptionally (Smith-Vikos and Slack, 2012). In eukaryotes and alsomammals, most portions of the genome transcribe to generate large
Fig. 4. DNA methylation and histone modifications, in both direct and indirect pathways, can affect autophagy. In the direct pathway, DNA methylation and histonemodifications directly and epigenetically regulate the expression of autophagy-related genes. In the indirect pathway, epigenetic regulation of nc-RNA expression byDNA methylation and histone modifications has a significant effect on controlling the expression of genes involved in autophagy since each specific miRNA or lncRNAcan regulate the expression of several genes.
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numbers of non-coding RNAs (ncRNAs) (Mercer et al., 2009). Non-coding RNAs have a variety of functions in biological processes at thetranscription and post-transcription levels. For example, they guidegenome rearrangement or DNA synthesis by protecting genomes fromexogenous nucleic acids (Cech and Steitz, 2014; Gupta et al., 2010;Zhao et al., 2016). More recently, studies have shown that non-codingRNAs not only act as a regulator in cell autophagy in vitro and in vivo butalso they contribute to disease phenotypes (Choudhry et al., 2016).
6.1. LncRNAs related to epigenetic regulation of autophagy
LncRNAs participate in a variety of pathophysiological processessuch as autophagy as an unusual regulator. They carry out this functionvia direct binding to DNA, RNA or protein (Cech and Steitz, 2014;Gupta et al., 2010). Regulating the ATG gene expression and also actingas a competing endogenous RNAs (ceRNAs) to regulate the miRNAs thatare related to autophagy are the other functions of lncRNAs in theprocess of autophagy (Yang et al., 2017). The cytoplasmic lncRNAsalter the expression of miRNAs and histone-modifying complexes suchas Xist, which are recruited by nuclear lncRNAs. Also, nuclear lncRNAsaffect the expression of a target gene by binding chromatin-modifyingcomplexes such as PRC2 (Khalil et al., 2009; Zhao et al., 2008).
Several investigations have tried to demonstrate the pivotal func-tions of lncRNAs in autophagy regulation. Here we highlight currentadvances in numerous molecular functions of regulatory lncRNAs in themodulation of cell autophagy, their specific autophagic targets andalso, the relationship between autophagy-associated lncRNAs andhuman disease such as cancer and heart disease (Xu et al., 2017).
Due to the increasing knowledge on autophagy and lncRNAs, animpression has been formed that a large number of autophagy-asso-ciated lncRNAs have functions in tumorigenesis; for instance, maternallyexpressed gene 3 (MEG 3) is a lncRNA that was recently introduced as atumor suppressor and also downregulated in bladder cancer. Also, it isnegatively associated with MAP1A/MAP1B-light chain 3 (LC3/ATG8),an autophagosome marker gene. Knockdown of MEG3 by short-inter-fering RNA (siRNA) can lead to important outcomes like activatingautophagy, enhancing cell proliferation and inhibition of cell apoptosisin human bladder cancer cell lines (Ying et al., 2013). Also, MEG3 in-hibition in macrophages infected with Mycobacterium bovis BCG en-hances the eradication of M. bovis BCG via autophagy induction (Pawaret al., 2016). Contrary to the above studies, another study on epithelialovarian cancer (EOC) demonstrates that MEG3 overexpression can in-duce autophagy and act as a tumor suppressor by modulating of ATG3activity. Overexpression of MEG3 induces autophagic flux by inter-acting with ATG3 and protecting its mRNA from degradation (Y-l et al.,2017). Due to the importance of MEG 3, further researches are neces-sary to thoroughly clarify the regulatory process of this gene in au-tophagy (Table 1).
The expression levels of HULC, a lncRNA which is remarkably up-regulated in liver cancer, have been observed to increase dramaticallyin primary and metastatic hepatocellular carcinoma. HULC can increasethe expression of USP22 (Ubiquitin Specific Peptidase 22) via inhibitingmiR-6886−3p, miR-6825−5p, and miR-6845−5p. Aberrant expres-sion of USP22 can reinstate SIRT1 protein and promote autophagy bysuppressing the acetylation of ATG5 and ATG7 (Xiong et al., 2017).
Studies have reported that the lncRNA HOX antisense intergenic RNAmyeloid 1 (HOTAIRM1), which is related to the myeloid cell differ-entiation, has a crucial role in the degradation of PML-RARA (PML-Retinoic Acid Receptor Alpha) oncoprotein through a miRNA-mediatedpathway that suppresses ATG genes. Reduction of HOTAIRM1 in acutepromyelocytic leukemia (APL) cells could suppress all-trans retinoicacid (ATRA)-induced PML-RARA degradation. HOTAIRM1 could induceautophagy and be involved in autophagy-mediated degradation PML-RARA through sponging miR-125b, miR-106b and miR-20a and,leading to augment the levels of their targets DRAM2, E2F1 and ULK1and, which are critical genes for autophagy machinery (Chen et al.,
2017).Low expression of both PTEN, a tumor-suppressor gene, and
PTENP1 lncRNA, a PTEN pseudogene, was reported in hepatocellularcarcinoma (HCC). The lncRNA PTEN1P serves as a CeRNA, by reg-ulating PTEN via competitive binding to miR-17, miR-19b and miR-20a.In addition to PTEN, pH domain leucine-rich repeat protein phosphatase(PHLPP), acting as negative regulators of Akt signaling, and also au-tophagy-related genes, including ULK1, ATG7 and p62/SQSTM1, arethe other targets of these miRNAs. Hence, PTENP1 overexpression re-presses PI3K/AKT axis, and induces autophagy and apoptosis in HCCcells (Chen et al., 2015a). In other words, The PTENP1 lncRNA mightexert an anti-tumor effect against HCC cells by autophagy modulation.
Another study showed that NBR2 lncRNA, located in the neighbor ofBRCA1 gene 2, can affect both autophagy and cancer. In response toenergy stress, NBR2 provokes AMPK kinase activity so that protectsnormal cells from tumor development by sustaining AMPK activation.However, reported down-regulation of NBR2 in human cancers couldinhibit AMPK activation, and autophagy and induce tumor develop-ment (Liu et al., 2016a, b). Moreover, the levels of NBR2 and AMPK arenegatively regulated by miR-19a in acute liver failure (ALF). MiR-19atargets NBR2 or PRAA1, the AMPK encoding gene; Hence, it negativelyaffects protective autophagy through mediating miR-19a-NBR2/AMPKaxis in hepatocytes (Liu et al., 2018a; Yin et al., 2019).
The other autophagy-related lncRNA is GAS5 (Growth Arrest-Specific 5), which is up-regulated in patients with osteoarthritis (OA).GAS5 can be involved in apoptosis and autophagic responses by down-regulation of miR-21 levels (Song et al., 2014). Furthermore, recentstudies have introduced GAS5-miR-23a-ATG3 axis, as a novel reg-ulatory pathway in autophagy. GAS5 can regulate ATG3 expression bysponging miR-23a. Down-regulation of GAS5 can reduce cell viability,promote tumor cells, and inhibit autophagy by affecting GAS5-miR-23a-ATG3 axis (Gu et al., 2018; Li et al., 2018). Accordingly, furthersuch studies may uncover many other mechanisms coupling lncRNA-miRNA crosstalks with autophagy and cancers, and also provide abroad framework to overcome metabolic stress.
The lncRNA BRAF-activated lncRNA (BANCR), overexpressedlncRNA in various types of cancer such as gastric cancers, melanoma,and cervical adenocarcinoma, promotes PTC (papillary thyroid carci-noma) cell proliferation by increasing the ratio of LC3-II/LC3-l andsubsequently autophagy activation. In other words, In vitro experi-ments have revealed that overexpression of BANCR could increase cellproliferation while acting to inhibit the apoptosis of PTC cells by in-ducing the activation of autophagy (Flockhart et al., 2012; Wang et al.,2014a; Li et al., 2015; Guo et al., 2019).
Since the mortality rate of heart disease has not been remarkablyreduced, attempts have been made to clarify the association betweenheart disease and new therapeutic targets such as lncRNAs. The cardiachypertrophy-associated transcript (Chast) is a lncRNA with a critical rolein cardiovascular diseases. According to the bioinformatics analysis,there is a relationship between Chast and PLEKHM1, a gene that reg-ulates autophagosome-lysosome fusion. Chast can inhibit autophagyand promote cardiomyocyte hypertrophy through down-regulation ofPLEKHM1 and presumably ATG5 expression (Viereck et al., 2016).Another study on rats with diabetic cardiomyopathy has suggested thatH19 lncRNA, a paternally imprinted and greatly conserved transcript,through directly binding with EZH2 and epigenetic silencing of DIRAS3,a potent autophagy inducer, could inhibit autophagy initiation. In ad-dition, reduced levels of H19 can up-regulate BECLIN1 and ATG7 ex-pression, resulting in increased autophagy induction (Zhuo et al.,2017). However conversely, a recent study has reported that H19through H19/SAHH/DNMT3B pathway, induces autophagy and re-sulting in promoted tamoxifen resistance in tamoxifen-sensitive breastcancer cells (Wang et al., 2019). Hence, further studies are required tomore precisely discuss the involvement of this lncRNA in autophagy.
In addition to cancer and cardiovascular diseases, lncRNAs asso-ciated with autophagy are implicated in some other diseases. MALAT1
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(Metastasis-Associated Lung Adenocarcinoma Transcript 1) is a lncRNAthat has attracted a lot of attention due to its elusive role in the reg-ulation of autophagy. MALAT1 is a powerful autophagy inducer thatcan protect BMEC (Brain Microvascular Endothelial Cells) underoxygen and glucose deprivation (OGD)/reoxygenation-mediated injuryby inducing BMEC autophagy. MALAT1, by functioning as a sponge formiR-26b, can down-regulate miR-26b and up-regulate ULK2 expression,the target of miR-26b, accordingly. The improvement of MALAT1/miR-26b/ULK2 pathway activity seems to be able to act as a mechanism ofself-defense against ischemic stroke (Wang et al., 2011). Moreover,MALAT1 can induce autophagy and improve HCC cells’ multidrug re-sistance by sponging miR-216b and possibly up-regulating the level ofBeclin-1 (Yuan et al., 2016). However, unlike the above studies, a studyhas indicated that MALAT1 inhibition can enhance the chemotherapysensitivity through induction of autophagy in DLBCL (Diffuse Large B-Cell Lymphoma) (Li et al., 2017b).
The lncRNA AK156230 quenches replicative senescences (RS) inMEFs (Mouse Embryonic Fibroblasts). The knockdown of AK156230 isassociated with the mTOR signaling pathway and thus down-regulationof ATG genes, including ULK2 (mammalian homologs of ATG1), ATG7
and ATG16L. In other words, AK156230 knockdown in MEFs mightprovoke aging by aberrations in both autophagy and cell cycle path-ways (Y-n et al., 2016).
The lncRNA regulator of insulin sensitivity and autophagy (RISA) canmodify the autophagy activation, leading to the alteration of insulinsensitivity. RISA knockdown can activate autophagy and reduce insulinresistance by increasing ULK1(mammalian homologs of ATG1) phos-phorylation (Yang et al., 2017).
Recently an RNA interference (RNAi) high-throughput screening(HTS) study is carried out using a siRNA library targeting 638 lncRNAsderegulated in tumors. The strongest hit from that screen was DRAIC(Downregulated RNA in Androgen Independent Cells), a previously de-scribed lncRNA that has been shown to involve in cell proliferation.Based on that findings, a novel role for DRAIC in the regulation ofautophagy has been suggested by affecting on the mTOR signalingpathway in MCF-7 cells (Tiessen et al., 2019) (Table 1).
Ultimately, both autophagy and lncRNAs take part in a broad rangeof diseases such as cardiovascular, diabetes and cancer diseases. Hence,further studies to find more lncRNA participating in the regulation ofautophagy may provide new therapeutic approaches to human disease
Table 1The related lncRNA involve in autophagy.
lncRNA Locus Relevant autophagy-related miRNAs
Relevant autophagy-related genes/proteins
Effect onautophagy
Autophagy stages Related diseases/phenotype References
HULC Chr6 miR-6886-3pmiR-6845-5pmiR-6825-5p
ATG5ATG7
Autophagyinduction
Autophagasme elongationand closure
Liver cancer (Xiong et al., 2017)
HOTAIRM1 Chr7 miR-20a miR-106bmiR-125b
ULK1E2F1
DRAM2
Autophagyinduction
Autophagyinitiation
APL (Chen et al., 2017)
BANCR Chr9 – LC3-II/LC3-lratio Autophagyinduction
Autophagasme elongationand closure
PTC (Wang et al.,2014a)
CHAST Chr11 – ATG5Plekhm1
Autophagysuppression
Autophagasme elongationand closure
Fusion
Cardiovascular diseases (Viereck et al.,2016)
NBR2 Chr17 miR-19a AMPK Autophagyinduction
Autophagyinitiation
ALF (Liu et al., 2016a,b)
H19 Chr11 – DIRAS3Beclin1(ATG)
ATG7
Autophagysuppression
Autophagyinitiation
Diabetic cardiomyopathy (Zhuo et al., 2017)
MALAT-1 Chr11 miR-26bmiR-216b
ULK2beclin1
Autophagyinduction
Autophagysuppression?
Autophagyinitiation
Brain microvascularendothelial cell (BMEC) injury
HCC
DLBCL
(Wang et al., 2011)(Yuan et al., 2016)(Li et al., 2017b)
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in future.
6.2. MiRNAs related to epigenetic regulation of autophagy
Apart from lncRNAs, miRNAs are the other important part of non-coding RNAs that participate in autophagy regulation. There are manypieces of evidence that miRNAs can regulate autophagy process invarious cells (Frankel and Lund, 2012). miRNAs are single-strandedRNAs binding to complementary mRNAs and making them susceptibleto degradation before translation, leading to the inhibition of the targetgenes. Until now, thousands of them have been found in different or-ganisms and also they are highly conserved, indicating their importance(Smith-Vikos and Slack, 2012). MiRNAs are epigenetic regulators thatlike other regulators can modulate autophagy at different stages. MiR-199a, miR-26b, miR-106b, miR-20a, miR-17−5p, miR-25, miR-595,miR-4487, miR-101, miR-7 and miR-409−3p are examples of the po-tential regulators of autophagy induction (Gozuacik et al., 2017). MiR-199a inhibits autophagy by positively controlling mTOR signalingpathway, resulting in cardiac hypertrophy (Li et al., 2017c). MiR-26bcan mediate autophagy suppression through ULK2 targeting in prostatecancer cells (Clotaire et al., 2016). MiR-106b and MiR-20a can suppressautophagy by down-regulation of ULK1 expression in C2C12 myoblasts(Wu et al., 2012). Studies on breast cancer cells have indicated thatmiR-25 regulates autophagy, which is associated with chemoresistance.MiR-25 performs its autophagy regulator by targeting ULK1. MiR-25suppression results in autophagic cell death by up-regulation of ULK1expression (Wang et al., 2014b). Also, miR-17−5p, an upregulatedmiRNA in Bacillus Calmette-Guérin (BCG) infected macrophages, in-hibits host cell autophagy by binding to ULK1 (Duan et al., 2015). MiR-4487 and miR-595 are the other miRNAs that target ULK1 and this way,they can contribute to the regulation of autophagy in neuroblastomaSH-SY5Y cells (Chen et al., 2015b). Studies on breast cancer and HCC
cells have shown that miR-101 could act as a powerful suppressor ofautophagy through RAB5A, ATG4D and STMN1 (Stathmin1) targeting(Xu et al., 2013). Furthermore, down-regulation of miR-101 can induceautophagy and reduce oligodendroglial Alpha-Synuclein (α-syn) ag-gregation in MSA (multiple system atrophy) (Valera et al., 2017). Also,miR-7, a tumor suppressor miRNA, has been reported to inhibit pro-liferation and enhance autophagy by targeting mTOR in HCC cells(Wang et al., 2017). In addition, miR-409−3p, down-regulated inovarian cancer (OC) cells, targets Fip200, a key autophagy-initiatingprotein. As a result, miR-409−3p down-regulation increases cisplatin-resistance in OC cells through inducing autophagy mediated by Fip200(Cheng et al., 2020) (Table 2).
On the other hand, several miRNAs have been reported, which areimplicated in controlling autophagy in other sequential steps. For in-stance, miR-30a inhibits autophagy in chronic myeloid leukemia (CML)and HCC cells by selectively blocking BECN1 (BECLIN-1/mammalianhomolog of yeast ATG6) and ATG5, which are needed for autophagynucleation and elongation stages, respectively (Fu et al., 2018b; Yuet al., 2012). Also, MIR-376a and miR-376b regulate autophagy bydirectly blocking ATG4C and BECN1 expression levels, which are cri-tical autophagy-related proteins (Korkmaz et al., 2012, 2013). In ad-dition, miR-129−5 P, depending on its promoter methylation, can in-hibit autophagy by targeting BECN1 in human nucleus pulposus (NP)cells (Zhao et al., 2017). A study on rats with CCH (chronic cerebralhypoperfusion), a critical risk factor for vascular dementia, has shownthat miR-96 inhibition could improve cognitive impairment by reg-ulating mTOR and autophagy inactivation (Liu et al., 2018b). Fur-thermore, a study on prostate cancer has indicated a dual role for miR-96, in a dose-dependent manner in hypoxia-induced autophagy reg-ulation by mTOR or ATG7 targeting (Ma et al., 2014). In addition tomiR-96, other miRNAs, including miR-137, miR-17, and mir-20a,contribute to autophagy regulation by ATG7 targeting. MiR-137 has a
Table 2The master miRNAs involve in autophagic pathway.
MiRNA Locus Relevant autophagy relatedgenes and proteins
Effect on autophagy Related diseases/phenotypes References
MiR-199a Chromosomes 1, 19 MTOR signaling pathway Autophagy suppresion Cardiac hypertrophy (Li et al., 2017c)MiR-26b Chromosome
2ULK2 Autophagy suppresion Prostate cancer (Clotaire et al., 2016)
MiR-20a Chromosome13
ULK1 , ATG7 and ATG16L1 Autophagy suppresion C2C12 myoblasts/MacrophagesBCG infection
(Wu et al., 2012).(Guo et al., 2016)
MiR-106b Chromosome7
ULK1 Autophagy suppresion C2C12 myoblasts (Wu et al., 2012).
MiR-25 ChromosomeX
ULK1 Autophagy suppresion Breast cancer (Wang et al., 2014b)
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remarkable function in regulating neuronal maturation and neurogen-esis and mediates inhibition of starvation-induced autophagy by ATG7blocking (Yin et al., 2014; Zeng et al., 2015). In addition, miR-17 tar-gets ATG7 and inhibits autophagy in human glioblastoma cells and it issuggested that down-regulation of miR-17 can enhance drug sensitivityand low-dose ionizing radiation (LDR) treatments (Comincini et al.,2013). The study on macrophages with M. tuberculosis infection hasdemonstrated that the levels of miR-20a increase in infected macro-phages, thereby suppressing autophagic response and enhancing BCGsurvival by targeting ATG7 as well as ATG16L1 (Guo et al., 2016). Inaddition, miR-874, miR-223, miR-142−3p, and miR-96 are othermiRNAs that can target ATG16L1 in different cell types (Gan et al.,2017; Huang et al., 2018; Zhang et al., 2018; Li et al., 2020). Moreover,miR-23a, miR-200b, and miR-378 are among the miRNAs that cantarget ATG12, and miR-216b has also been suggested to target theimportant players of autophagy, such as BCLN1, UVRAG, and ATG5 tomodulate autophagy process (Guo et al., 2017; Luo et al., 2018; Panet al., 2015; Tan et al., 2018) (Table 2).
Also, recent studies have shown that dysregulated miRNAs play asignificant role in both autophagy and cancer chemoresistance. Forinstance, miR-199a-5p, miR-22, and miR-361−5p influence chemo-sensitivity by negatively regulating autophagy-related genes. MiR-199a-5p could inhibit autophagy-induced chemoresistance of acutemyeloid leukemia (AML) cells by targeting DRAM1 (Li et al., 2019).MiR-22 could decrease chemoresistance in osteosarcoma(OS) cells byrepressing autophagy through inactivation of PI3K/Akt/mTORpathway (Meng et al., 2020). Likewise, miR-361−5p can also inhibitautophagy and chemoresistance by directly targeting forkhead boxprotein M1 (FOXM1) via the PI3K/Akt/mTOR axis in gastric cancercells (Tian et al., 2017). Such studies are providing greater insight intothe mechanism study and treatment of chemoresistance in cancer pa-tients (Table 2).
In summary, owing to the significant role of miRNAs in regulatingautophagy, a better understanding of microRNA-modulated autophagicprocesses under different cellular stresses, can accelerate and improvethe clinical use of miRNAs-mediated autophagic networks as emergingtherapeutic strategies for many human diseases.
7. The new perspective of the interaction between epigenetic andnon-coding RNAs in autophagy
Altogether, more and more evidences have shown that miRNAs andlncRNAs are significantly related and applied to control autophagicresponses of cells and tissues. Based on in silico and experimental stu-dies, a lot of autophagy-related miRNAs and lncRNAs are located andembedded in CpG islands and histone modification regions for makingepigenetic signatures, and their expression can be affected via epige-netic mechanisms. In turn, each non-coding RNAs especially miRAscould control multiple targeted genes in various intracellular signalingof autophagy in the downstream of themselves (Zare et al., 2018). As aresult, epigenetic phenomena could indirectly control expression ofmany genes through controlling of the related miRNAs and lncRNAs inautophagy signaling. As known, the epigenetic signatures could directlyregulate expression of related genes in autophagy process. Overall,epigenetics potentially regulates autophagy-related genes via both di-rectly and indirectly controlling autophagic cell fate. It seems the role ofepigenetics could be highlighted in network intracellular autophagysignaling and could open new horizons in autophagy-related diseasessuch as neurodegenerative, cardiovascular and cancer diseases. Thementioned idea could be applicable for therapeutic aims to controlautophagy and be very useful for early prediction and prognosticationof autophagy-related diseases that need to declare more clinical docu-ments. Furthermore, many questions remain; since autophagy has beenstudied more extensively in the yeast and animal models, it is veryimportant to revise of autophagy-related human diseases throughclinical studies (Fig. 4).Ta
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S. Talebian, et al. Mechanisms of Ageing and Development 190 (2020) 111309
11
8. Conclusion
Autophagy is a lysosomal catabolic pathway of cellular componentsregulated by evolutionarily conserved ATG genes in physiological re-sponses to starvation, aging, and some environmental factors. To thisaim, special epigenetic signatures, related miRNAs, and lncRNAs sig-nificantly regulate ATG genes to control autophagic responses involvedin cellular physiology, pathology, and etiology of age-related diseasessuch as neurodegenerative, cancer, and inflammatory diseases. Despitethe association between mutations in ATG genes and Alzheimer’s,Parkinson’s, and Crohn’s diseases in some documents, epigeneticallydysregulation of ATG genes, autophagy-related miRNAs, and lncRNAsplay a significant role in etiology of age-related and metabolic diseases.Altogether, the control of the ATG genes by epigenetic modifier agentsand miRNAs or lncRNAs regulators with their mutual interactions are ahelpful strategy to manage autophagic processes in related diseases.Based on some pieces of evidence, epigenetic phenomena located inupstream of both miRNAs and lncRNAs regulate them. On the otherhand, miRNAs and lncRNAs, directly or indirectly, regulate ATG genesto adjust autophagy. Owing to this fact, these factors are applicable forprognostic and diagnostic biomarkers and are candidates to designtherapeutic strategy for affected by autophagic diseases that needsfurther basic and clinical studies in this regard.
Funding/Support
This study was supported by a grant from Immunology ResearchCenter (Grant No. 109/97) of Tabriz University of Medical Sciences,Tabriz, Iran.
Authors contribution statement
M. R. Alivand contributed to the design subtitles, the first writtensections of manuscript was colledted and finalized through S. Talebian,H. Daghagh and K. Ilkhani contributed to search and prepare the firstdraft of manuscript. B. Yousefi, F. Seif and Y. Ȍzkul contributed to thefinalize and edit of written manuscript.
Declaration of Competing Interest
The authors declare that they have no conflict of interest.
Acknowledgments
We would like to extend our gratitude to the Dep. Of MedicalGenetics, Faculty of Medicine, Tabriz University of Medical Sciences,Tabriz, Iran.
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