The novel regulatory role of lncRNA‐miRNA‐mRNA axis in cardiovascular diseases

Abstract Long noncoding RNAs (lncRNAs) are RNAs longer than 200 nt in length that are characterized by low levels of sequence conservation and expression; lncRNAs modulate various biological functions at epigenetic, transcriptional and post‐transcriptional levels, or directly regulate protein activity. As a family of small and evolutionarily conserved noncoding RNAs, microRNAs (miRNAs) are capable of regulating physiological and pathological processes via inhibiting target mRNA translation or promoting mRNA degradation. A number of studies have confirmed that both lncRNAs and miRNAs are closely associated with the development of cardiovascular diseases (CVDs), such as cardiac remodelling, heart failure, myocardial injury and arrhythmia, and that they act as biomarkers, potential therapeutic targets or strong indicators of prognosis; however, the underlying molecular mechanism has not been elucidated. Recently, emerging evidence showed that the novel regulatory mechanism underlying the crosstalk among lncRNAs, miRNAs and mRNAs plays a pivotal role in the pathophysiological processes of CVDs in response to stress stimuli. In this review, I comprehensively summarized the regulatory relationship of lncRNAs, miRNAs and mRNAs and highlighted the important role of the lncRNA‐miRNA‐mRNA axis in CVDs.

greater that 200 nt, and some have a poly (A) tail. According to the NONCODE database (http://www.noncode.org), there are 102 783, 87 553 and 27 793 lncRNA genes for human, mouse and rat, respectively; in comparison, human have 19 836 human protein-coding genes (Ensemble 90). Alternative splicing during differentiation causes lncRNAs to exhibit lower levels of sequence conservation and expression than protein-coding genes, but growing evidence has confirmed that lncRNAs maintain pivotal and multiple functions in a vast array of biological processes despite their limited expression levels. To date, multiple lncRNAs have been well documented due to the development of high-throughput RNA sequencing (RNA-seq) techniques; they can be divided into five subclasses as sense, antisense, bidirectional, intergenic and intronic lncRNAs, based on their position in the genome or relation to the coding gene. 9 Functional analysis showed that lncRNAs could regulate genes expression at epigenetic, 10 transcriptional 11 and post-transcriptional levels, 12 or could directly modulate protein activity. 13 Unlike lncRNAs, miRNAs are a family of evolutionarily conserved, endogenous, ncRNAs approximately 22 nt in length, that can bind to the 3′-untranslated region (3′-UTR) of mRNAs through imperfect complementarity. 14 miRNAs may regulate more than 30% of genes and play a significant role in the regulation of biological activities in cells by inhibiting target mRNA translation or promoting mRNA degradation. 15,16 It is of note that lncRNA and miRNA can modulate the initiation and progression of CVDs via regulating different molecular mechanisms. For instance, the inhibition of Meg3 suppressed cardiac fibrosis and hypertrophy by decreasing MMP-2 expression. 17 The up-regulation of miR-29 could reduce lesion size and necrotic zones via targeting Col1A and Col3A. 18 Recently, a novel regulatory mechanism among lncRNAs, miRNAs and their mRNA targets has been annotated in CVDs by pull-down assay, chromatin immunoprecipitation (ChIP), luciferase reporter gene assay and quantitative reverse transcription polymerase chain reaction (qRT-PCR) methods, although a plurality of evidence has revealed that lncRNA-miRNA-mRNA cascades participated in various pathophysiological processes in the field of oncology. 7,[19][20][21] In this review, four kinds of regulatory

REGULATING THE BIOLOGICAL ACTIVITIES OF CARDIOMYOCYTE S AND CARDIAC FIBROBLASTS
In 2014, the lncRNA AK048451, named cardiac hypertrophy related factor (CHRF), was first identified as an endogenous sponge of miR-489 that could directly bind to miR-489 in a sequence-specific manner and restrain miR-489 expression; this ability was demonstrated by analysis with the bioinformatics programme RNA hybrid, luciferase reporter gene activity assay and pull-down assay. The enforced expression of CHRF promoted hypertrophic responses, and the inhibition of CHRF retarded cardiac hypertrophic effects induced by angiotensin II (Ang II) treatment by regulating the miR-489 activity and the expression of its target, myeloid differentiation primary response (Myd88); these effects suggested that CHRF served as the upstream negative regulator of the miR-489/Myd88 axis under the hypertrophic condition. 22 Other lncRNAs have also been confirmed to act as miRNA sponge in order to modulate cardiomyocyte hypertrophy. The lncRNA growth arrest-specific 5 (GAS5) was generally regarded as a tumour suppressor, acting as a miR-21 sponge, that could inhibit the proliferation and promote the apoptosis of various cancer cells. 23,24 Interestingly, the study showed that GAS5 contained a binding site for miR-23a and acted as a sponge of miR-23a.
Up-regulated GAS5 expression inhibited cardiomyocyte hypertrophy through negatively regulating miR-23a and its target forkhead box O3 (Foxo3a). 25 Additionally, a previous study confirmed that myocardial infarction-associated transcript (MIAT) possessed binding sites for miR-150 and could serve as a miR-150 sponge in modulating cell proliferation, apoptosis and migration. 26 In cultured H9C2 cells, the overexpression of MIAT triggered an Ang II-induced hypertrophic response via inhibiting the level of miR-150 level; however, the possible target of miR-150 was not identified in this study. 27 Mitochondrial fission and fusion are associated with cardiomyocyte apoptosis. 28 The lncRNA AK017121, named cardiac apoptosis-related lncRNA (CARL), was identified as a functional sponge of miR-539. The overexpression of CARL suppressed mitochondrial fission, apoptosis and ischaemia/reperfusion (I/R) injury by directly inhibiting the expression of miR-539 and subsequently up-regulating the level of its target prohibitin 2 (PHB2). 29 The modulation of CARL-miR-539-PHB2 expression might provide a novel approach for the treatment of ischaemic heart disease (IHD). Another lncRNA AK009271, named mitochondrial dynamic related lncRNA (MDRL), is also involved in mitochondrial fission and fusion under stress condition. MDRL, which contains a target site for miR-361, could directly interact with miR-361 and suppressed the expression and activity of miR-361, thus reducing mitochondrial fission and apoptosis upon anoxia/reoxygenation treatment. Although the specific target mRNA of miR-361 was not discussed, a novel regulatory mechanism between miR-361 and miR-384 was explored. miR-361 could directly bind to the primary transcript of miR-484 and hampered the processing of pri-miR-484 into pre-miR-484 by Drosha in the nucleus. The overexpression of MDRL reduced the level of pri-miR-484 and increased the expression of pre-miR-484, while these effects were reversed when MDRL expression was knocked down. These data implied that different lncRNAs could suppress the mitochondrial fission and apoptosis by modulating different molecular pathways. 30 Cardiac differentiation is essential to maintain normal heart development and function. The molecular mechanism underlying H19-modulated cardiac differentiation was examined in P19CL6 cells, which are a functional in vitro model for exploring cardiomyocyte differentiation. 31 During the late stage of cardiac differentiation of P19CL6 cells, miR-19b was negatively regulated by H19, as demonstrated by luciferase activity assay and qRT-PCR. The overexpression of H19 significantly inhibited cell proliferation and promoted cell apoptosis via modulating miR-19b and its target sox6, while the down-regulation of H19 reversed these effects. 32 Cardiomyocyte autophagy is an evolutionarily conserved process in face of stress stimuli, but abnormal autophagy contributes to cell death. 33 The lncRNA AK079427, named APF, played a crucial role in the regulation of cardiomyocyte autophagy by modulating miR-188-3p and its target ATG7. The knockdown of APF reduced autophagic vesicle formation and cell death in cardiomyocytes upon anoxia/reoxygenation treatment, as well as in I/R injury heart via inhibiting miR-188-3p expression and augmenting the level of ATG7. 34 Unveiling the functional role of APF in cardiomyocyte autophagy is helpful for protecting against cardiac dysfunction. In an I/R injury model, compared to control groups, APF-siRNA mice demonstrated a decreased diastolic left ventricular internal diameter (LVIDd) but increased fractional shortening (FS), indicating that APF may be viewed as a therapeutic target to preserve cardiac function.
Cardiomyocyte necrosis mainly contributes to heart failure, while the underlying mechanism is not fully explained. 35 H19, which contains three potential miR-103/107 binding sites, prevented cardiomyocyte necrosis via inhibiting the level of miR-103/107 and its target Fas-associated protein with death domain (FADD) in response to H 2 O 2 treatment, while these effects were offset by silencing H19. 36 Another lncRNA, named necrosis-related factor (NRF), was closely related to necrotic death of cardiomyocytes by acting as an endogenous RNA sponge that interacted with miR-873 in the cytoplasm.
Silencing of NRF increased miR-873 expression and decreased the levels of the miR-873 targets receptor-interacting serine/threonineprotein kinase 1 (RIPK1) and RIPK3, which led to a sharp reduction in myocardial necrosis. 37 Strikingly, NRF could also be regulated at the transcriptional level, and a binding site of p53 was detected in the promoter region of NRF. H 2 O 2 treatment exacerbated the association of p53 with the NRF promoter, as determined by ChIP assay, indicating that p53 promoted the activity of NRF in response to stress. A functional assay showed that silencing p53 reduced necrotic cell death and inhibited NRF promoter activity upon H 2 O 2 treatment, and the increased expression of miR-873 and decreased level of RIPK1/RIPK3 were also induced by the down-regulated P53 level. 37 50 It has been verified that miR-133 could prevent cardiac hypertrophy via inhibiting its targets RhoA and Cdc42. However, it is unclear whether these mRNAs also act as effect targets of miR-133 or are directly regulated by ROR in this situation. 51 Atrial fibrillation (AF), which is a result of abnormal electrical activities in atrial tissues, is the characteristic of electrical remodelling contributing to cardiac failure. 52 LncRNAs and miRNAs could F I G U R E 1 H19 as a crucial modulator in the regulation of diverse pathological processes. H19 suppressed miR-103/107-FADD and miR-19b-sox6 cascades resulting in preventing cardiomyocyte necrosis, inhibiting proliferation and promoting apoptosis in P19CL6 cells. H19 coexpression with miR-675 attenuated cardiac hypertrophy and potentiated CPC senescence through inhibiting CaMKIId and USP10 expressions HUANG | 5771 modulate the progression of AF via targeting diverse mRNAs. For instance, the lncRNA AK055347 contributed to the pathogenesis of AF via the regulation of Cyp450, ATP synthase and MSS51. 53 Upregulated miR-208b abolished the expression and function of CAC-NA1C, CACNB2 and SERCA2 during atrial remodelling. 54 lncRNA could mediate electrical remodelling during AF through interacting with miRNA and its target mRNA. AF was induced in three of five rabbits in the TCONS_00075467 knockdown group, and silencing of TCONS_00075467 could markedly shorten the atrial effective refractory period (AERP), action potential duration (APD), and L-type calcium current (ICaL) density of primary atrial myocytes, suggesting that inhibiting the level of TCONS_00075467 was associated with the development of AF. 55 As a lincRNA, TCONS_00075467 may act in cis to regulate the expression of neighbouring protein-coding genes or may modulate target genes through trans-mechanism, but the location of TCONS_00075467 is far from the upstream or downstream sequences of the known protein-coding transcripts, and in addition,

REGULATOR OF lncRNAs TO PRE VENT TH E PROLIFERATION AND MIGRATION OF ECs
In previous studies, MIAT was identified as a sponge of miR-150 in regulating the biological activities of epithelial cells and myocyte cells. 24,25 Conversely, miR-150 could negatively modulate the expression of MIAT to suppress the proliferation, migration and tube formation of ECs. Vascular endothelial factor (VEGF) was a target of miR-150-5p, but its level was up-regulated when MIAT was overexpressed, implying that an interplay among MIAT, miR-150-5p and VEGF is crucial to modulate the microvascular function. 56 As a lincRNA, retinal noncoding RNA3 (RNCR3) was supposed to act as competing endogenous RNAs by suppressing target miRNA level, and to ultimately further influenced mRNA expression. Nevertheless, miR-185-5p was identified as a negative regulator of RNCR3 in the proliferation of ECs and VSMCs. The level of RNCR3 was up-regulated in aortic atherosclerotic lesions, and the   (Table 1); however, the journey of exploring the function and mechanism of the lncRNA-miRNA-mRNA axis in the regulation of cardiovascular physiology and pathology remains long.

CONFLI CT OF INTEREST
The author confirms that this article content has no conflict of interest.

ACKNOWLEDG EMENTS
The project was supported by the Natural Science Foundation of Anhui Province (No. 1608085QH196).