Role of LncRNAs in the Pathogenesis of Coronary Artery Disease

Coronary artery disease (CAD), caused by coronary artery occlusion, is a common cardiovascular disease worldwide. Long non-coding RNAs (lncRNAs) are implicated in the regulation of endothelial cell injury, angiogenesis, plaque formation, and other pathological mechanisms in CAD by acting on different cell types. Some lncRNAs are significantly upregulated in CAD patients; however, other lncRNAs are significantly downregulated. Differential expression of lncRNAs in CAD patients enables them to be exploited as potential biomarkers to evaluate disease progression and diagnosis/prognosis in CAD patients. In this study, we reviewed the role of lncRNAs in the development of different clinical subtypes of CAD.


Introduction
Coronary artery disease (CAD) is one of the most deadly diseases worldwide [1].Clinically, CAD is divided into stable angina pectoris, unstable angina pectoris, and acute myocardial infarction (AMI).AMI is associated with inflammation, fibrosis, and angiogenesis, and can lead to heart failure in severe cases [2].The typical mechanism of AMI is the formation and progression of intracoronary thrombus, which in turn produces varying degrees of thrombotic stenosis/occlusion, ultimately leading to myocardial necrosis and the formation of circumferential myocardial scarring in the area of coronary artery supplied [3,4].Molecular mechanisms such as mitochondrial dysfunction, inflammation, oxidative stress, and excessive fibrosis can lead to adverse cardiac remodeling in the late stage of an AMI, which results in increased morbidity and mortality [5].Studies have found that several long noncoding RNAs (lncRNAs) are abnormally expressed in biological samples extracted from CAD patients.LncRNAs can regulate pathological mechanisms and disease progression through different target genes or signaling pathways, making them essential biomarkers [6].LncRNAs, a noncoding RNA larger than 200 nucleotides, play a biological role in CAD by acting on downstream target molecules such as microRNAs (miRNAs, miRs), mRNAs, or transcription factors [7,8].Most lncRNAs act as competing endogenous RNAs (ceRNAs) and regulate the expression and activation of downstream mRNAs by competitive binding with miR-NAs, thus affecting the cardiovascular system's biological functions, such as cell proliferation, migration, and apop-tosis.Therefore, lncRNAs may have an important regulatory role in the pathophysiology of CAD [9].The lncRNA-miRNA-mRNA pathway is a classic ceRNA mechanism.Learning more about lncRNAs and their role in CAD will help to develop new treatment and diagnostic methods for CAD patients.In this review, we aim to explore the biological role of lncRNAs in various subtypes of CAD.

LncRNAs in CAD
The mechanism and function of lncRNAs in CAD have been widely studied in various cell types, most extensively in endothelial cells.Wang et al. [10] demonstrated that lncRNA p21 acts on miR-221 through the ceRNA mechanism, forming the miR-221/SIRT1/Pcsk9 axis.LncRNA p21 overexpression can inhibit endothelial cell apoptosis and promote endothelial cell proliferation, migration, and tube formation, thus reducing subcutaneous lipid deposition to prevent the progression of atherosclerosis (AS).LncRNA has been downregulated in AS patients and AS mouse models.Similarly, compared with healthy subjects, the expression level of lncRNA TONSL-AS1 in the plasma of CAD patients is also downregulated.Forced overexpression of this lncRNA in primary human coronary artery endothelial cells has been shown to promote proliferation and inhibit apoptosis by upregulating B-cell lymphoma-2 (BCL-2) expression levels through the negative regulation of miR-197 in these cells [11].Furthermore, Kai et al. [12] have reported that lncRNA NORAD (NO-RAD, non-coding RNA activated by DNA damage) expression levels were significantly upregulated in CAD patients and oxidized low-density lipoprotein (ox-LDL)-treated hu-man umbilical vein endothelial cells (HUVECs).LncRNA NORAD is closely related to the occurrence and development of AS.NORAD recruits HDAC6 by enriching for FUS (FUS RNA binding protein), then HDAC6 binds to the promoter region of the VEGF gene, enhancing the level of H3K9ac deacetylation in this region and thereby inhibiting VEGF gene transcription.Li et al. [13] have shown upregulation of lncRNA uc003pxg.1 and downregulation of miR-25-5p in peripheral blood mononuclear cells from CAD patients.LncRNA uc003pxg.1 has been shown to promote the proliferation and migration of HUVECs by upregulating cyclinD1 and CDK6 via negatively downregulating miR-25-5p in an in vivo study.Interestingly, two different transcripts of lncRNA ANRIL exert diametrically opposing effects on CAD endothelial cells, which highlights the molecular characteristics of lncRNAs and their diverse biological roles [14].It is unknown whether other CAD-related lncR-NAs have multiple transcripts and different functions.Table 1 (Ref.[10][11][12][13][15][16][17][18][19][20][21][22]) shows the functional roles of lncRNAs, as assessed in endothelial cells, in the occurrence and development of CAD.
Kang et al. [23] have reported overexpression of lncRNA AL355711 in atherosclerotic plaques and animal models of AS, and matrix metalloproteinase-3 (MMP3) has been associated with vascular smooth muscle cell (VSMC) migration in cardiovascular disease.Furthermore, knockdown of lncRNA AL355711 has also been demonstrated to inhibit AS progression by regulating VSMC migration through the ABCG1/MMP3 pathway.Upregulation of lncRNA CDKN2B-AS1 has been proven to upregulate PTPN7 through competitive binding with miR-126-5p, attenuating VSMC proliferation and promoting apoptosis.However, this lncRNA showed decreased expression in ox-LDL-induced VSMC models and serum samples from CAD patients.Transfection of pcDNA-CDKN2B-AS1 into ox-LDL-induced VSMCs has been demonstrated to result in the upregulation of lncRNA CDKN2B-AS1, which may play an important biological role; however, these findings have not been studied in vivo [24].In addition, lncRNA Kcnq1ot1 has been found to stimulate the expression of Tead1 by competitively acting on miR-466k and miR-466i-5p, thus promoting injury and apoptosis of cardiomyocytes.However, this specific mechanism still needs to be clarified in further studies [25].Knockdown of lncRNA Mirt2 has been shown to exacerbate hypoxia/reoxygenation-induced H9C2 myocardial cell damage and promote the progression of ischemic myocardial infarction in AMI rat models, while there was no significant effect on the normal or sham group.Upon further study of this mechanism, it was found that overexpression of Mirt2 could upregulate PDK1 levels through the negative regulation of miR-764 and inhibit myocardial apoptosis and injury.Thus, a signaling axis of lncRNA Mirt2/miR-764/PDK1 protecting cardiomyocytes was formed [26].Table 2 (Ref.[15,[24][25][26][27][28][29][30][31][32][33][34]) lists those CAD-related lncRNAs whose functions have been evaluated in cardiomyocytes or VSMCs.Some studies have shown that vascular aging is a specific risk factor for CAD.VSMCs play an important role in the pathological processes of vascular remodeling and stiffness associated with vascular aging [35,36].Therefore, there may be some potential correlations between the effects of lncRNAs on VSMC functions and vascular aging that have yet to be explored.
Compared with the control group, lncRNA MHRT levels in the border region of myocardial infarction in AMI mice were significantly increased, and miR-3185 was proven to be a direct target gene of MHRT regulating fibrosis.Overexpressed lncRNA MHRT promotes myocardial fibrosis after myocardial infarction by negatively regulating miR-3185 and increasing TGF-β1-induced proliferation of myocardial fibroblasts and intracellular deposition of collagen fibers Ⅰ and Ⅲ.Therefore, MHRT may be a therapeutic target for myocardial fibrosis [37].Zou et al. [38] found that the migration and proliferation of cardiac fibroblasts (CFs) in lncRNA ZFAS1 knockdown mice were significantly inhibited after hypoxia treatment, and cardiac function was also improved.The Wnt/β-catenin signaling pathway is closely related to the pathological mechanism of myocardial infarction.This study further confirmed that inhibition of the Wnt/β-catenin signaling pathway can reverse the effects of shZFAS1 on cardiac fibroblasts and cardiac function, revealing a potential regulatory network.In AMI mouse heart tissue and Ang Ⅱ-induced CFs, lncRNA SNHG7 gene knockout can significantly reduce Ang Ⅱinduced apoptosis, collagen synthesis, and inflammatory responses of CFs.Therefore, lncRNA SNHG7 depletion exerts its functions through binding miR-455-3p, thus playing a protective role via regulating the platelet-activating factor receptor [39].Luo et al. [40] found that lncRNA 554 at least partially regulates collagen synthesis and myocardial fibrosis after myocardial infarction by activating the TGF-β1 signaling pathway.Although its downstream target molecules still need to be further studied, it is clear that the knockdown of the lncRNA 554 gene will become a target for inhibiting cardiac fibrosis.Table 3 (Ref.[37][38][39][40][41][42]) summarizes the studies between lncRNA and cardiac fibrosis in CAD.These results show the role of lncRNAs in CFs.The increased proliferation of CFs and deposition of extracellular matrix proteins have been described as cardiac fibrosis, which severely affects the prognosis of CAD [43].One study suggests that the classical TGF-β and WNT signaling pathways display information crosstalk that appears to regulate the fibrosis process in CAD [44].The signaling pathways listed in Table 3 include only TGF-β and WNT signaling pathways involved in regulating myocardial fibrosis.Whether other signaling pathways are potentially involved through these pathways is unknown.The specific mechanisms by which lncRNA's impact cardiac fibrosis by regulating these two signaling pathways needs to be further determined.

Diagnostic/Prognostic Significance of LncRNAs in CAD
Changes in circulating lncRNA expression levels in patients with CAD make them potential biomarkers for diagnosis and prognosis.Liu et al. [45] found that compared with a normal coronary artery group, there were 98 differentially expressed lncRNAs in peripheral blood mononuclear cells of unstable angina patients.A ROC curve was used to reflect the relationship between the sensitivity and specificity of these lncRNAs.The AUC, which was between 0.1 and 1, can be used to directly evaluate the diagnostic value of the lncRNA; the larger the value, the greater the diagnostic potential.
Among 98 differentially expressed lncRNAs, the AUC values of MALAT1 and LNC_000226 were 0.81 and 0.799, respectively.Both have very high diagnostic values, distinguishing unstable angina patients from the normal group.However, the specificity of LNC_000226 was not very high [45].In addition, compared with healthy subjects, lncRNA MIAT was significantly overexpressed in the peripheral blood of CAD patients, which is likely to be an important biomarker for the diagnosis of CAD.LncRNA MIAT had an AUC of 0.908 and sensitivity and specificity of 0.700 and 0.714, respectively, and is one of the independent risk factors for CAD patients [46].Studies have shown that the inflammatory mediators tumor necrosis factoralpha (TNF-α), monocyte chemotactic protein-1 (MCP-1), vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and interleukin-6 (IL-6) positively correlate with risk stratification in patients with coronary heart disease, but these inflammatory mediators were negatively correlated with plasma lncRNA FA2H-2 levels.LncRNA FA2H-2, as an independent risk factor for coronary heart disease, was expressed with an area under the ROC curve of 0.834 and sensitivity and specificity of 0.85 and 0.82, respectively.Therefore, lncRNA FA2H-2 is likely to distinguish the control group from the CHD group [47].The increased number of coronary lesion vessels was an independent risk factor for poor prognosis in patients with CAD.ROC curve analysis indicated that the AUC of lncRNA FGF9-associated factor (FAF) had a prognostic value in CAD of 0.916 in addition to a high diagnostic value of 0.935.Additionally, compared with controls with no major adverse cardiac events, the expression of FAF in patients with major adverse cardiac events group was lower [48].The diagnostic/prognostic effects of lncR-NAs in CAD are summarized in Table 4 (Ref.[22,[45][46][47][48][49][50][51][52][53]).All the lncRNAs listed in the table have high diagnostic val-ues; however, the specificity and sensitivity of some lncR-NAs have not been determined.The biomarkers of CAD lncRNAs and their mechanisms of action are summarized in Fig. 1.

Conclusions and Perspectives
Our review has found that most lncRNAs act as ceR-NAs, competing with downstream target miRNAs to regulate the pathophysiological process of CAD through different mechanisms, such as regulation of vascular endothelial cells, regulation of vascular smooth muscle cell activity, regulation of myocardial cell proliferation and apoptosis, collagen fiber production, and myocardial fibroblast function.The JAK1/STAT3 signaling pathway, AKT signaling pathway, and other signaling pathways are all lncRNA signaling pathways involved in CAD.lncRNAs affect different aspects and different targets in this process.For example, lncRNA MIAT regulates the expression of miR-181a-5p and miR-10a-5p; both are involved in cardiomyocyte apoptosis.The knockdown of both lncRNA MALAT1 and lncRNA 554 can inhibit myocardial fibrosis through the TGF-β1 signaling pathway.This suggests that there may be a rich regulatory network linking lncRNAs with CAD.Most importantly, differences in circulating lncRNA expression levels can also be used to distinguish healthy individuals from patients with CAD, as well as ST-elevation myocardial infarction and UA patients, serving as markers for diagnosis and prediction of disease progression.The stability of lncRNAs and their easy extraction from serum and body fluids make them easier to detect.Increased myocardial fibrosis (MF) following a myocardial infarction is a major cause of heart failure (HF).Studies have shown that several lncRNAs are differentially expressed in the transition from MI to MF to HF.This may have the potential for predicting disease progression and prognosis in patients with an MI, but the detailed molecular and pathological mechanisms involved in the progression from MI-MF-HF have not been thoroughly validated.
The mortality rate in CAD remains high, largely because of the lack of effective drugs to prevent or decrease myocardial ischemic necrosis in clinical practice.LncR-NAs may become the prototype to create effective drugs.In-depth exploration of the complex splicing process, differential transcriptional processing, differential intracellular expression, and subcellular localization of lncRNAs will contribute to the understanding of the pathogenesis and regulatory mechanism of CAD and lead to improvements in the clinical diagnosis and treatment of CAD.In summary, lncR-NAs are involved in regulating many aspects of the pathogenesis of CAD and can be used as specific/sensitive markers for this disease.The diagnostic/prognostic/therapeutic role of lncRNAs in CAD will need to be explored in future studies.original data; JY and YP were responsible for the first review of the manuscript; XB and LW provided the overall design of the manuscript and the final review of the manuscript.All authors participated in the editorial revision of the manuscript.All authors read and approved the final manuscript.

Fig. 1 .
Fig. 1.LncRNAs as biomarkers of CAD participate in pathological processes and then affect the occurrence and development of CAD.Abbreviations: ECs, endothelial cells; VSMCs, vascular smooth muscle cells; CM, cardiomyocyte; CF, cardiac fibrosis.

Table 2 . CAD-related lncRNAs whose function has been assessed in myocardial cells or vascular smooth muscle cells.
Abbreviations: HVSMCs, human vascular smooth muscle cells; VSMCs, vascular smooth muscle cells; HA-VSMC, human aortic vascular smooth muscle cells; HCM, human cardiomyocytes.