The biological function and potential mechanism of long non‐coding RNAs in cardiovascular disease

Abstract Long non‐coding RNAs (lncRNAs), as part of the family of non‐protein‐coding transcripts, are implicated in the occurrence and progression of several cardiovascular diseases (CVDs). With recent advances in lncRNA research, these molecules are purported to regulate gene expression at multiple levels, thereby producing beneficial or detrimental biological effects during CVD pathogenesis. At the transcriptional level, lncRNAs affect gene expression by interacting with DNA and proteins, for example, components of chromatin‐modifying complexes, or transcription factors affecting chromatin status. These potential mechanisms suggest that lncRNAs guide proteins to specific gene loci (eg promoter regions), or forestall proteins to specific genomic sites via DNA binding. Additionally, some lncRNAs are required for correct chromatin conformation, which occurs via chromatin looping in enhancer‐like models. At the post‐transcriptional level, lncRNAs interact with RNA molecules, mainly microRNAs (miRNAs) and mRNAs, potentially regulating CVD pathophysiological processes. Moreover, lncRNAs appear to post‐transcriptionally modulate gene expression by participating in mRNA splicing, stability, degradation and translation. Thus, the purpose of this review is to provide a comprehensive summary of lncRNAs implicated in CVD biological processes, with an emphasis on potential mechanisms of action.

| 12901 ZHANG et Al. and non-coding genes, leading to post-transcriptional silencing or infrequent activation. 2,3 It is acknowledged that miRNAs are involved in a variety of physiological and pathological processes. In contrast, lncRNA research is in its infancy. However, thanks to the development of RNA-sequencing technologies and genome-wide analyses, thousands of lncRNAs have been unveiled. Despite intense technological efforts, only a small proportion of identified lncRNAs have been functionally annotated. In the following, we review the generally accepted categorization and function of ln-cRNAs, and explore the potential regulatory mechanisms of cardiovascular-related lncRNAs in cardiovascular physiology and pathology.

| LncRNA CL A SS IFI C ATI ON: LO C ATI ON AND FUN C TION
LncRNAs represent a subgroup of ncRNAs ranging from 200nt to ~100 kilobases (Kb) in length. However, they lack open reading frames (ORFs). 4 Until now, lncRNA classification criteria have not been unified. LncRNA nomenclature is primarily based on their empirical features, including origin of transcription, molecular function as well as cellular localization. 5 Through greater understanding of RNA-sequencing data/technologies, more than 50,000 lncRNAs, from intronic, exonic or intergenic regions, have been identified in many different human tissues. 6 The majority of lncRNA genomic loci rely on intergenic regions; some are found in introns of coding genes, 7 or they originate from enhancer gene regions (enhancer-derived lncRNAs, elncRNAs). With the identification of circular RNAs (circRNAs), which are processed by the intron back-splicing, it has been recognized that the genomic structure of lncRNAs is more than just linear. 8 Similarly, lncRNAs are also categorized by their molecular function: (a) Signal lncRNAs can serve as signalling molecules in response to cellular cues or particular stimuli; they participate in tissue molec- Enhancer lncRNAs gather at promoter and enhancer regions via chromosomal loops, to enhance transcription. 9 Typically, lncRNAs are transcribed by RNA polymerase II or III (RNA pol II/ III) molecules, which are matured by selective cleavage and subjected to 5' end capping and polyadenylation processing. 10 Based on their cellular localization, lncRNAs can be divided into two major categories: nuclear and cytoplasmic. Nuclear-localized lncRNA transcripts have structural and regulatory roles and affect chromatin status by interacting with chromatin-modifying complexes, thereby controlling gene transcriptional activities. 11,12 Cytoplasmic lncRNAs complement with mRNAs to form double-stranded RNA molecules, which interfere with mRNA stability and translation, protein localization and turnover and other signalling pathways. 13 They also function as endogenous sponges for miRNAs, thereby preventing translational inhibition or mRNA degradation. 14

| LncRNA S A S EMERG ING REG UL ATOR S IN C ARD I OVA SCUL AR B I OLOGY
Far removed from the concept of 'junk RNA', lncRNAs are believed to be functional regulators that modulate normal development and disease processes in the cardiovascular system. By monitoring lncRNA differential expression, studies have identified a number of lncRNAs that play crucial roles in regulating gene expression programmes during normal cardiovascular development and CVD-related pathogenesis. 15,16 Globally, CVDs are a major cause of mortality and generate huge health and financial burdens worldwide. 17

| MECHANIS MS OF AC TI ON OF C ARDIOVA SCUL AR-REL ATED LncRNA S
In recent years, a number of studies have shown that lncRNAs play pivotal mechanistic roles in regulating gene expression, through multiple mechanisms, at different levels. The underlying molecular mechanisms underpinning several lncRNAs have not yet been elucidated. However, the exploration of cardiovascular-related lncRNAs is still in its early stages. Therefore, understanding mechanisms behind these functional transcripts is a major disease challenge. In the following sections, we summarize recent discoveries linking lncRNA biological functions to potential CVD mechanisms, at transcriptional and post-transcriptional levels.

| LncRNAs transcriptional regulation via the recruitment of multiple chromatin modifiers
The regulation of chromatin structure is of great importance for gene transcription in eukaryotes. Enzymes that catalyse chromatin structural changes mainly include histone-modified and chromatinremodelling complexes. In recent years, lncRNAs have been shown to regulate the access or dismissal of multiple chromatin-modifying complexes from chromatin, and activating or inhibiting gene expression at the transcriptional level. Such regulatory modes are common for lncRNAs, as approximately 40% of these molecules interact directly with diverse chromatin-modifying complexes. 20 Mechanistically, lncRNAs function as scaffolds to assemble chromatin-modifying enzymes into complexes, facilitating lncRNAs as guides to recruit these complexes to specific genomic loci, thereby altering chromatin expression. An alternative scenario could be that other lncRNAs interact with chromatin-modifying complexes by acting as decoys to impede the binding of chromatin modifiers to target genomic loci, in turn affecting transcriptional processes ( Figure 1). 21

| Interactions with histone-modifying complexes
Approximately 40% of lncRNAs are directly related to diverse histone-modifying complexes, including PRC2, MLL, LSD1, WDR5 and others. 22,23 It is accepted that lncRNAs serve as scaffolds to coordinate the recruitment of protein complexes to target loci, to change the chromatin or DNA state. 21  subunit of polycomb repressor complex 2 (PRC2), which is a specific histone methyltransferase (core subunits are EZHZ and EED) that catalyses histone H3 lysine 27 trimethylation. By acting as a molecular decoy, Chaer bound to PRC2 and impeded its targeting to loci, thereby inhibiting H3K27me at promoter regions of target genes implicated in cardiac hypertrophy. 24 In other work, Chaer in patients with atherosclerosis was highly expressed when compared with healthy individual, suggesting that Chaer promoted atherosclerosis by mediating PRC2 activity via the mTOR signalling pathway. 25 Additionally, Klattenhoff et al identified a mouse-specific, heart-related lncRNA termed Braveheart (Bvht), which was required for cardiovascular lineage commitment and cell differentiation.
LncRNA-Bvht appeared to interplay with PRC2 to regulate target gene transcriptional programmes. 26 The lncRNA, Fendrr, also plays a critical regulatory role in embryonic cardiac differentiation. 27

| Chromosome conformation regulation and enhancer activity
Some lncRNAs are required for correct chromatin conformation and act through chromatin looping in enhancer-like models. 21 These lncRNAs appear to organize higher-order chromatin interactions between promoters and distal enhancers, or enhancer-like noncoding gene loci, thereby activating transcription. These elncRNA are newly annotated lncRNAs, encoded by functional enhancers as transcriptional units. 34 This novel class of lncRNAs orchestrates long-range gene activation by modulating chromatin organization and structure, as recently described. 35 These researchers identified LEENE, an enhancer-associated lncRNA encoded in a distal enhancer region, which forms proximal associations with the endothelial nitric oxide synthase (eNOS) promoter locus. This study not only confirmed that LEENE positively regulated eNOS transcription, which is a marker of endothelial cell (EC) homeostasis and vascular function, but was also implicated in cardiovascular endothelial regulation. By Additionally, the KCNQ1 overlapping transcript 1 (Kcnq1ot1), which is an lncRNA derived from the KCNQ1 locus, was shown to modulate Kcnq1 expression by establishing a repressive chromatin conformation. 36 The Kcnq1 gene is essential for heart development and function, encoding voltage-gated potassium channels in cardiac myocytes during heart development. Kcnq1 defects contribute to Long QT Syndrome (LQTS), a cardiac disorder associated with arrhythmia. 37 Similarly, in late embryogenesis, the paternally expressed lncRNA-Kcnq1ot1 loses its imprinted expression and converts to biallelic expression during foetal heart development. This expression occurs simultaneously with Kcnq1. In parallel with this transition, conformational chromatin changes are also detected, involving spatial proximity between the Kcnq1 promoter and distant heart-specific enhancers. 38

| LncRNAs as sources of cardiovascular miRNAs
As the two major subgroups of ncRNAs, lncRNAs and miRNAs exert significant roles in cardiovascular pathophysiology. LncRNAs serve as sources and endogenous inhibitors of miRNAs. 39 Genomic analyses have revealed that approximately 50% of human miR-NAs may be produced from the introns of coding genes, while the remainder is expressed from introns or exons of non-coding transcripts. 10 Initially, RNAPII transcribes miRNA genes into F I G U R E 2 Models of enhancer activity of lncRNAs. In the chromatin looping model, lncRNA-LEENE (red line) is transcribed from enhancer non-coding genes. LEENE RNA transcripts recruit RNAP II to the eNOS promoter and bridge the enhancer of the non-coding LEENE gene and the promoter of the eNOS gene. As a result, eNOS transcription levels are enhanced. lncRNA, long noncoding RNA; eNOS, endothelial nitric oxide synthase; RNAP II, RNA polymerase II primary transcripts (pri-miRNAs), which are longer hairpin-containing RNAs. 33 Subsequently, in the nucleus, the miRNA-processing enzyme complex, comprised of Drosha and its cofactor Dgcr-8, severs these pri-miRNAs into precursors (pre-miRNAs). These pre-miRNAs are then transferred to the cytosol where they are processed into mature miRNAs by a second enzyme complex, Dicer and its transactivation response RNA-binding protein (TRBP) complex. 40 Thus, mature miRNAs are derived from pri-miRNAs via the sequential processing of miRNA-processing enzymes, Drosha and Dicer in the nucleus and cytoplasm, respectively. 41 For example, lncRNA-H19 has been shown to act as a primary miRNA precursor, and miR-675 is the mature variant of the transcript. 42 As the pri-miRNA template for miR-675, H19 is highly transcribed in the mouse embryo, while miR-675 expression is restricted to the placenta. MiR-675 inhibition was shown to be mediated by the RNA-binding protein HuR, which binds to H19 transcripts, thereby inhibiting miR-675 processing at the Drosha stage. 43

| LncRNAs as negative modulators of cardiovascular miRNAs
LncRNAs possess mRNA-like structures and therefore exert sponge-like effects on multiple miRNAs to competitively sequester them from target mRNAs. Thus, lncRNAs relieve miRNA suppression on their target mRNAs, facilitating mRNA expression and function. 45 In the study by Salmena et al, the competitive endogenous RNA (ceRNA) hypothesis identified a new post-transcriptional regulatory mechanism for lncRNAs. These authors proposed that lncR-NAs contained miRNA-binding sites, also known as miRNA response elements (MREs), thus facilitating lncRNA-binding to miRNAs. 46 It is noteworthy that miRNAs suppress target mRNAs, by inhibiting translational activities or promoting degradation. 47 Once miRNAs and lncRNAs are complemented, these effects are reversed, meaning that target mRNA abundance and function are up-regulated. In a similar manner, cytoplasmic circRNAs are also sponges for miRNAs via sequence-specific binding. 48,49 As described below, cumulative studies have shown that lncRNA and miRNA are dynamically regulated during cardiac conditions via this sponge mechanism. (Figure 3; Table 1).

| Cardiomyocyte apoptosis
Several studies have highlighted the functional role of lncRNAs as miRNA sponges in the regulation of cardiomyocyte apoptosis. 50,51 More recently, the newly identified lncRNA, the small nucleolar RNA Moreover, SNHG1 also promoted BCL2L2 expression, which was a target of miR-195. 52 Another cardiac apoptosis-related lncRNA, the metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), was increased during hypoxia or ischaemia. 53

| Cardiomyocyte necrosis
Emerging studies have shown that lncRNAs are also involved in the regulation of cardiomyocyte necrosis, by targeting miRNAs.
As a major type of cardiomyocyte death mechanism in cardiac disease, programmed necrosis appears to be mediated by the F I G U R E 3 Cardiac lncRNAs, which act as miRNAs sponges, are involved in the regulation of cardiomyocyte apoptosis, necrosis, autophagy, hypertrophy and cardiac fibrosis. A solid arrow indicates promotion; a dashed arrow represents inhibition; Cardioprotective lncRNAs are in bold type; Heart-damaging lncRNAs are in italics receptor-interacting serine/threonine-protein kinases (RIPK) 1 and

3, which are antagonized by the FADD (death domain) containing
Fas-associated protein. 57 A recent study revealed that miR-873 diminished cardiomyocyte necrosis in the mouse ischaemic/reperfusion (I/R) model by targeting and repressing RIPK1/RIPK3 expression. 58 To explore further molecular mechanisms, researchers searched for upstream regulators of miR-873 and identified a novel lncRNA, necrosis-related factor (NRF). Using a quantitative real-time PCR (qRT-PCR) approach, they discovered that NRF levels were increased in the ischaemic zone upon mouse I/R injury. Other studies have suggested that NRF lncRNA directly binds to miR-873 and represses its inhibitory effects on RIPK1/RIPK3, thus intensifying RIPK1/RIPK3-dependent programmed necrosis in cardiomyocytes.
The loss-of-function of NRF attenuated myocardial necrosis and infarction during cardiac I/R injury. In addition, a previous study reported that lncRNA H19 competitively bound miR-103/107, impairing FADD expression and ameliorating necrosis. 57 Enforced expression of H19 was shown to counteract cardiomyocyte necroptosis through modulation of the miR-103/107-FADD pathway.

| Cardiomyocyte autophagy
Recent studies have suggested that lncRNAs are also implicated in the regulation of autophagy in cardiomyocytes, via several miRNAs.
For example, lncRNA-APF (autophagy promoting factor) enhanced autophagy in I/R mouse hearts by targeting miR-188-3p, thereby elevating autophagy-related protein 7 (ATG7) translation, resulting in increased myocardial ischaemic injury. 59 As well as being an important autophagy-promoter, ATG7 is a specific target of miR-188-3P. 60 By directly binding to miR-188-3p, lncRNA-APF lost its cardioprotective function on autophagy through ATG7 targeting. APF knockdown exhibited prominent reductions in myocardial infarction upon I/R-injured mice.

| Cardiomyocyte hypertrophy
Several research findings have highlighted important functional links between lncRNA and miRNA in terms of modulating cardiac hypertrophy. In 2018, researchers discovered that the X-inactive-specific transcript (XIST), which regulates X-chromosome inactivation (XCI) in mammals, was up-regulated in mice with hypertrophic myocardium.
XIST lncRNA was shown to serve as a 'sponge' to miR-330-3p, which targeted and inhibited S100B mRNA expression. XIST elevated S100B

| Cardiac fibrosis
Several studies have reported functional lncRNA-miRNA interac-

| Post-transcriptional control of cardiovascular-related mRNAs
Besides the well-established ceRNAs mechanisms of action, recent studies have also reported that lncRNAs modulate mRNA alternative splicing, stability, degradation and translation efficiencies.
LncRNAs primarily base-pair with target mRNAs to form doublestranded complexes, or they interact with RBPs to interfere with mRNA splicing and translation processes. 74 Indeed, evidence also suggests that some lncRNAs can directly target mRNA for degradation. Blum et al discovered a natural anti-sense lncRNA for tyrosine kinase containing immunoglobulin and epidermal growth factor homology domain-1 (tie-1AS), which was formed at the Tie-1 locus.
Tie-1 is a receptor tyrosine kinase expressed in ECs and is essential for EC growth during vascular formation. The lncRNA tie-1AS selectively bound tie-1 mRNA and down-regulated tie-1 transcript levels resulting in EC contact junction defects. 75 Furthermore, in the absence of base-pairing with mRNAs, lncRNAs also interact with RBPs to interfere with pre-mRNA splicing. For example, the nuclear lncRNA MALAT1 alters pre-mRNA splicing patterns via interactions with serine/arginine (SR) splicing factors. 76 Interestingly, MALAT1 depletion decreased the accumulation of SR splicing factors in nuclear speckles, while SR protein expression was increased. However, more transcriptome-wide studies are required to understand how lncRNAs affect mRNAs in cardiovascular physiological processes.

| LncRNA s COULD B E A S THER APEUTI C TARG E TS OR B I OMARK ER S
With more in-depth investigation of cardiovascular-related lncRNAs, new molecular genetic insights can be generated on CVD biology, potentially creating unique pharmacological and therapeutic target opportunities. LncRNAs appear to regulate many cellular processes, as they act in specific tissues and cell types, making them excellent candidates for therapeutic applications. 77 However, promising approaches targeting lncRNAs are still in their infancy; major challenges to lncRNA research are their high species-specificity and poor conservatism. Even with major lncRNA successes in animal models, these findings are not necessarily transferable to humans. Therefore, it is particularly important to transfer the therapeutic targeting of lncRNAs from animal models to human diseases.
The lncRNA field is promising in terms of its biomarkers of various diseases in body fluids. The abnormal expression patterns of lncRNAs are often correlated with specific disease types, making these molecules appropriate for clinical diagnostics and prognostics.
Therefore, circulating lncRNAs are increasingly considered as new, non-invasive, highly sensitive biomarkers for the diagnosis, prognosis and risk stratification of CVDs. 78 The combination of traditional biomarkers with novel lncRNA biomarkers might overcome certain deficiencies in prognostic assessments, for example, a mitochondrial-derived lncRNA, LIPCAR was investigated in patients with and without left ventricular remodelling after MI, and was used to predict future cardiac remodelling and risk of death from HF. 79 Recently, Zhang et al observed that the expression of the plasma-based ln-cRNA, myosin heavy-chain-associated RNA transcripts (MHRT) was significantly down-regulated in HF patients. 80 Follow-up studies showed that plasma MHRT levels were directly proportional to the survival conditions of HF patients. In view of this work, circulating MHRT shows promising diagnostic and prognostic use for HF treatment; however, validation in larger patient populations will be required to confirm its application as a clinical biomarker in the future.

| CON CLUS IONS
Increasing evidence suggests that lncRNAs are regulated during normal and pathological cardiac physiological processes and may be clinically advantageous as therapeutic targets for the reasons previously mentioned. In this review, we focused on their mechanism of action, functional roles in pathophysiological processes and their potential as biomarkers or novel therapeutic targets in the cardiovascular system. LncRNAs regulate gene expression at the transcriptional and post-transcriptional level through interaction with nucleic acids, and with proteins in both sequence-and structure-specific manners.
Our review provides an understanding of their mode of action and establishes a theoretical basis for their study as biologically relevant molecules in CVD. Indeed, more molecular insights underpinning their mechanism of action will be required to translate lncRNA research findings into clinical practice.

CO N FLI C T S O F I NTE R E S T
The authors confirm that they declared no conflict of interest.

AUTH O R S ' CO NTR I B UTI O N S
Chengmeng Zhang: wrote the manuscript; Bing Han: prepared the revised the manuscript. All authors reviewed the manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
Research data are not shared in this article, because no new data were created or analysed in this study.