{"paper_id":"cce1d3ec-679f-48f6-91ca-d8b129940e3f","body_text":"The discovery of functional noncoding RNAs has opened a kaleidoscopic world of unanticipated mechanisms extending far beyond the DNA-RNA-protein paradigm; noncoding RNAs may in fact outnumber coding RNAs ( 1 ). Long noncoding RNAs (lncRNAs) have been discovered throughout the genome; scientists are working to explore their functions in health and disease. The  ANRIL  lncRNA was first identified in a melanoma kindred with a large (403 kb) deletion at the  CDKN2A/B  locus ( 2 ).  ANRIL  has attracted broad attention because it is located at a genomic hotspot for disease heritability, the  CDKN2A/B  locus. Although protein coding genes at this locus have important well-studied roles in cell cycle regulation, data suggest that some locus disease-associated single nucleotide polymorphisms (SNPs) act through effects on  ANRIL  itself. Intriguingly, studies suggest  ANRIL  not only impacts the biology of cancer, but also has cell-type-specific roles in metabolic disease. Although  ANRIL  has been reviewed in the past ( 3 ,  4 ), knowledge has exponentially increased in recent years. Here we review advances in ANRIL SNPs, gene regulation, cell biology, and disease roles of  ANRIL .\n\nANRIL , or  CDKN2B-AS1 , is located at the human  CDKN2A/B  locus at 9p21.3. This gene cluster, extending over a nearly 350 kb genomic region housed within a single topologically associated domain (TAD) ( 5 ), contains three protein coding genes and, antisense to them, the  ANRIL  lncRNA (Figure  1 ). The protein coding genes include  S-methyl-5 ′ -thioadenosine phosphorylase  ( MTAP ),  CDKN2A , which encodes splice variants p16 INK4A  and p14 ARF , and  CDKN2B , which encodes p15 INK4B  ( 9 ,  10 ).  MTAP  lies at one end of the locus, 192 kb telomeric to the 5′ start of  ANRIL . At the centromeric end of the locus, the  ANRIL  gene contains 19–21 reported exons over a 126 kb region.  CDKN2A  lies between  MTAP  and  ANRIL , near the first exon of  ANRIL ;  CDKN2B  is located within the first intron of  ANRIL , in an antisense direction. The proteins encoded by  CDKN2A  and  CDKN2B  are tumor suppressors with well-established roles in cell proliferation, apoptosis, senescence and aging ( 11 ,  12 ). p16 INK4A  and p15 INK4B  are cyclin dependent kinase (CDK) inhibitors, inhibiting retinoblastoma phosphorylation by CDK4/6. The p14 ARF  protein, a splice variant of  CDKN2A  which due to a frame shift has no amino acid homology to the principal other  CDKN2A  splice variant, p16 INK4A , modulates p53 activity.  ANRIL  is transcribed by RNA polymerase II and spliced into multiple linear and circular isoforms in a tissue-specific manner. In general,  ANRIL  roles, explored in detail below, include gene regulation in  cis  and in  trans  through interaction with polycomb repressive complex (PRC) histone modifiers, as well as RNA-RNA interactions such as microRNA (miRNA) sponge activity ( 3 ,  13 ). Known biological impact of  ANRIL  activities include modulation of proliferation, apoptosis and cellular adhesion pathways ( 14 ).\nCDKN2A/B locus and  ANRIL  isoforms.  (A)  At the CDKN2A/B locus, the  ANRIL  lncRNA is antisense to the protein coding genes. The p15INK4B gene is contained within intron 1 of  ANRIL .  (B)  To date,  ANRIL  has 21 reported exons.  (C,D)  Numerous linear  (C)  and circular  (D)  exons have been reported. Due to the discovery of additional exons, distal exons in some circular isoforms have been renumbered based on the current 21 reported exons.  * Exons 15-16 refer to exons 14-15 in Holdt et al. ( 6 ).  ** Exon 16 refers to exon 14 in Sarkar et al. ( 7 ) and Burd et al. ( 8 ).\n\nThe evolutionary development of the human  ANRIL  gene has been studied by comparative analysis of the genomes of 27 organisms including non-mammalian vertebrates, non-placental mammals, non-primate placental mammals, and primates ( 15 ).  ANRIL  originated in ancestors of the Eutherian (placental) mammalian clade. Initially the gene contained only a few exons; over time,  ANRIL  underwent clade-specific evolution, adding exons in many mammals but losing exons in rodents. The full 21 exon gene is present only in simians.  ANRIL  genes contain many repeat elements, both intronic, and exonic; evidence suggests that transposon activity has mediated many of the observed evolutionary changes in exon presence or absence, location, sequence, conservation, and structure, as well as introduction of splice sites ( 15 ).\nEarly  ANRIL  variants were likely not transcribed or functional ( 15 ). One hallmark of functional RNAs is splice signals at intron/exon boundaries. In simians, 191 intron/exon boundaries contained canonical splice signals, while 20 did not. In lower mammals, however, only about half of intron/exon junctions contained identifiable splice signals ( 15 ). This finding suggests that as  ANRIL  gained exons, and exon sequences became more conserved across species, it also increased the number of splice signals and gained functionality. Taken together, data suggest that  ANRIL  may be functional only in simians, and that functionality may have been introduced by transposon activity ( 15 ).\n\nWith at least 21 exons (new exons discovered as recently as 2017 ( 7 )), the  ANRIL  gene can potentially generate a large number of splice variants. In fact, many  ANRIL  isoforms have been reported (Figure  1 ) ( 16 ). Exon numbering has changed over time as new exons were discovered. Studies observe multiple isoforms in any given cell type, mostly at low abundance. A different range of isoforms may be identified from one cell type to another, but tissue-dependent isoform expression in primary cells or tissues has not yet been comprehensively quantified using the same reagents and techniques. Intriguingly, many studies have now identified both linear and circular  ANRIL  isoforms ( 6 – 8 ). The longest open reading frame identified in any  ANRIL  variant is 86 codons, supporting the concept that functionality of this gene is through RNA activity ( 16 ).\nMany conventional linear polyadenylated  ANRIL  isoforms are detected in different cell types. Circular  ANRIL  (circ ANRIL ) isoforms, without polyadenylation, have also been described. Circular RNAs, which are formed by “back-splicing” in which a downstream splice donor site is joined to an upstream splice acceptor site, were discovered in 2012 to be a broadly occurring phenomenon across developmental stages and tissues, arising from at least 14% of human transcribed genes ( 17 ,  18 ). CircRNAs enjoy distinct properties from linear RNAs, including, in general, enhanced stability and longevity, cytoplasmic localization, and lack of translation (although if an IRES is engineered, circRNAs can support translation) ( 19 ). Traditional PCR using antisense-oriented primers cannot distinguish between linear and circular isoforms; other methodology, such as PCR using “outward-facing” primers directed away from each other, detection of specific exon-exon junctions, or protection from RNAse R digestion can quantify circular RNAs.\nCareful examination of melanoma cell lines showed that the abundance of individual  ANRIL  exons is non-uniform, supporting the presence of different isoforms ( 7 ). In both transformed cell lines and in human brain derived cells, abundance of  ANRIL  exons was lower than exons from locus protein-coding genes  CDKN2A  and  CDKN2B  ( 8 ). Linear isoforms tend to include proximal exons (1-2), whereas isoforms with only central exons (4-16) are more likely to be circular ( 8 ). In melanoma cells, proximal exons (exon 1 and exon 5-6) were more highly expressed than distal exons. This suggests that short isoforms of  ANRIL , which tend to include proximal exons, are more abundant than longer isoforms in this cell type ( 7 ). In human peripheral blood mononuclear cells and a monocyte cell line, four major groups of  ANRIL  transcripts were found, all with common proximal exons including exons 1, 5, and 6 but with different distal exons, of various lengths ( 8 ). Multiple circular  ANRIL  isoforms have been detected. A circ ANRIL  isoform with an exon 14-5 head-to-tail junction was reported to be the predominant form in both an immortalized fibroblast cell line ( 8 ) and in a majority of melanoma cell lines ( 7 ). Other non-canonical back-spliced junctions observed in melanoma samples included exon 14-5, 7-4, 10-5, and 14-4 ( 7 ). The exons most commonly observed in circ ANRIL  in melanoma cells were 4, 5, 6, 7, 10, 13, and 14; in varied human cell types, the majority of circ ANRIL  species were exon 5-6-7 containing ( 6 ).  ANRIL  exons 1, 2, 3, 8, 9, 11, and 12 were rarely included in circular RNA products ( 8 ). In melanoma lines, no correlation was observed between abundance of linear and circ ANRIL  ( 7 ). However, circ ANRIL  expression was inversely correlated with linear  ANRIL  expression in peripheral blood mononuclear cells in a cardiovascular cohort ( 6 ). Circ ANRIL  was found to be resistant to RNAse R digestion compared with linear  ANRIL , and an actinomycin D time course confirmed enhanced stability of the 14-5 circ ANRIL  isoform compared with linear isoforms ( 7 ,  8 ).\nStructure and function of lncRNAs is of high interest in the scientific community, given the increasing recognition of lncRNA roles in cancer and the normal biology of higher organisms. As such, prediction of lncRNA structures is an important computational challenge. One approach is to identify structural elements through comparison of related lncRNAs. The MONSTER tool was used to compare  ANRIL  to two lncRNAs with similar biological function:  HOTAIR  and  COLDAIR  ( 20 ). MONSTER identifies sequence-predicted secondary structure, such as regions likely to be single stranded RNA, double stranded RNA, hairpin loops, interior loops and bulges. Comparing predictions of two lncRNAs with similar functions is proposed as a mechanism to identify structural motifs. When  HOTAIR, COLDAIR , and  ANRIL  were compared, several common structures were identified, putative structural motifs related to their common function in epigenetic regulation, which could lead to a molecular understanding of mechanism of action in future studies ( 20 ). Another study identified the region of ANRIL that interacts with CBX7, a polycomb repressor component; secondary structure analysis revealed hairpin structural motifs with significant binding affinity to CBX7. Fluorescence anisotropy suggested a ternary complex between a particular loop of ANRIL, CBX7 and a H3K27me3 methylated histone peptide ( 21 ).\nRNA localization impacts function. In melanoma cells, linear  ANRIL  species containing proximal (exon 1) and distal (exons 13b, 19) exons were predominantly found in the nucleus. However, middle exons (exons 5, 6, and 7), which are found in both linear and circ ANRIL , were observed in cytoplasmic fractions, suggesting that  circANRIL  species may be predominantly cytoplasmic ( 7 ). Nuclear localization suggests linear isoforms may be responsible for the known  ANRIL  function of regulating gene transcription via chromatin modulation (see below). Conversely, cytoplasmic localization suggests  circANRIL  forms may participate in post-transcriptional functions. In gastric ( 22 ), prostate ( 21 ), and urothelial ( 23 ) cancer cells, ANRIL was predominantly nuclear. In a beautiful high-resolution analysis of single-molecule lncRNA localization, ANRIL was found to be mostly localized to cell nuclei, in one or several bright foci. Like other lncRNAs analyzed, ANRIL nuclei foci were lost in mitotic cells ( 24 ). Physiological stimuli that change ANRIL localization may provide clues as to ANRIL functions. Intriguingly, in a retinal cell line  ANRIL  isoforms were observed by fluorescence in situ hybridization to localize to the peri-nuclear cytoplasmic space.  ANRIL  abundance was induced by glucose, but  ANRIL  localization did not change with high glucose exposure ( 25 ). On the other hand, a study in HUVEC cells, using primers predicted to detect both linear and circular isoforms, found  ANRIL  to be mostly nuclear; nuclear  ANRIL  was increased after exposure to TNF-α ( 26 ). At least one study has used  ANRIL  as a nuclear positive control to test localization of other transcripts ( 27 ). Future cell type specific studies of ANRIL localization under basal, stimulated, and stress conditions may lead to clues as to ANRIL roles in tissue health and disease.\n\nAbundance of  ANRIL  species is determined by promoter transcriptional activity, splicing decisions, and RNA stability (Figure  2 ). Like other genes,  ANRIL  promoter activity is influenced by epigenetic control and transcription factor occupancy. Intriguingly, epidemiological findings suggest that epigenetic regulation of  ANRIL , through promoter methylation, has important long-lasting consequences for tissue function ( 29 – 31 ). As such,  ANRIL  regulation is one mediator of the impact of early life environmental signals on adult human health.\nSummary of regulation and functions of the ANRIL lncRNA.  (Left)  Some of the known mechanisms by which ANRIL abundance is regulated, at the transcriptional and post-transcriptional levels. On the  (Right) , a selection of known ANRIL cellular functions are depicted. We apologize for observations not included in this summary image. The ANRIL structural prediction in the center is of a common long-isoform of ANRIL, and was generated from Gruber et al. ( 28 ).\nEpidemiological and experimental findings demonstrate that methylation of the  ANRIL  promoter region regulates  ANRIL  gene expression and has functional importance. The first exons of  ANRIL  and  p14ARF  are separated by only 300 bp, in head-to-head antisense orientation; the intergenic region between them is a bidirectional promoter ( 4 ,  32 ,  33 ).  In silico  analysis of ENCODE ChromHMM data ( 34 ) revealed that this region is enriched for both promoter and enhancer activity, and DNAse I hypersensitivity, across multiple cell types, suggesting this is a regulatory region. This region is bound by CTCF, usually considered to be a transcriptional repressor, insulating promoters from enhancer activity. Oddly, CTCF binding at the  ANRIL  promoter was associated with active-chromatin mark histone H3K4 trimethylation ( 35 ). CTCF binding, and  ANRIL  and p14ARF expression, were inhibited by methylation of local CpG islands and increased by demethylation. Knockdown of CTCF prevented the demethylation-induced expression of  ANRIL  and p14ARF, confirming that CTCF is a methylation-sensitive positive regulator of  ANRIL  promoter activity ( 35 ).\nAdditional evidence supports the functional importance of CpG sites for  ANRIL  promoter activity, locus gene expression and transcription factor binding. Mutagenesis of the CpG sites affects both  ANRIL  and p14ARF promoter activity ( 29 ,  30 ). Methylation status of several CpG differentially methylated regions at  ANRIL  in umbilical cord tissues was positively associated with abundance of linear but not circular  ANRIL , but inversely associated with p14ARF and p16INK4a expression ( 29 ). CpG methylation also affects other transcription factors binding at the  ANRIL  promoter to regulate downstream gene expression, such as interferon gamma, SMAD3/4 and ERα ( 29 ,  30 ). Methylation status of CpG islands around the  p16INK4A  transcription start site was also shown to coordinate transcription of  ANRIL  and p16INK4A in human cells ( 36 ). Given the multiple protein complexes binding across different CpG sites, and variable quantitative impact of individual CpG region mutagenesis on  ANRIL  isoforms and locus gene expression, regulation appears to be complex.\nANRIL  expression is influenced by cellular processes such as genotoxic stress, tumorigenesis, senescence, and inflammation. Activity at the bidirectional promoter region upstream of both  ANRIL  and  p14ARF  genes is influenced by the critical cell cycle regulator E2F1 ( 32 ,  33 ). In response to the genotoxic stress of DNA damage, E2F1 transcriptionally activates  ANRIL  in an ATM-dependent manner ( 33 ,  37 ). In this case,  ANRIL  is thought to promote cell growth by suppressing locus INK-family inhibitors after DNA repair is complete, allowing re-entry into cell cycling ( 33 ). The  ANRIL  promoter was also responsive to E2F1 in cancer cells ( 32 ). In addition to E2F1, several known potent oncogenes regulate  ANRIL  expression in various cancers. In lung cancer, c-MYC binds to an E-box in the  ANRIL  promoter and induces  ANRIL  expression ( 38 ). In nasopharyngeal carcinoma, transcription factor SOX2 was shown to bind directly to the  ANRIL  promoter and activate transcription of  ANRIL  and its downstream effector β-catenin ( 39 ). In liver cancer, SP1 binds the  ANRIL  promoter and positively regulates  ANRIL  transcription ( 40 ). On the other hand, TET2, a tumor suppressor in human gastric cancer, binds to the promoter region of  ANRIL  and regulates expression of  ANRIL  as well as p16INK4a, p15INK4b, and p14ARF ( 41 ). Transcription regulation of  ANRIL  is involved not only in cell DNA damage and oncogenesis, but also in disparate processes such as cell senescence and inflammation. In senescence, oncogenic Ras was found to reduce expression of  ANRIL  ( 13 ,  21 ,  42 ). In inflammation, STAT1 activates the  ANRIL  locus in vascular endothelial cells has been reported; CAD-associated  ANRIL  SNP rs10757278, located in a known downstream enhancer region, disrupts the STAT1 binding site and modulates IFN-γ induced  ANRIL  expression via stimulation ( 43 ). Intriguingly, the binding of STAT1 at this enhancer exerts cell-type specific regulation of  ANRIL  expression: repression in lymphoblastoid cells lines, but activation in HUVEC cells ( 43 ). In sum, data support an important role for cell-type specific transcriptional regulation of the  ANRIL  lncRNA in a range of cellular processes and outcomes.\nCell type dependent variation in abundance of different  ANRIL  isoforms suggests that splicing may be a point of regulation ( 8 ,  44 ). Almost nothing is known about  ANRIL  splicing decisions. Disease-associated  ANRIL  gene polymorphisms have shed light on this process. In lymphocytes, the coronary artery disease (CAD) associated SNP rs10757278 (intron 12) correlates with abundance of certain circular (14-5 and 4-6) and linear (exon 1-2, but not 18-19, containing) isoforms ( 8 ). The rs10757278 A allele was found to inhibit skipping of exon 15, promoting circ ANRIL  species ending in exon 14 ( 8 ). Mechanisms regulating  ANRIL  splicing require further study.\nDeterminants of  ANRIL  transcript longevity and stability remain uncertain, but miRNAs can participate.  ANRIL , downregulated following Kaposi's sarcoma associated herpesvirus (KSHV) infection, contains multiple seed matches for KSHV miRNAs. Forced miRNA expression decreased  ANRIL  abundance, and miRNA pull-down experiments confirmed a direct interaction. In addition, KSHV latency associated proteins vFLIP and vCyclin also decreased  ANRIL  abundance, suggesting post-transcriptional miRNA-dependent and independent regulation ( 45 ).\n\nMany studies show that ANRIL functions in cells to regulate gene expression via chromatin modification. Acting  in cis, ANRIL  interacts with both PRC-1 and−2 to mediate epigenetic transcriptional repression of neighboring genes  CDKN2A  and  CDKN2B , through mechanisms involving histone modification and chromatin remodeling ( 13 ,  21 ,  33 ).  ANRIL  interacts with PRC1 component CBX7 to recruit PRC1 to the p14ARF and p16INK4A loci, silencing the  CDKN2A  locus by H3K27-trimethylation ( 21 ). At  CDKN2B, ANRIL  was shown to recruit SUZ12, a subunit of the PRC2 ( 13 ).  ANRIL  also interacts with PRC-associated protein YY1 ( 46 ). Intriguingly, the structural conformation of the methyl-lysine binding pocket in the chromodomain of CBX7, which interacts with H3K27-trimethylation to cause chromatin compaction, is influenced by allosteric RNA-protein binding with  ANRIL  ( 47 ). However, despite this well-documented repression of other locus genes by  ANRIL , a positive correlation between  ANRIL  (both short and long isoforms),  CDKN2A  and  CDKN2B  RNA abundance has been frequently reported, suggesting transcriptional co-regulation of these genes predominates in many tissues ( 8 ,  10 ,  14 ,  16 ,  44 ,  48 – 51 ).\nANRIL  also acts in a PRC1/2 dependent mechanism to repress distant genes  in trans  ( 32 ,  46 ). Trans regulation by  ANRIL  may be dependent on Alu motifs, which are found both in  ANRIL  transcripts and in the promoters of  ANRIL  target genes ( 46 ). This mechanism was shown to regulate the CARD8 gene in endothelial cells ( 52 ). Polycomb group proteins, which are highly enriched near Alu motifs across the genome, are recruited to target gene promoters upon  ANRIL  over-expression. In support of this concept, silencing  ANRIL  impacts expression of a large number of genes across the genome ( 14 ). Separate from chromatin modification,  ANRIL  is reported to regulate Wnt signaling by binding to SOX2, increasing transcriptional activity of the WNT/β-catenin pathway ( 39 ).\nANRIL  also influences gene expression via miRNA networks.  ANRIL  regulates miRNAs both at the epigenetic level, through regulation of miRNA transcription, and through direct binding to miRNAs, acting as a miRNA “sponge.” In gastric cancer cells,  ANRIL  epigenetically silences miR-99a/miR-449a through a PRC2 mechanism ( 22 ). In general, expression of  ANRIL  and its target miRNAs are negatively correlated in tissues and cell lines ( 22 ,  53 – 57 ).  ANRIL  has been described as having pro-oncogenic effects by sponging miRNAs (see below for more details). On the other hand, circ ANRIL  containing exons 5-6-7 was found to lack miRNA sponge activity ( 6 ). Inhibition of miRNAs can reverse the effects of  ANRIL  knockdown.\nANRIL  has broad impacts on cell biology, including influence over proliferation, senescence, apoptosis, extracellular matrix remodeling, and inflammation ( 14 ). In cancer,  ANRIL -miRNA interactions regulate networks of downstream targets of miRNAs, promoting an oncogenic role for  ANRIL  in cell proliferation, metastasis, invasion, radio-resistance, drug-induced cytotoxicity and apoptosis, involving many different signaling pathways ( 22 ,  53 – 57 ). Specifically, repression of cell cycle inhibitors p14ARF, p15INK4B, and p16INK4A increases proliferation, decreases senescence, and contributes to the DNA damage response ( 13 ,  21 ,  33 ). PRC-mediated epigenetic repression of Kruppel-like factor 2 (KLF2) influences proliferation and apoptosis ( 40 ,  58 ). Cooperation between  ANRIL  and PRC-associated YY1 increases TNF-alpha dependent inflammatory mediators (IL-6, IL-8) through NF-kB ( 26 ).  ANRIL  influences the cellular response to oxidative stress through a miR-125a regulation of MCL-1 ( 59 ). Circular  ANRIL  species were found to regulate ribosome biogenesis in vascular smooth muscle cells ( 6 ).\n\nA primary driver of interest in  ANRIL  is the large body of genomic data linking the  ANRIL  gene with risk of human disease. Genome-wide association studies (GWAS) have identified many disease-associated SNPs in or near the  ANRIL  gene ( 60 ). The  CDKN2A/B  locus is remarkable for the large number of associated diseases, ranging from aging and frailty to cancer to metabolic disease. Perhaps surprisingly given the validated importance of the products of the  CDKN2A  and  CDKN2B  genes in cell biology, in some cases  ANRIL  expression shows stronger phenotype association than protein-coding  CDKN2A/B  locus genes ( 4 ,  48 ), linking  ANRIL  itself to a range of important human diseases.\nStudies indicate that SNPs in the ANRIL gene can impact ANRIL expression and function. The  CDKN2A/B  locus is associated with risk of cancer, atherosclerotic disease, type 2 diabetes, stroke, aneurysm, periodontitis, Alzheimer's disease, aging, frailty, glaucoma, endometriosis, multiple sclerosis, hypertension ( 10 ,  61 ). Reviewed here are only SNPs within or downstream of the  ANRIL  gene; broader  CDKN2A/B  locus disease associations have been reviewed previously ( 10 ,  62 ). Integrating information from published observations and the NCBI linkage disequilibrium database ( 63 ), we find that disease-associated SNPs in the  ANRIL  gene that modulate locus gene expression fall into approximately six groups (Table  1 ) defined loosely by linkage block and reported effects. Exceptions outnumber the rules, however; for nearly all groups there are reports of SNPs with different or even opposite effects. Summarized here is a generalized synopsis of the majority of reports. Group A SNPs, while located in  ANRIL  introns, generally impact  CDKN2A/B  but not  ANRIL  biology ( 48 ,  50 ,  64 ). All other SNP groups have reported impacts on  ANRIL  itself, but reports often describe conflicting direction of change. Some SNPs are reported to fall in enhancer regions ( 43 ,  49 ,  64 ) or to impact  ANRIL  splicing ( 8 ) or secondary structure ( 69 ,  74 ). The data are incomplete. A particular weakness of the field is that although tissue-specific effects are likely to determine how polymorphisms impact disease risk, in many cases the relevant primary tissue has not been tested.\nDisease associated SNPs in/near the  ANRIL  gene that modulate locus gene expression.\nGroups A–F are defined loosely based on linkage disequilibrium (defined as LD>0.8 in Caucasian population in LDHap) and by predicted or tested impact on ANRIL expression or structure. Intron and exon numbers are based on 21 exons .\nThere is no consistent global pattern with respect to SNP impact on  ANRIL  abundance. For most SNP groups, risk-SNPs are reported that both increase and decrease  ANRIL  levels in different studies. Variability may be related to differences in technique used to detect  ANRIL  that favor one isoform over others, cell type studied, acute and chronic biology and genetic origin of the cellular material studied, and of course the individual biology of each polymorphism. Most  ANRIL  SNPs fall in large linkage blocks, which are variable among different human genetic groups; in many cases the SNP tested may not be the causative SNP in the linkage block, and published linkage blocks may not apply to the material tested if not carefully matched by origin. It is entirely possible that all conflicting results are correct; for example, a CAD risk-SNP could increase pro-proliferative  ANRIL  isoforms in endothelial, macrophage or vascular smooth muscle cells to drive atherosclerosis, whereas a diabetes risk-SNP at the same position could decrease proliferative  ANRIL  isoforms in beta cells to limit beta cell mass. The complexity of the human system necessitates testing the relevant  ANRIL  isoforms in the relevant cell type, preferably in primary cells, in tissue- and disease-specific manner.\nA comprehensive review of all SNP effects is beyond the scope of this review. Some  ANRIL  located disease-associated SNPs impact both  ANRIL  expression and  CDKN2A/CDKN2B  expression ( 14 ,  48 ); others impact  ANRIL  but not  CDKN2A  or  CDKN2B  ( 14 ,  48 ,  60 ), and still others impact  CDKN2A/CDKN2B  but not  ANRIL  ( 48 ,  50 ,  64 ). Some SNPs are located within predicted or proven enhancer regions ( 10 ,  43 ,  48 ,  49 ,  64 ,  75 ) or miRNA binding sites ( 65 ,  66 ), providing possible mechanisms of cell type specific gene regulation.\nBeyond regulation of  ANRIL  transcription, polymorphisms could impact  ANRIL  function by influencing relative abundance of different isoforms through RNA splicing or stability, or through altering the secondary structure or interactions of any given isoform. Several studies have identified  ANRIL  isoform-specific effects ( 50 ,  53 ,  67 ,  71 ,  72 ); for example, four SNPs forming an atherosclerosis risk haplotype were associated with increased expression of some, but not all,  ANRIL  isoforms ( 44 ). SNPs may influence the relative abundance of linear compared to circular isoforms ( 8 ). Several SNPs are reported to impact  ANRIL  free energy of folding, resulting in a predicted change in secondary structure, with implications for function and stability ( 48 ,  69 ,  74 ).\n\nANRIL  was initially identified in a kindred of familial melanoma-neural system tumor with a germ-line deletion of the entire  CDKN2A/B  locus ( 2 ). Although the  CDKN2A/B  locus is deleted or silenced in approximately 40% of human cancers, related to the tumor suppressive actions of  CDKN2A  and  CDKN2B  ( 76 ),  ANRIL  itself has pro-oncogenic properties.  ANRIL  is implicated in many malignancies, including cancers of the bladder ( 77 ), ovary ( 78 ,  79 ), lung ( 38 ,  58 ,  80 – 82 ), liver ( 40 ,  54 ,  83 ), stomach ( 22 ), breast ( 57 ,  84 ,  85 ), esophagus ( 86 ), nasopharyngeal cavity ( 39 ,  87 ,  88 ), thyroid ( 89 ), bone ( 90 ), cervix ( 91 ), colon ( 92 ), prostate ( 21 ,  56 ), glioma ( 55 ), and others ( 76 ). High tissue abundance of  ANRIL  in cancers is associated with aggressive clinicopathologic features such as high histological grade tumor size, advanced tumor-node-metastasis stage, and poor overall survival ( 22 ,  38 ,  40 ,  58 ,  78 ,  79 ,  83 ,  87 ,  89 ,  91 – 93 ). Certain SNPs within the  ANRIL  gene are associated with  ANRIL  and  CDKN2A/B  locus gene expression and clinical parameters ( 4 ,  48 ,  70 ,  94 – 96 ).  ANRIL  may be useful as a prognostic biomarker and a therapeutic target for clinical cancer management.\nAccumulating evidence suggests that  ANRIL  participates in tumorigenesis by influencing cell proliferation, apoptosis and metastasis. Depletion or overexpression of  ANRIL  changes expression levels of many genes involved in proliferation, cellular adhesion and apoptosis ( 14 ,  32 ,  46 ).  ANRIL  overexpression promotes proliferation, migration, invasion, and epithelial-mesenchymal transformation but inhibits cell apoptosis;  ANRIL  loss-of-function represses tumor size and growth rate, cell proliferation, migration, invasion, metastasis, and enhances apoptosis and senescence ( 22 ,  38 ,  55 ,  56 ,  58 ,  77 ,  80 ,  81 ,  84 ,  89 – 92 ). Suppression of  ANRIL  is required for Ras-induced senescence ( 13 ,  21 ,  42 ). High  ANRIL  levels are associated with resistance to chemotherapy, and  ANRIL  knockdown may promote chemosensitivity ( 37 ,  79 ,  88 ,  97 – 99 ). On the other hand,  ANRIL  mediated anti-oncogenic effects of phospholipase D in lung cancers ( 82 ).\nANRIL  may promote carcinogenesis through a number of mechanisms. Canonical  ANRIL  transcriptional mechanisms may play a role, such as by  in cis  suppression of the  CDKN2A/CDKN2B  tumor suppressor genes ( 80 ,  81 ,  100 ), or through PRC-mediated  in trans  gene regulation ( 40 ,  58 ,  80 ).  ANRIL  miRNA regulation has been implicated in cancers as well, including mechanisms involving let-7a and miR-125a in nasopharyngeal and oral carcinoma ( 56 ,  88 ,  101 ), miR-99a/miR-449a in gastric cancer ( 22 ), miR-122-5p in hepatocellular carcinoma ( 54 ), miR-186 in cervical cancer ( 91 ), and miR-199a in breast cancer ( 57 ), miR-34a in glioma ( 55 ), and miR-323 in pediatric medulloblastoma ( 102 ). Transcription factors affected by  ANRIL  in cancers include KLF2 ( 40 ,  58 ), SMAD ( 56 ,  86 ,  89 ) and β-catenin.  ANRIL  interacts with signal transduction pathways in cancers such as PI3K/AKT, p38 MAPK, TGF-β, ATM-E2F1, and MTOR ( 33 ,  55 ,  56 ,  86 ,  89 ,  99 ,  103 ).  ANRIL  can also drive cancer progression by increasing glucose uptake for glycolysis ( 87 ), through lymphangiogenesis via LYVE-1, VEFG-C, and VEGFR-3 ( 92 ), and through invasion and metastasis via MET and MMP3 ( 78 ). An intriguing but mostly unexplored phenomenon is breakpoint fusion transcripts including exons from  ANRIL  fused with exons from  MTAP , a neighboring protein-coding gene, which were identified in 20% of screened melanoma cell lines ( 104 ).\n\nIn addition to cancer, genome-wide association studies have repeatedly and confidently identified links between the genomic region containing  ANRIL  and risk of developing cardiometabolic disease, including type 2 diabetes and manifestations of atherosclerosis such as CAD and stroke ( 10 ,  62 ). This locus influences risk not only of classic type 2 (obesity-related) diabetes, but also with related syndromes such as gestational diabetes, transplant-associated diabetes, and cystic fibrosis related diabetes, but not risk of type 1 (autoimmune) diabetes ( 10 ). Although diabetes is a clinical risk factor for atherosclerosis, the genetic influence for these conditions at the  ANRIL  locus is mostly non-overlapping, with atherosclerosis SNPs located throughout the  ANRIL  gene, and T2D SNPs located distal to the last  ANRIL  exon (7). One exception is a SNP located in  ANRIL  exon 2, rs564398, which is associated with both T2D and CAD ( 105 ). Since  CDKN2A/B  locus genes are known for their roles in cell cycle regulation and cancer, and not metabolism, many questions remain as to how this locus impacts metabolic disease.\nSince  ANRIL  locus SNPs influence risk of atherosclerosis, many studies have now tested whether  ANRIL  gene expression is related to atherosclerosis-associated diseases. In subjects with angiographically confirmed CAD in the Leipzig heart study, specific  ANRIL  isoforms were positively correlated with CAD risk SNP haplotype in PBMCs, whole blood, and atherosclerotic plaque tissue ( 44 ). In the Framingham heart study,  ANRIL  SNPs were associated with multiple CAD-related outcomes, and showed isoform-specific  ANRIL  correlation in leukocytes, with short isoforms predicted to contribute to CAD pathogenesis ( 50 ). CAD risk-SNPs may regulate the relative abundance of linear and circular  ANRIL  isoforms ( 8 ). Intriguingly, abundance of  ANRIL  in circulating plasma was positively correlated with in-stent restenosis ( 53 ), but in PBMCs harvested at the time of angioplasty/reperfusion,  ANRIL  levels were lower in subjects with myocardial infarction, but higher in subjects with older age, diabetes, hypertension. In this cohort,  ANRIL  levels in PBMCs improved model prediction of subsequent left ventricular dysfunction ( 106 ).  ANRIL  promoter methylation may mediate an epigenetic influence on future cardiac risk; higher CpG methylation at birth was associated with higher pulse wave velocity, a marker for increased arterial stiffness indicating greater cardiovascular risk, at 9 years of age ( 31 ).\nMechanisms by which  ANRIL  impacts atherosclerotic disease remain debated. In aortic smooth muscle cells, knockdown of  ANRIL  using siRNA targeting exon 1 or exon 19 revealed altered gene expression networks impacting cell proliferation, apoptosis, extracellular matrix, and inflammation ( 14 ). Atherogenic gene expression networks were regulated by  ANRIL  via the Alu mechanism, in which Alu motifs target  ANRIL  to particular gene locations, recruiting PRC complexes and altering gene methylation status ( 46 ).  ANRIL  may impact risk of ischemic stroke by regulating the Caspase recruitment domain 8 (CARD8) gene in endothelial cells ( 52 ). A known CAD-associated miRNA, miR-92a, may mediate some  ANRIL  effects;  ANRIL  targets GATA2, MAP1B, and ARG1 were found to require miR-92a, placing this miRNA downstream of  ANRIL  for some atherogenic effects ( 69 ). Finally,  ANRIL  is related to inflammation:  ANRIL  is increased by pro-inflammatory factors NF-κ B and TNF-α in endothelial cells, and  ANRIL  was found to bind directly to the YY1 transcription factor to mediate TNF-a induction of cytokines IL-6 and IL-8 ( 26 ).\nAlthough GWAS studies do not suggest a link between  CDKN2A/B  locus SNPs and obesity risk in adult populations, intriguingly,  ANRIL  may be a genomic site of environmental epigenetic influence on obesity. The  ANRIL  promoter contains CpG methylation sites that are differentially regulated across samples. In human tissues taken at birth, lower CpG methylation in infancy predicted higher fat mass at 6 years of age, as well as increased bone size, mineralization and density ( 29 ,  30 ).  ANRIL  promoter methylation was also negatively correlated with BMI in contemporaneous samples of peripheral blood from adolescents and in adipose tissue from adults ( 29 ). Methylation of these CpG sites increased tissue abundance of  ANRIL  RNA, in a mechanism that might include increased activity of an estrogen response element. Functional studies in a liposarcoma cell line showed that transcription factor binding to an adjacent ERE was enhanced by methylation, and estradiol increased  ANRIL  expression ( 29 ).\nMultiple SNPs in different linkage blocks at the  CDKN2A/B  locus are associated with T2D risk; evidence in human populations suggests these SNPs impact pancreatic islet mass or function ( 10 ). Despite the fact that the T2D risk SNPs are located in or near the  ANRIL  gene, the field has largely assumed the effect was mediated by the protein coding genes at the locus, due to extensive published work implicating p16INK4A in the regulation of beta cell mass ( 10 ). However, although studies have found no association between  CDKN2A/B  T2D SNPs and transcript level of  p14ARF, p15INK4B , or  p16INK4A  in human islets ( 10 ,  107 ), an age-dependent positive association was identified between distal T2D risk-SNPs (group F in Table  1 ) and  ANRIL  expression ( 68 ). On the other hand, a T2D risk-SNP in  ANRIL  exon 2 (group C in Table  1 ) was associated with reduced  ANRIL  expression, again with no change in  p14ARF, p15INK4B , or  p16INK4A  expression ( 14 ,  48 ); however, these studies were carried out in blood rather than islets. In human islets, this exon 2 SNP was shown to remove a CpG methylation site; risk allele was associated with reduced islet insulin content but no change in locus gene expression ( 108 ). Risk allele at this SNP was associated with impaired beta cell proliferation response to high glucose ( 68 ). In a study relevant to diabetic retinopathy, high glucose exposure increased  ANRIL  expression in human retinal epithelial cells ( 25 ). In  ANRIL  was found to increase expression of VEGF, a critical element of the neovascularization that is central to damage from retinopathy, via a mechanism involving PRC2 and miR200b ( 25 ,  109 ).\n\nStudies suggest the  ANRIL  lncRNA influences risk of a number of diseases, including many types of cancer as well as metabolic disease. Current understanding of  ANRIL  biology indicates the primary function of this lncRNA is to regulate gene expression, both locally at  CDKN2A/B  as well as across the genome, via mechanisms including chromatin modulation, transcription factor binding, and miRNA regulation. Knowledge concerning ANRIL function in cancers is more solid and advanced than for metabolic tissues. Mechanisms by which SNPs influence ANRIL abundance remain uncertain and require more study; how DNA methylation regulates ANRIL in cancers also will benefit from more study. Much remains to be learned about the structural complexity of  ANRIL ; how the various identified linear and circular isoforms impact tissue biology to modulate disease risk is mostly unknown. There is an urgent need for deeper understanding of how  ANRIL  isoforms modulate cellular function in human organs and tissues, and to explore the differing roles of ANRIL in cancer and metabolic disease. Given the advent of RNA therapeutics, and the broad disease relevance of  ANRIL , it is possible that these studies may lead to future disease prevention and treatment.\n\nYK, C-HH, and LA wrote and revised the manuscript.\nThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.","source_license":"CC-BY-4.0","license_restricted":false}