Non‐coding RNAs in aortic dissection: From biomarkers to therapeutic targets

Abstract Aortic dissection (AD) is the rupture of the aortic intima, causing the blood in the cavity to enter the middle of the arterial wall. Without urgent and proper treatment, the mortality rate increases to 50% within 48 hours. Most patients present with acute onset of symptoms, including sudden severe pain and complex and variable clinical manifestations, which can be easily misdiagnosed. Despite this, the molecular mechanisms underlying AD are still unknown. Recently, non‐coding RNAs have emerged as novel regulators of gene expression. Previous studies have proven that ncRNAs can regulate several cardiovascular diseases; therefore, their potential as clinical biomarkers and novel therapeutic targets for AD has aroused widespread interest. To date, several studies have reported that microRNAs are crucially involved in AD progression. Additionally, several long non‐coding RNAs and circular RNAs have been found to be differentially expressed in AD samples, suggesting their potential roles in vascular physiology and disease. In this review, we discuss the functions of ncRNAs in AD pathophysiology and highlight their potential as biomarkers and therapeutic targets for AD. Meanwhile, we present the animal models previously used for AD research, as well as the specific methods for constructing mouse or rat AD models.


| INTRODUC TI ON
Aortic dissection (AD) is a fatal vascular disease defined as the intimal tear of the aorta, which causes the blood in the cavity to pass into the middle of the artery wall, and eventually forms the dissected haematoma and the true and false lumen expanding along the long axis of the artery. 1 Accumulating evidence indicates that the annual incidence of AD is 4.586 per 100 000 persons aged 65-75 years, with more than 35 deaths per 100 000 persons. 2 If left untreated, about 24% of the patients die within the first 24 hours and 50% die within 48 hours. 3 In short, AD is an acute and severe disease with a high mortality rate. There are two classification systems for AD: Stanford, which is the most commonly used, and DeBakey. The Stanford classification is further divided into two types-A and B ( Figure 1). Stanford type A AD originates from the ascending aorta and is divided into two subtypes according to the involvement of the abdominal aorta or the limitation to the ascending aorta. It is usually treated by sternotomy, especially for emergency surgical intervention, whereas Stanford type B AD does not involve the ascending aorta and is usually treated using drugs, unless the condition is complex. 4 AD can also be categorized as acute, subacute and chronic, specifically at two time-points (2 weeks and 3 months). 3 Studies have demonstrated that several factors can influence AD. For example, various genetic mutations were reported to lead to the occurrence and progression of ADs, such as Marfan syndrome (FBN1 mutations), 5 Ehlers-Danlos syndrome (COL3A1 mutations) 6 and Loeys-Dietz syndrome (TGFBR1 or TGFBR2 mutations). 7 Moreover, infections and vascular inflammation, which involves some inflammatory factors including matrix metalloproteinases (MMPs) and vascular endothelial growth factor (VEGF), and vascular smooth muscle cell (VSMC) phenotypic transition are also significantly involved in AD. Additionally, bicuspid aortic valve, one of the most common cardiovascular malformations with an incidence of 1%-2% in the general population, is also reported to be closely related to AD development 8 mainly owing to lack of elastic fibre components and increased release of matrix metalloproteinases. 9 Recently, increased studies focus on the participation of non-coding RNAs (ncRNAs) in various diseases. [10][11][12][13] ncRNAs are mainly divided into two main categories according to their length: small ncRNA and long ncRNA. They normally cannot encode proteins, which were regarded as 'junk' transcriptional products.
However, recent studies have found that ncRNAs are functional regulatory molecules that can regulate gene expression at the transcription and post-transcription levels. Particularly, numerous studies have reported that non-coding RNAs (ncRNAs) play a significant role in the development of cardiovascular diseases, such as atherosclerosis, restenosis after stent and aneurysm. [14][15][16][17][18] Likewise, several reports have suggested that ncRNAs also play a crucial regulatory role in the occurrence and development of AD. [19][20][21] In this review, we discuss the role of ncRNAs and their target genes, as well as their regulatory mechanisms in AD. In this review, we highlight some previously identified ncRNAs that are involved in multiple processes that lead to AD. In-depth research of these ncRNAs can provide new insights into the treatment F I G U R E 1 The Stanford classification of aortic dissection. There are mainly two types: Stanford A with dissection range limited to ascending aorta or dissection range involved abdominal aorta, and Stanford B with dissection located in the descending aorta and prevention of AD and serve as potentially effective biomarkers and therapeutic targets for predicting the risk of AD and evaluating prognosis in clinical settings. Furthermore, smoking, hypertension, atherosclerosis, hyperlipidaemia and age are certain risk factors that contribute to the occurrence of AD. 22

| THE PATHOG ENE S IS OF AD
The pathogenesis of AD includes vascular inflammation, activity of MMPs and phenotype switching of VSMCs. 23 Damage to the endothelial cells (ECs) triggers vascular inflammation, which is regulated by the immune response mechanism, and it subsequently activates MMP, an enzyme used to degrade the extracellular matrix (ECM).
The imbalance between MMPs and TIMPs directly contributes to the remodelling of the aortic wall, which is a central link in the formation of AD. 24 It has also been reported that the macrophages and their related products localized within the walls of the aorta can trigger and maintain the thoracic aortic dissection (TAD) inflammation and matrix degradation. 25 Collagen synthesis and MMP-2 (gelatinase A) production are elevated in the synthetic phenotype of VSMCs and can promote collagen deposition and type IV collagen and elastin degradation. 26 These evidences suggested that AD is a very complex process involving inflammation and matrix degradation.
Media degradation is a major histopathological feature of AD, which includes the severe degradation of the ECM that is associated with the depletion of the smooth muscle cells (SMCs), rupture of elastic fibres and collagen degradation, consequently weakening the artery wall and eventually leading to the formation of AD. 27 The VSMCs in the middle layer of the aorta play an important role in maintaining aortic wall homeostasis. Notably, dysfunction in the proliferation and migration of VSMCs has been reported to be associated with vascular diseases. 28 Therefore, VSMCs are also believed to be responsible for the formation of AD. During AD pathogenesis, the dysfunctional VSMCs are considered to be one of the most important factors, mainly including apoptosis and phenotype switching. 29 VSMC phenotypic transformation is a biological process which is characterized by the transformation of a contractile (differentiated) phenotype into a synthetic (dedifferentiated) phenotype that could occur under the influence of the environment including signal transduction, gene transcription and epigenetic modification. 30 Several molecular mechanisms are involved in this process, such as serum response factor (SRF), Krüppel-like factor 4 (KLF4), forkhead box O 4 (FOXO4), microRNAs, ten-eleven-translocation 2 (TET2), Rho-actin and transforming growth factor-β (TGF-β). 31 Two VSMCderived ECM proteins, collagen and elastin, have been implicated with apoptosis and phenotype switching during AD pathogenesis.
Apoptosis of VSMCs and elastin fibre fragmentation can lead to the degeneration of the ECM, thereby weakening the strength and elasticity of the vessel wall, 9 whereas abnormal phenotypic switching can promote collagen deposition and elastin degradation. 26 These two processes directly influence the development of AD; however, their underlying molecular mechanisms remain to be elucidated.
Different stimuli, such as growth factors, chemical factors, cell adhesion molecules, ECM enzymes and damage-stimulating signals, can also result in the occurrence of AD. 32 Hypertension has also been confirmed as a risk factor that contributes to the occurrence of AD, which represents an important haemodynamic basis. 9 For example, miR-21 has been shown to be involved in the development of hypertension and target organ damage (TOD) by regulating renin-angiotensin-aldosterone system (RAAS) and peripheral blood mononuclear cells. 33 miR-505 promotes the development of hypertension by affecting the function of endothelial cells. 34 Particularly, vascular endothelial cells and haemodynamics were reported to be significant for vascular homeostasis and hypertension. 35 Shear stress is a force per unit area, which is generated when tangential force (blood flow) acts on the inner membrane of vessel (endothelial cells), and plays an important role in determining the pathological origin of arterial disease. 36 In addition, shear stress is also associated with endothelial phenotype changes, which are related to atherosclerosis. 37 Recently, a large number of literature reported that a series of microRNAs can regulate haemodynamics and endothelial cell behaviour, thus participating in the occurrence of aortic diseases. microRNA-126 was shown to be involved in the signal transmission from endothelial cells to SMC, thereby inducing SMC turnover which was affected by haemodynamic shear stress. 38 It was also reported that Krüppel-like factor 2 (KLF2) can regulate the gene expression pattern of endothelial cells under the stimulation of atheroprotective flow. KLF2 transduction or shear stress stimulation can induce endothelial cells to secrete extracellular vesicles to enrich miR-143/145 and control the target gene expression of co-cultured SMC, which is of great significance in combating atherosclerosis. 39 Moreover, haemodynamic changes and endothelial cell dysfunction are also crucially involved in the pathogenesis of AD. Recent reports demonstrated that EC dysfunction participates in the development of AD through endoplasmic reticulum (ER) stress depending on microparticles derived from SMCs, and eventually leads to EC apoptosis and inflammation. 40 The modification of protein S-nitrosylation (SNO) in endothelial cells can result in the destruction of the endothelial barrier, which ultimately leads to the formation of TAD. 41 Currently, some studies revealed the regulatory roles of microRNAs on endothelial cells behaviour during aortic dissection. For example, miR-27a promotes the activation of apoptotic pathway by targeting the expression of fas-associated protein with death domain (FADD), thereby promoting the apoptosis of EC, whereas the treatment of miR-27a activator targeting ECs apoptosis can significantly reduce the incidence of AD. 42 Together, these evidences provide us with new insights into the occurrence and development of AD, and suggest more comprehensive prevention and diagnostic methods.

| CLINI C AL E VALUATI ON OF MIRNA S IN AD PATIENTS
As mentioned above, AD is an acute and severe disease with a high mortality rate. The annual incidence of AD is 4.586 per 100 000 of AD regulated by ncRNAs including miRNAs (Table 1), lncRNAs (Table 2) and circRNAs (Table 3), and the diagnostic and therapeutic potential of some statistically different ncRNAs detected in the tissue or blood samples of AD patients and healthy people. We also summarized the information regarding the identified ncRNAs and their diagnostic values, particularly their sensitivity and specificity, and the four miRNAs (miR-25, miR-29a, miR-155 and miR-26b) showed more reliable diagnostic value with a sensitivity of 92%, a specificity of 93.33% and area under curves (AUC) of 0.973 44,45 ( Table 4). And we listed novel potential biomarkers that need to be further validated in Table 5. [46][47][48][49][50] The expression of these non-coding RNAs in the peripheral blood or aortic tissues of AD patients and healthy people was obviously different (fold change > 2), which has potential clinical significance as early diagnostic biomarkers.
However, their regulatory function and underlying mechanisms in the occurrence and development of AD need to be further explored.
We believe that these ncRNAs can be developed into reliable   60 Here, we introduce several specific miRNAs that have been reported to play definite roles in the development of AD, and could be considered as potential diagnostic biomarkers therapeutic targets of AD ( Figure 2) ( Table 6).
The overexpression of miR-134-5p in aortic smooth muscle cells was also confirmed to inhibit tunica media degeneration and TAD evolution in vivo. 19 In an AngII-induced and high-fat diet TAD model, Ad-miR-134-5p significantly inhibited aortic dilatation and vascular media degeneration, thereby reducing the incidence of AD by 39%.
These results suggest that the miR-134-5p, STAT5B and ITGB1 in VSMCs may be potential therapeutic targets for AD.

| microRNA-320 (miR-320), microRNA-320d (miR-320d) and microRNA-582 (miR-582)
The expression of miR-320 was found to be highly down-regulated in peripheral blood of AD patients and confirmed to be negatively correlated with the expression of MMP, which is produced by monocytes or macrophages. 69 MMPs, which are enzymes with similar structures but different functions, are classified into five groups: collagenases, gelatinases, stromelysins, matrilysins and membrane type. 70 Specific types of MMPs can be produced by different cells, whereas activated monocytes and macrophages can produce multiple MMPs. 71 Physiological remodelling of the ECM and vascular system requires MMP expression; however, its overexpression has adverse effects. For example, MMP-2 is related to the increased risk of AD. 26 Collectively, results suggest that miR-320 can regulate several genes were discovered to be related to the apoptotic pathway.
In particular, TRIAP1 and NET1 may be downstream targets of miR-320d, whereas COL1A1 and SPP1, which are related to extracellular matrix degradation, may be downstream targets of miR-582. 21 The apoptotic pathways in VSMCs play an essential role in the development of AD. Das et al confirmed that the enrichment of S100A12, which is a pro-inflammatory protein, can activate caspase 3 (CASP3) and promote apoptosis in human AD tissues. 72 In summary, miR-320d and miR-582 can regulate the progression of AD by participating in the apoptotic pathway and serve as potential biomarkers for the diagnosis and treatment of different diseases. However, this hypothesis needs further validation in human or mouse models.

| microRNA-144-3p (miR-144-3p)
miR-144-3p has increased expression levels in AoSMCs derived from AD patients dissection specimens. Notably, the down-regulation of miR-144-3p expression through adeno-associated viruses can reduce the incidence and severity of AD. Bioinformatic analysis and dual-luciferase reporter assay confirmed that miR-144-3p binds to the 3′-UTR of the tropoelastin (TE) mRNA to inhibit protein translation. 73 TE is a soluble monomer of elastin, a polymeric extracellular protein that determines the ductility and elastic rebound of many cells, accounting for more than 50% of the AoSMC dry weight. The polymerization of TE and other protein components forms the extracellular elastic matrix, which determines the mechanical stability and physical properties of tissues. 74 Moreover, TE polymorphism defects greatly contribute to the initiation of AD. 75 In a BAPN-induced mouse AD model, the inhibition of miR-144-3p expression resulted in the reduced incidence of AD from 90% to 50%. This indicates that miR-144-3p plays a vital role in the pathogenesis of AD and may serve as a potential therapeutic target.

| microRNA-146b (miR-146b) and microRNA-146a-5p (miR-146a-5p)
It was reported that miR-146b expression was significantly increased in peripheral blood and aortic wall tissues of the TAAD group compared with the control group (P < .001). In addition, the differential expression of miR-146b positively correlated with the high risk of AD. Many ways were used to predict miR-146b-related target genes, identifying NF-κB1, tumour necrosis factor receptor-associated factor 6 (TRAF6), MMP16 and actin alpha 2 (ACTA2). 76 It has been previously confirmed that atherosclerosis and autoimmune inflammation are associated with the excessive activation of NF-κB1. 77 Therefore, NF-κB1 may also be involved in the occurrence of AD through the immune-inflammatory response. miR-146b can also target the 3′-UTR of TRAF6 and promote vascular EC inflammation by regulating the Toll-like receptor (TLR) immune signalling pathway. Hence, these predicted gene targets may also be involved in the formation of AD. 78 MMPs, which can degrade the ECM and regulate immune-inflammatory responses, are associated with apoptosis and inflammatory signalling pathways, and therefore, MMPs play an important role in vascular remodelling. 24 Mutations in ACTA2 have been reported to regulate the TGF-β signalling pathway; thus, miR-146b can participate in the development of AD by regulating the TGF-β signalling pathway. 79 In summary, the increased expression of miR-146b may be related to the incidence and severity of TAAD. Therefore, miR-146b may be a potential biomarker that can predict the risk and severity of TAAD rupture and become a basis for selecting appropriate treatment methods.
miR-146a-5p was discovered to participate in the regulation of the immune system and myeloid tumorigenesis, 80 as well as cell proliferation and migration. 81 SMAD4-mediated regulation resulted in significantly increased miR-146a-5p expression (P < .05) and elevated VSMC proliferation and migration rates via in Stanford type A AD patients plasma samples and tissue specimens. 82 However, the small sample size and unknown association between circulating miR-146a-5p levels and AD severity limit the potential of miR-146a-5p as a biomarker for predicting prognosis.

| microRNA-30a (miR-30a)
miR-30a expression was found to be significantly up-regulated in human AD ascending aorta specimens, consequently reducing the lysyl oxidase (LOX) expression via translation inhibition in the aortic wall. 83 LOX is an extracellular, copper-dependent enzyme that cross-links collagen and elastin. LOX inactivation inhibits the crosslinking of collagen and elastin, leading to aortic aneurysm (AA) in inbred mottled mice. 84 In addition, LOX knockout or reduced LOX expression of in mice, turkeys and rats was associated with AD. 85 In AngII-induced AAD and aortic angiography animal model, the rats pre-treated with agomiR-30a have a significantly higher probability of developing AD. Collectively, these results provide new insights into the molecular mechanism of AD formation and highlight the potential of miR-30a as a potential therapeutic target for AD.

| microRNA-143/145 (miR-143/145) gene cluster
In AngII-induced AD model, the down-regulated expression of the miRNA-143/145 gene cluster and the switch of VSMCs from a contractile to a synthetic phenotype were associated with the P38-MAPK signalling pathway, and in AD ascending aorta tissues, the expression of phosphorylated p38-MAPK increased obviously. 52 Additionally, the down-regulation of the miR-143/145 gene cluster during the pathogenesis of AD can promote VSMC phenotypic switching and aortic media degeneration through the TGF-β1 signalling pathway. 12 The miR-143/145 gene cluster, including miR-143 and miR-145, is limited to the adult SMC lineages during development. 86 Notably, AngII regulates the expression of miR143/145 and promotes the transition of murine VSMCs from a synthetic to a contractile phenotype during the formation of AD. 87 It was reported that AngII has elevated levels in the serum of AD patients and the overexpression of AngII can induce aortic atherosclerosis and AA by modulating the proliferation, differentiation, apoptosis and hypertrophy of VSMCs. 88 Recently, it was confirmed that AngII can also activate MAPKs, including signalregulated kinase (ERK1/2), JNK and p38, by activating the signalling cascades specifically related to the proliferation, migration, differentiation and fibrosis of VSMC. 89 However, further studies are needed to verify whether the down-regulation of the miR-143/145 gene cluster activates p38 signals before or after the onset of AD.
PKD1 and TGF-β1 were suggested to be associated with the development of aortic diseases, and these two were observed to have decreased expression levels in AAD patients. 90,91 In summary, miR-4787-5p and miR-4306 may play important roles in the early diagnosis of AAD. However, more samples need to be studied to verify the diagnostic value of miR-4787-5p and miR-4306 as potential biomarkers in AAD.

| microRNA-26b (miR-26b)
High-mobility group AT-hook 2 (HMGA2) plays an essential role in cellular proliferation and differentiation during embryonic development and is involved in the development of cardiovascular diseases, including atherosclerosis and cardiac lipomas. 92 Furthermore, HMGA2 overexpression in AAD occurs in a let-7d-independent manner, which influences AAD through epithelial-mesenchymal transition (EMT). 93 It was confirmed that the miR-26b/HMGA2 axis contributes to TAAD development through the TGF-β signalling pathway. miR-26b regulates the expression of HMGA2, thereby indirectly regulating VSMC proliferation and apoptosis, and activates the TGF-β/SMAD3 signalling pathway to promote VSMC proliferation. 94 miR-26b was found to be negatively correlated with the risk and severity of TAAD, and miR-26b expression was decreased in ascending aorta tissue of TAAD patients. It was confirmed as a biomarker for TAAD diagnosis, which can predict the risk of AD, assess the prognosis and serve as a basis for choosing the timing of surgery. 44

| lncRNA-XIST, ENSG00000269936 and lncRNA-1421
The lncRNA-miRNA-mRNA network revealed that lncRNA-XIST directly binds to miR-17-5p to regulate p21 expression. lncRNA-XIST and p21 were up-regulated, whereas has-miR-17-5p was down- which participate in AD development through different ways. [103][104][105] Collectively, these studies provide new insights into the molecular mechanisms of AD and may reveal the important function of lncRNAs in creating future prevention strategies.

| PTENP1
The lncRNA PTENP1 is a pseudogene of the tumour suppressor gene phosphatase and tensin homologue deleted on chromosome ten (PTEN). 106  Some TAD-associated miRNAs can be regulated by several circR-NAs. For example, miRNA expression profile analysis revealed that the expression of hsa-miR-320a, hsa-miR-320b and hsa-miR-320c was inhibited by hsa-circRNA-101238 in TAD aortic specimens. 85 Therefore, the higher the expression level of circRNA-101238 in human TAD tissues, the lower the expression level of the downstream target miR-320a, leading to increased MMP9 expression.

| circ-MARK3
Tian et al also performed RNA-Seq on human acute Stanford type A aortic dissection (AAAD) diseased ascending aortic specimens and discovered that 506 circRNAs were significantly differentially expressed. 47  To understand the biological and pathophysiological mechanisms associated with AD occurrence and progression, the abnormally expressed miRNAs, lncRNAs and circRNAs must be identified and validated in related animal models or human AD tissues. Extracting the RNA from key cell types, such as ECs, SMCs and immune cells, will help obtain specific insights into the cell-type specific changes during AD development. After verifying their differential expression, the molecular mechanisms through which these ncRNAs regulate AD development must also be tested in vivo and in vitro.
In most cases, AD has very poor prognosis; however, the development of interventional therapy has markedly improved the current therapeutic efficacy. Recently, a new method for AD treatment previously used for the nucleic acid therapy of thoracic aortic dissection (TAD) through a multifunctional cationic nanoparticle system has been reported. 121 This system has no therapeutic effect on its own and is only a microRNA (miRNA) carrier that can be stored in the blood and will not damage the organs. This treatment strategy is suitable for people with TAD susceptibility, such as patients with Marfan syndrome. However, this delivery system has low specificity because its target is exposed to the pathological tissue of type IV collagen. Currently, an effective treatment for AD is still unavailable.
Therefore, in-depth studies focusing on the pathogenesis, new interventions and treatment strategies are required.
The rapid growth of miRNA research provides new insights for future studies on the identified potential biomarkers and therapeutic targets. However, some research gaps still need to be addressed. In conclusion, in-depth research of ncRNAs will provide potential therapeutic targets and biomarkers to monitor the progress and severity of AD in patients. Early and correct diagnosis of AD can significantly reduce mortality in human patients. Therefore, further efforts to identify other candidate biomarkers and to elucidate their underlying mechanisms, as well as their diagnostic and therapeutic potentials, are required.

ACK N OWLED G EM ENT
This work was supported by the National Natural Science Foundation of China (grant no. 81870331, 31701208), the Natural Science Foundation of Shandong Province (grant no. ZR2017MC067) and the Qingdao municipal science and technology bureau project (grant no. 18-2-2-65-jch,19-6-1-7-nsh).

CO N FLI C T O F I NTE R E S T S
None.