Crosstalk between ubiquitin ligases and ncRNAs drives cardiovascular disease progression

Cardiovascular diseases (CVDs) are multifactorial chronic diseases and have the highest rates of morbidity and mortality worldwide. The ubiquitin–proteasome system (UPS) plays a crucial role in posttranslational modification and quality control of proteins, maintaining intracellular homeostasis via degradation of misfolded, short-lived, or nonfunctional regulatory proteins. Noncoding RNAs (ncRNAs, such as microRNAs, long noncoding RNAs, circular RNAs and small interfering RNAs) serve as epigenetic factors and directly or indirectly participate in various physiological and pathological processes. NcRNAs that regulate ubiquitination or are regulated by the UPS are involved in the execution of target protein stability. The cross-linked relationship between the UPS, ncRNAs and CVDs has drawn researchers’ attention. Herein, we provide an update on recent developments and perspectives on how the crosstalk of the UPS and ncRNAs affects the pathological mechanisms of CVDs, particularly myocardial ischemia/reperfusion injury, myocardial infarction, cardiomyopathy, heart failure, atherosclerosis, hypertension, and ischemic stroke. In addition, we further envision that RNA interference or ncRNA mimics or inhibitors targeting the UPS can potentially be used as therapeutic tools and strategies.


Introduction
Cardiovascular diseases (CVDs) are a wide spectrum of disorders affecting hearts or vessels, including myocardial ischemia/reperfusion (I/R) injury, myocardial infarction (MI), cardiomyopathy, myocarditis, heart failure (HF), atherosclerosis, hypertension (HT), and ischemic stroke (1)(2)(3)(4).Myocardial ischemia and MI can induce excessive ventricular remodeling and the development of cardiomyocyte hypertrophy, leading to HF (2,3,5).Myocarditis is an inflammatory disease that damages the tissue of the heart muscle, and the main reasons are autoimmune response, viral infections, or immune reactions after infection (5).In addition, vascular diseases are progressive inflammatory pathologies, that can result in endothelial injury, atherosclerotic plaque formation, and HT, especially pulmonary arterial hypertension (PAH) (6).
The ubiquitin-proteasome system (UPS) is the main nonlysosomal pathway for targeted protein degradation in eukaryotic cells (7)(8)(9).The UPS consists of three components: a ubiquitination mechanism (ubiquitin, E1, E2 and E3 enzymes), deubiquitinating enzymes and the 26S proteasome (10).First, E1 activates the C-terminal glycine residues of ubiquitin by using ATP, and then activated ubiquitin is transferred to E2 by a high-energy thioester bond linking the C-terminal carboxyl group of ubiquitin to the active cysteine in E2.Third, E3 catalyzes the covalent attachment of ubiquitin to the lysine side chain of the substrate protein (11,12).Subsequently, substrate proteins modified by polyubiquitin chains are degraded via the 26S proteasome.The 26S proteasome consists of two 19S regulatory complexes and one 20S complex (13,14).E3 ubiquitin ligases regulate various aspects of eukaryotic biology by promoting ubiquitination degradation of target proteins (15).For example, smad ubiquitin regulatory factors (Smurfs) come from the HECT family of E3 ubiquitin ligases and consist of two members, Smurf1 and Smurf2 (16).Additionally, ubiquitination is invertible, and deubiquitylation can remove ubiquitin from specific substrate proteins by deubiquitinating enzymes, which maintains ubiquitin system homeostasis (10,17,18).The ubiquitin-specific protease (USP) family, which includes USP3, USP7, USP14, and USP28, is one of the major deubiquitinating enzymes (19).Encouragingly, both ubiquitination and deubiquitinating enzymes, such as Smurf1, Smurf2, carboxyl terminus Hsp70-interacting protein (CHIP), Nedd4, WWP2, and YOD1, are involved in the regulation of cardiovascular function and may allow for new targeted therapeutic opportunities (7).Notably, the UPS plays an essential role in many aspects of biological processes, such as cell cycle control, cell differentiation and survival, protein turnover, protein quality control and signal transduction, some of which involve ncRNAs (7)(8)(9).
Noncoding RNAs (ncRNAs), such as microRNAs (miRNAs), long noncoding RNAs (lncRNAs), circular RNAs (circRNAs) and small interfering RNAs (siRNAs) are an increasing spectrum of transcriptional regulatory factors with no coding potential but significant structural and regulatory functions (20)(21)(22).MiRNAs are single-stranded RNAs consisting of 20-22 nucleotides, and their primary function is to combine complementary sequences on mRNAs to repress their translation (5).LncRNAs are a class of ncRNAs that are more than 200 nucleotides in length and can regulate the expression of specific miRNAs and consequently modulate miRNA downstream target genes (23,24).Long intergenic noncoding RNAs (lincRNAs) function as essential regulatory factors of gene expression (25).CircRNAs are stable single-stranded ncRNAs with closed-loop structures with miRNA sponges that interact with RNA-binding proteins as the main molecular mechanism (26,27).Moreover, siRNAs mediated by RNA interference (RNAi) are significantly versatile tools to target corresponding mRNA and silence protein-coding genes and have been used clinically for tissue targeting based on complex chemical modifications and carrier/ligand coupling (28).One of the roles of ncRNAs is the precise control of protein stability via UPS.
There are emerging reviews about the interaction of ncRNAs and UPS on cancer, especially non-small cell lung cancer, hepatocellular carcinoma, colorectal cancer, and gastric cancer (29)(30)(31), while implications of protein ubiquitination modulated by ncRNAs in cardiovascular aspects have not been systematically elaborated and comprehensively summarized although multiple studies have demonstrated that ncRNAs have a profound impact on the genetic and proteinic programming of CVDs through their interaction with the UPS (32).Fine-regulation between ncRNAs and the UPS is critical in the epigenetics of CVDs.Overall, this review paper emphasizes the mechanisms of ubiquitination in the context of the widespread association of ncRNAs with CVDs and attempts to elucidate the perturbations of the crosstalk between the UPS, especially ubiquitin ligases, and ncRNAs in CVDs.Overall, we provide the latest advances and look forward to future cardiovascular treatment strategies targeting ncRNAs and ubiquitin ligases.

Crosstalk between ncRNAs and the UPS
In recent years, multiple crosstalk between ncRNAs and the UPS has been demonstrated to play essential regulatory roles in various diseases, particularly CVDs (Figure 1) (33).First, the positive or negative regulation of miRNA on ubiquitination has gradually attracted considerable research attention.(1) MiRNAs directly inhibit E3 ubiquitin ligase expression and subsequently suppress ubiquitination and degradation of target proteins.(2) MiRNAs directly inhibit ubiquitin-conjugating enzyme (E2) expression.(3) MiRNAs directly inhibit the expression of deubiquitinating enzymes and disrupt the ubiquitination balance of deubiquitinating enzymes and target proteins.(4) MiRNAs regulate multiple UPS components.(5) MiRNAs target some proteins that are involved in the regulation of E3 ubiquitin ligases.
Furthermore, the study of upstream regulatory pathways of miRNAs, such as lncRNAs and circRNAs, is also a meaningful research direction (29,31).(1) LncRNAs/circRNAs promote the ubiquitination and degradation of target proteins.a) LncRNAs/ circRNAs interact with target proteins and promote their ubiquitination and degradation.b) LncRNAs/circRNAs enhance the interaction between the E3 ubiquitin ligase and its target.c) LncRNAs combine with RNA binding proteins or chaperone proteins that are involved in the interaction between the E3 ubiquitin ligase and target protein (20).( 2) LncRNAs inhibit the ubiquitination and degradation of target proteins.(3) LncRNAs/ circRNAs act as competitive endogenous RNAs (ceRNAs) for miRNAs and thus regulate the expression of E3 ubiquitin ligases/ deubiquitinating enzymes.
In addition to being regulated by ncRNAs, the UPS can also regulate ncRNA levels.On the one hand, E3 ubiquitin ligases directly regulate ncRNAs (Figure 2), thereby promoting ubiquitination and degradation of target proteins.On the other hand, the UPS indirectly regulates ncRNAs through the involvement of transcription factors/proteins.Specifically, knockdown of the E3 ubiquitin ligase by siRNA has been indicated to be involved in the pathology of CVDs via regulation of downstream protein levels and could serve as a potential therapeutic strategy.
3 Crosstalk between ubiquitin ligases and ncRNAs drives cardiac disease progression

Apoptosis and myocardial infarction
Ischemic conditions during myocardial I/R cause anoxic damage to cardiomyocytes, reducing mitochondrial ATP synthesis via oxidative phosphorylation, altering cell membrane permeability, and finally inducing apoptosis; therefore, timely restoration of blood flow is abstractly crucial for alleviating acute MI (34).Untimely reperfusion can produce further damage and exacerbate cardiac dysfunction, which is referred to as myocardial I/ R injury, anoxia/reoxygenation (A/R) injury or hypoxia/ reoxygenation (H/R) injury (34,35).Some miRNAs, particularly miRNA-424(322) and miR-378a-3p, were shown to be significantly decreased in myocardial I/R mice or rats, while overexpression of these miRNAs improved myocardial I/R-induced apoptosis and injury by inhibiting ubiquitin ligase (FBXW7, Smurf2 and TRIM55) expression and downstream protein degradation (Figure 3).In detail, Chen, Dong et al. (36) found that enhanced expression of miR322 prevented reperfusion-induced cardiomyocyte apoptosis by reducing the expression of the ubiquitin ligase FBXW7 and increasing N1-ICD protein in the hearts of mice in vivo.Similarly, miR-322/503 directly binds to and inhibits the translation of Smurf2 (an E3 ubiquitin ligase), thereby suppressing Smurf2 ubiquitination-mediated EZH2 degradation and activating the Akt/GSK3b pathway, ultimately reducing myocardial apoptosis and infarct size in the I/R rat model in vivo and in vitro (37).In addition, miR-378a-3p suppresses ubiquitinated degradation of DUSP1 by decreasing expression of the E3 ubiquitin ligase TRIM55 and then inactivates JNK1/2, eventually preventing myocardial I/R-related apoptosis and injury in rat myocardial I/R models in vivo (38).Conversely, miR-379 could facilitate cardiomyocyte apoptosis by inhibiting Smurf1 expression; meaningfully, exogenous Smurf1 protein could rescue miR379-induced apoptosis in cardiomyocytes (39).Additionally, circUSP39 promotes cardiomyocyte apoptosis and accelerates H/R-induced injury by sponging miR-362-3p and upregulating TRAF3 expression (40).
Interestingly, lncRNAs are also emerging as an essential class of regulators in the pathology of myocardial I/R.Collectively, lncRNA-CIRPIL, lncRNA Fendrr and LINC01588 were shown to be downregulated in myocardial I/R mice or rats and to function as negative mediators of myocardial I/R-induced cardiomyocyte apoptosis by enhancing E3 ubiquitin ligases (COP1, CHIP, MDM2 and WWP2) in vivo.First, lncRNA-CIRPIL interacts with p53 and sequesters it in the cytoplasm, directly accelerating the ubiquitin-regulated degradation of p53 by upregulating the expression of E3 ubiquitin ligases COP1, CHIP and MDM2, thereby attenuating myocardial A/R-induced apoptosis and injury (41).Similar to lncRNA-CIRPIL, lncRNA Fendrr suppresses myocardial H/R-induced apoptosis by facilitating the ubiquitination degradation of p53 via COP1 (42).Moreover, LNC01588 can make direct contact with the HNRNPL protein and upregulate the E3 ubiquitin ligase WWP2, which then reduces the levels of downstream SEPT4, thereby ameliorating myocardial oxidative stress and MI during myocardial I/R damage (43).On the other hand, inhibition of p53 ubiquitination degradation and inhibition of WWP2 attenuate the protective effects of lncCIRPIL and LINC01588 (41,43).Conversely, the ZFP36L2-lncRNA PVT1-miR-21-5p-MARCH5 axis positively affects the fission and fusion of mitochondria and cardiomyocyte apoptosis following myocardial I/ R. Specifically, lncRNA PVT1 can reduce miR-21-5p expression by directly binding to and being positively regulated by zinc finger protein 36-like 2 (ZFP36L2), which then elevates the E3 ubiquitin ligase MARCH5, exerting a deteriorating effect on myocardial mitochondrial fission and myocardial I/R injury in vivo and in vitro (44).
In addition to myocardial I/R-induced apoptosis, siRNA and E3 ubiquitin ligase were also associated with ischemia and cardiotoxic-induced apoptosis.Furthermore, Qiu et al. (45) revealed that knockdown of the E3 ubiquitin ligase RNF4 by siRNA could exacerbate cardiac oxidative stress-induced apoptosis and ischemia-induced injury by upregulating the PML nuclear body and promoting p53 activity in mouse models in vivo.Moreover, the knockdown of the E3 ubiquitin ligase CHIP by siRNA promoted doxorubicin (DOX)-induced cardiac oxidative stress and injury in the mouse heart in vivo (46).Further investigations have illustrated that CHIP can ameliorate cardiac toxicity by facilitating ubiquitinmediated degradation of SHP-1 and p53 and activating the STAT3 and ERK1/2 pathways.
In addition to ischemia-or I/R-induced cardiomyocyte apoptosis, miRNAs and circRNAs are also involved in the progression of MI through interactions with the UPS.It was demonstrated that miRNA-1 was overexpressed in male mice in vivo and in vitro during MI (47).Mechanistically, miRNA-1 extensively induces the expression of UPS components, such as 19S, 20S and ubiquitin ligase E3, and accumulates ubiquitinpositive proteins, ultimately exacerbating cardiac apoptosis and remodeling in cardiomyocytes.In contrast, suppression of miR-32-3p enhances coronary atherosclerotic plaque instability, promotes endoplasmic reticulum stress-regulated cardiomyocyte apoptosis, and aggravates acute MI by increasing ubiquitin ligase RNF13 expression in rats in vivo (48).Moreover, circNfix was validated as a proapoptotic factor for MI and inhibited cardiomyocyte proliferation and angiogenesis in rats and mice in vivo (49).Regarding the regulatory mechanism, circNfix enhances the ubiquitinated degradation of Ybx1 via the E3 ubiquitin ligase Nedd4l, and circNfix also sponges miR-214 and facilitates the expression of Gsk3b, a downstream effector associated with cardiac angiogenesis.Frontiers in Immunology frontiersin.org

Cardiomyopathy and myocarditis
Cardiomyopathy can be classified as primary (genetic) and secondary, resulting in different phenotypes, including dilated, hypertrophic, diabetes and age-related cardiomyopathy (50,51).Dilated cardiomyopathy is typically characterized by left ventricle dilation, which reduces systolic function and appears as an HF symptom (32,52).Diabetic cardiomyopathy represents a metabolic disease associated with independent risk factors such as hyperinsulinemia, hyperglycemia and insulin resistance (53).Moreover, hypertrophic cardiomyopathy is a heterogeneous genetic disease resulting in ventricular hypertrophy, hypercontractility and fibrosis (54), while cardiac hypertrophy is an acquired compensatory reaction to both physiological and pathological overload (55).
It was verified that the miR-199/214 cluster was downregulated during dilated cardiomyopathy and that this downregulation exacerbated the loss of heart mass via the activation of the UPS, including ubiquitin E2-ligases Ube2i and Ube2g1 and E3-ligases MuRF-1 and MAFbx, in vitro (56).Notably, miR-199/214 cluster expression was positively regulated by the transcription factor TWIST1, which might be used as a preregulator for miR-199/214/ UPS-involved dilated cardiomyopathy.In addition, miR-195-5p was speculated to inhibit SGK1 and hERG protein expression by enhancing the activity of the E3 ubiquitin ligase Nedd4-2 during high-glucose induced cardiomyopathy in rat cardiomyocytes in vitro (57).
Moreover, miR-485-5p and lncRNAs (NONRATT054243 and NONRATT057160) have been shown to relieve myocardial apoptosis and cardiac hypertrophy through different types of ubiquitination (58, 59).Specifically, miR-485-5p can suppress mitochondrial fission and phenylephrine (PE)-induced cardiac hypertrophy by decreasing the expression of the SUMO E3 ligase MAPL and thus elevating the level of Mfn2 in an in vivo mouse model (58).In addition, the E3 ubiquitin ligase Nrdp1, which positively regulates the two core lncRNAs, NONRATT054243 and NONRATT057160, was also revealed to mitigate Ang IIinduced cardiac hypertrophy by reducing downstream PIK3R3 expression and AKT phosphorylation (59).Conversely, UBE3A knockdown by siRNAs exacerbated isoproterenol-induced cardiac hypertrophy by activating the TLR4/MMP-9 pathway (60).
In addition to dilated cardiomyopathy, high glucose-induced cardiomyopathy and cardiac hypertrophy, ncRNA (especially miRNA) and the UPS are associated with septic and viral myocarditis.Remarkably, Gong, Li et al. (61) found that transcription factor sex-determining region Y (SRY)-Box 9 (SOX9) negatively regulated miR-96-5p and was significantly increased in sepsis-induced myocarditis in mice in vivo.USP7 deubiquitination modification upregulated SOX9, inhibited miR-96-5p expression and facilitated NLRP3 expression, myocardial pyroptosis and injury exacerbation.Furthermore, overexpression of miR-30a facilitates coxsackievirus B3 (CVB3) replication and induces viral myocarditis by suppressing the expression of the E3 ubiquitin ligase TRIM25, inhibiting RIG-I ubiquitination and thus reducing IFN-b activation (62).Additionally, in CVB3-infected cardiomyocytes in vivo and in vitro, upregulated miR-21 impairs cell-cell junctions via disruption of desmosome and fascial adhesion by reducing the deubiquitinating enzyme YOD1 and enhancing K48-linked polyubiquitination-mediated desmin degradation and subsequently impairing cell-cell junctions (63).

Heart failure
HF is a clinical syndrome that results from long-term cardiac dysfunction, cardiomyocyte hypertrophy and structural remodeling caused by hypoxia, ischemia, metabolic disorders, etc (2,32).
The following four ncRNAs, miR-379 and miR-199a, miR-146a and lncDACH1 were shown to be upregulated during HF and to aggravate HF via inhibition of the E3 ubiquitin protein Smurf1, ubiquitin-binding enzymes Ube2i and Ube2g1, and SUMO1 and upregulation of Smurf1 in vivo and in vitro (39, 64-66).Chen's groups suggested that miR-379 could facilitate cardiomyocyte apoptosis and possibly cause HF due to deficiency of the antiaging gene Klotho by inhibiting Smurf1 expression.Interestingly, exogenous Smurf1 protein could rescue miR379induced apoptosis in cardiomyocytes (39).Likewise, miR-199a can be negatively regulated by signal transducer and activator of transcription 3 (STAT3), and its overexpression inhibits the expression of Ube2i and Ube2g1, thereby leading to deterioration of cardiomyocyte ultrastructure and HF (64).Specifically, miR-146a is increased in extracellular vesicles derived from failing hearts.The key regulatory pathway involves extracellular vesicle-associated miR-146a transfer into cardiomyocytes from fibroblasts and suppression of SUMO1 expression, thereby impairing cardiac contractile function and aggravating HF (65).Moreover, lncDACH1 was demonstrated to decrease cell shortening and calcium transients, impair myocardial function and consequently lead to HF by directly promoting Smurf1-induced ubiquitination degradation of sarcoplasmic reticulum calcium ATPase 2a (SERCA2a) in mouse hearts (66).
In contrast, Wang et al. (67) observed that miR-454 was significantly decreased in HF rat models in vivo and oxidative stress-damaged cardiomyocyte H9c2 cells in vitro.Specifically, miR-454 can suppress cardiomyocyte apoptosis by inhibiting TrkA ubiquitination and degradation and activating the cAMP signaling pathway by negatively targeting the ubiquitin E3 ligase NEDD4-2.Similarly, downregulation of miR-129-5p impairs myocardial structure and function during chronic HF by promoting Smurf1 expression and then accelerating ubiquitination degradation of PTEN in chronic HF rat models (68).

Other cardiac diseases
In addition to MI, cardiomyopathy and HF, several research groups have demonstrated a combined role for ncRNAs and ubiquitination in cardiac diseases such as heart aging, myocardial fibrosis, and calcified aortic valve (AV) disease.
In mouse models in vivo, lncRNA LOC105378097 suppressed cardiomyocyte mitophagy and induced heart aging by promoting Parkin ubiquitination and reducing Parkin protein stability (69) (Figure 4).CircHIPK3 attenuates heart aging by promoting the binding of E3 ubiquitin ligase b-TrCP to HuR, enhancing ubiquitination and degradation of HuR and ultimately decreasing the activity of p21 in mice in vivo and in vitro (70).Additionally, it was verified that circ-sh3rf3 inhibited fibroblast-to-myofibroblast differentiation and consequently alleviated myocardial fibrosis by directly enhancing GATA-4 ubiquitination degradation via the E3 ubiquitin-protein ligase sh3rf3 and then abolishing GATA-4 inhibition of miR-29a expression (71).Furthermore, loss of miR-483 contributes to endothelial inflammation and consequently calcific AV disease and exerts deteriorating effects by increasing the expression of Ube2c, enhancing ubiquitination-induced pVHL degradation and activating the HIF-1a pathway (72).

Crosstalk between ubiquitin ligases and ncRNAs drives vascular disease progression 4.1 Atherosclerosis
Atherosclerosis is a chronic inflammatory disease of the blood vessel wall characterized by maladaptive endothelial responses, VSMC structural changes, and macrophage lipid deposition (3, 5, 28).During atherosclerosis development, the major pathological processes are endothelial cell inflammation injury and overgrowth and migration of human VSMCs, in which ubiquitination and ncRNAs are implicated.
The lncRNA NEAT1 and miR-19b mentioned below were shown to aggravate endothelial cell injury and induce an inflammatory response via upregulation of the expression of the deubiquitinating enzyme BRCC3 and inhibition of the expression of the deubiquitinating enzyme CYLD (73, 74) (Figure 5).In detail, Yao, Song et al. (73) elucidated that lncRNA NEAT1, as a powerful miR-204 sponge, promoted BRCC3 expression in human umbilical vein endothelial cells (HUVECs) and significantly aggravated H/Rmediated NLRP3 inflammasome-activated endothelial cell injury and pyroptosis.In addition, miR-19b exerted a proinflammatory effect on vascular endothelial cells by reducing the expression of CYLD and further enhancing the accumulation of TRAF6 in spontaneously hypertensive rats in vivo (74).
Conversely, Zong et al. (83) verified that miR-30e could exert an inhibitory effect on the proliferation and migration of human VSMCs and act as an antiatherosclerotic agent by negatively targeting Ube2i and reducing the IkBa/NFkB signaling pathway in rats in vivo.Similarly, lincRNA-p21 functioned as a negative regulator of VSMC proliferation during atherosclerosis and induced VSMC apoptosis by directly binding to the E3 ubiquitin ligase

Stroke
Ischemic stroke is a typical clinical condition of cerebrovascular disease due to neuronal damage and destruction of supporting structures caused by cerebrovascular embolism and cerebral infarction (85).
Therapeutically, lithium improved neurovascular remodeling by upregulating miR-124 expression, decreasing REST abundance and inhibiting deubiquitination of peri-infarct brain tissues and proteins on the 4th day post-stroke (102).Inhibition of FBXO3 by siRNA alleviates cerebral I/R injury by suppressing ubiquitination

Crosstalk between ubiquitin ligases and ncRNAs drives in hypertension progression
Presented as an increase in localized pulmonary arterial blood pressure, PAH is characterized by endothelial cell dysfunction, VSMC proliferation, vascular remodeling, and right ventricular overload (106,107).
VSMCs form the middle layer of arteries and have regulatory functions in the vasculature, such as regulating blood flow, blood pressure and vascular tension (108).Accumulating evidence has illustrated that several miRNAs and lncRNAs are associated with the progression of PAH through the involvement of ubiquitination.Three ncRNAs, miR-27b-3p, miR-21 and lncRNA SOX2-OT, promoted the proliferation of pulmonary artery smooth muscle cells (PASMCs) via downregulation of FBXW7 and WWP1 and upregulation of SUMO1 (109)(110)(111).The researchers revealed that overexpression of miR-27b-3p could significantly elevate GLI1 expression through inhibition of ubiquitin ligase FBXW7 expression and accelerating KLF5 accumulation by attenuating its ubiquitination degradation in rats in vivo (109).Notably, miR-21 overexpression facilitated chronic hypoxia-induced PASMC proliferation and pulmonary vascular remodeling, and the possible mechanism was that miR-21 negatively regulated the E3 ubiquitin ligase WWP1 and then activated TGF-b1 signaling in mice in vivo and in vitro (110).Furthermore, Jiang, Hei, et al. (111) identified that increased lncRNA SOX2-OT promoted PASMC growth and migration by negatively reducing downstream miR-455-3p expression and elevating SUMO1 levels.In contrast, miR-92b-3P suppressed the proliferation of PASMCs under hypoxic conditions and improved PAH by inhibiting USP28 expression in vitro (112).
In addition, miR-140-5p expression was reduced in rat models in vivo and in PAH patients, and knockdown of miR-140-5p could facilitate the expression of the E3 ubiquitin ligase SMURF1 and activate BMP signaling, thereby promoting PASMC proliferation and pulmonary artery endothelial dysfunction and possibly exacerbating PAH (113).Conversely, miR-146-5p facilitated hypoxia-induced proliferation of pulmonary artery endothelial cells and PASMCs by negatively regulating USP3 (114).Specifically, Xu et al. (115) performed a control experimentknockdown of the 26S proteasome regulatory subunit PA700 by siRNA and modifications of PA700 by tyrosine nitration in vitroand the results indicated that PA700 nitration could promote the combination with the 20S proteasome, activate the 26S proteasome, NcRNAs and ubiquitination in stroke.
and reduce GTPCH I and vascular protective enzyme BH4 expression, ultimately damaging endothelial cells in Ang IIinduced hypertension.Notably, Baptista et al. discovered that miR-424(322) functioned as a messenger connecting PAH and right ventricle hypertrophy; mechanistically, overexpression of miR-424(322) suppressed SMURF1 expression and activated the BMPR2 pathway in a PAH rat model in vivo and in PAH patients, thereby inducing right ventricle overload (116).
6 Prognostic and therapeutic targets

Diagnostic indicators of the UPS involving ncRNAs
Increasing evidence has revealed that ncRNAs are in different biofluids, which suggests that ncRNAs have the potential to be used as diagnostic indicators by detecting their dynamic expression (117,118).Unlike sampling ncRNAs via invasive techniques such as biopsy, it is easy and inexpensive to detect ncRNAs from blood samples (119).However, the current obstacle is the lack of some ncRNAs with sensitivity and specificity (120).
Intriguingly, numerous clinical studies have shown that ncRNAs serve as diagnostic markers for CVDs.By using various screening approaches, such as single-cell sequencing and functional high-throughput screening, ncRNA molecules identified in body fluids can be validated in large cohorts of patient samples and used as novel biomarkers for CVDs (121).For example, miR-423-5p and lncRNA LIPCAR might be biomarkers for heart failure in patients' plasma; miR-1, miR-133, miR-423, miR-208b, miR-499 and lncRNA UCA1, LIPCAR might be used as biomarkers for acute myocardial infarction; miR-155 and miR-17 might serve as biomarkers for coronary artery disease in blood or urine (28,122,123).Circ-STAT3 might be a predictor for stroke functional outcomes in 982 patients' analysis for stroke recovery (124).However, ncRNA-based diagnostic strategies for CVDs are still in the experimental and preclinical stages due to ncRNA variability, analytical and technical factors, relevant isolation methods, crossplatform standardization and accuracy (28), therefore, ncRNAbased diagnostic markers for CVDs may be a supplement and development program for early screening and accurate and comprehensive diagnosis of CVDs in the future.

Therapeutic target of UPS and ncRNAs
Advanced studies have shown that therapeutic strategies based on specific ncRNA modulation are promising candidates to improve CVDs (28, 125,126).NcRNAs are also considered to be key novel regulators of cardiovascular function and risk factors, potentially serving as small-molecule targets (e.g., miRNAs, lncRNAs, circRNAs and lincRNAs) or gene silencing tools (e.g., siRNAs) for cardiovascular treatment and prognosis assessment (23,28).Specifically, RNA interference (RNAi) drugs and further novel drugs that mimic or inhibit endogenous ncRNAs may lay the blueprint for the treatment of CVDs at the small molecule level by utilizing molecular interactions, particularly ubiquitination or deubiquitination.
On the fourth level, targeting upstream proteins, which are regulators of crosstalk between ncRNA and the UPS, represents a promising and novel approach to CVD treatment.Specifically, ZFP36L2 silencing improves myocardial I/R injury via regulation of the lncRNA PVT1/miR-21-5p/MARCH5 axis (44).In the treatment of dilated cardiomyopathy, TWIST1 acts as an advantageous target to improve heart quality by increasing miR-199/214 cluster expression and decreasing UPS activity (56).Interestingly, SOX9 is regulated by USP7 and has therapeutic value for sepsis-induced myocardial damage by regulating the downstream miR-96-5p/NLRP3 pathway (61).For vascular diseases, soybean-derived vasoactive peptide protects vascular endothelial cells from inflammatory damage via manipulation of the miR-19b/CYLD/TRAF6 axis (74).TGR5 has an anti-neuroinflammatory role in the clinical treatment of poststroke via the TGR5/Pellino3/caspase-8 and NLRP3 axes (104).Furthermore, PA700 inhibitors are effective for Ang II-induced hypertension therapy via regulation of 20S,26S proteasomes/ GTPCH I and BH4 (115).
Nucleic acid drugs based on various disease-associated ncRNAs provide a new perspective in disease therapy.NcRNA-based drugs are designed to exert therapeutic effects by interacting with proteins or nucleic acids (127).However, this type of drug has two notable limitations.First, RNA molecules may evoke the host's immune system and result in adverse immune responses.Second, because of the general nature of their targets, ncRNA-based drugs might not target specific sites (125).Therefore, current studies have proposed that ncRNAs combined with biocompatible nanoparticles (NPs) can offer better therapeutic properties.Using NPs as delivery vectors can reduce drug toxicity, facilitate targeted drug release, and increase drug bioavailability, which provides an optimized therapeutic strategy for the cardiovascular field (125,(128)(129)(130).In addition, with the production of proteasome inhibitors, the UPS offers a new therapeutic frontier.Drugs targeting ubiquitin ligases were found to have few side effects and significant treatment outcomes (15).
Notably, there have been various clinical trials using ncRNA or ubiquitin ligases for the treatment of CVDs.For instance, MiR-132 inhibitor CDR132L as a novel antisense therapy drug was demonstrated to alleviate heart failure and cardiac remodelling in first clinical trial (131).In the PREDIMED randomized controlled trial, a high-unsaturated fat medicine diet intervention, decreasing triglycerides, possibly reducing stroke risk in miRNA-410 target rs13702 C allele carriers (132).Moreover, the randomized controlled study found that the concentrations of HDL-carried miR-223 and miR-135a were elevated in healthy men following intake of trans fatty acids, preventing worsening of cardiovascular risk (133).Furthermore, siRNA therapy, Olpasiran, was applied to decrease lipoprotein(a) concentrations, thereby effectively ameliorating atherosclerotic CVDs (134).Similarly, Nissen, Wolski et al. revealed that plasma lipoprotein(a) concentrations are doserelated reduced in the siRNA SLN360 single ascending dose study (135).In addition, the ubiquitin E3 ligase NEDD4 rs4149601 can serve as a predictor of adverse cardiovascular consequences in whites without hydrochlorothiazide intervention, and NEDD4 rs292449, rs75982813 and rs4149601 can act as a predictor of blood pressure response after hydrochlorothiazide administration in Pharmacogenomic Evaluation of Antihypertensive Responses (PEAR) clinical trial (136).
In conclusion, known clinical trials using ncRNAs or ubiquitin ligases for the treatment of CVDs may provide a basis for emerging relevant laboratory studies of crosstalk between ncRNAs and ubiquitin ligases for CVD treatment, moving towards further clinical trials.For example, NEDD4 was found to predict adverse cardiovascular outcomes in PEAR clinical trial (136), and miR-454/ NEDD4-2/TrkA/cAMP axis was demonstrated to exert antiapoptosis effects and improves HF in rats, thus potentially laying the foundation for clinical trials.

Conclusion and future perspectives
Undoubtedly, CVDs constitute enormous health and socioeconomic burdens worldwide.The UPS is involved in the posttranscriptional modification of proteins.NcRNAs are unique RNA transcripts and play crucial roles in cellular processes and the development of various diseases, including CVDs.In this review, we creatively focused on the role of crosstalk between ncRNAs and the UPS in CVD processes.The main mechanism is that ncRNAs regulate E3 ubiquitin ligase levels and ubiquitination degradation of targeted proteins, and vice versa.A small number of E3 ubiquitin ligases can also regulate ncRNA expression.The crosstalk between ncRNAs and the UPS can affect the development of CVDs, such as myocardial I/R injury, MI, cardiomyopathy, HF, myocarditis, atherosclerosis, HT, and ischemic stroke.
A better knowledge of the role of ncRNAs and the UPS can finally provide promising therapeutic strategies for the treatment of CVDs.(1) Targeting ncRNAs can regulate E3/E2/deubiquitinating enzyme expression and subsequently promote or inhibit ubiquitination degradation of targeted proteins.(2) E3 ubiquitin ligases can act as therapeutic targets by regulating ncRNA expression or directly promoting ubiquitination and degradation of downstream proteins.(3) NcRNAs, UPS components and target proteins can serve as cotherapeutic targets.(4) Targeting upstream regulatory proteins or directly knocking down of E3 ubiquitin ligases by siRNA can have a therapeutic impact on CVD.Specifically, the levels of miR-424(322), miR-140-5p, circDLGAP4, and lncRNA LOC105378097, as well as SMURF1, HECTD1, and Parkin ubiquitination can be measured in clinical PAH, stroke, and cardiac aging patient blood, suggesting their diagnostic value.
Despite growing evidence from experimental studies demonstrating the potential mechanism and significant implication for ncRNAs and the UPS in CVDs, there are still many limitations.First, the specific mechanism is still incomplete, and there are few evidence-based epidemiologic and clinical studies.Second, individual ncRNA or the UPS has a large number of regulatory roles in CVDs, but some of them remain unclear.Third, compared to the crosstalk between miRNAs and the UPS, knowledge on the relationship between lncRNAs/circRNAs/siRNAs and the UPS is far scarcer.Fourth, it is also unknown whether the mechanism of the interaction between ncRNAs, the UPS and CVDs is indeed widely effective in cardiomyocytes.Fifth, further studies are needed to examine the effects of genetic variability among individuals (e.g.DNA and RNA methylation, ATP-dependent chromatin remodelling, and post-translational histone modifications) on the relationship between ncRNAs, ubiquitin ligases, and CVD progression (141).Sixth, the role of other protein degradation pathways regulated by ncRNAs (e.g.calpain and autophagy) in CVDs needs to be further summarized and expanded.Finally, therapy targeting ncRNAs or the UPS alone is not yet mature, and cross-linking CVD therapy with the two seems far off.
On the one hand, ncRNA-based therapy offers the advantage of targeting traditionally undruggable targets and a potential role in biomarker-guided therapy.However, there are key challenges to overcome in developing effective ncRNA therapies, such as (1) obtaining sufficient stability and delivery in vivo, (2) acquiring high specificity of therapeutic ncRNA for molecular targets, and (3) attaining the ability to target intended cell types or tissues via appropriate vectors and transcriptional targeting and minimizing the side effects of the therapeutic ncRNA itself or drug delivery systems.To reduce these shortcomings, lipid NPs and viral vectors have been widely studied and serve as novel vectors for the delivery of nucleic acid drugs.On the other hand, proteasome, ubiquitin ligase E3 inhibitors and deubiquitinating enzyme inhibitors are undergoing clinical trials and have promising roles in CVD treatment.Nevertheless, proteasome inhibitors may cause selective downstream cell death, the mechanism of which is unclear.The effectiveness of proteasome inhibitor treatment is impaired by innate or acquired resistance mechanisms.
In addition to basic research on signaling pathways, the specific crosstalk of ubiquitin ligases and ncRNAs in CVDs needs to be further investigated with epidemiological evidence and clinical trials.We believe that advanced therapy based on ncRNAs and ubiquitination or deubiquitination can be more effective in treating CVDs and reducing the high mortality rate of CVD for the benefit of all humankind.

FIGURE 1
FIGURE 1Relationship of ncRNAs, UPS and CVDs.The crosstalk of the UPS and ncRNAs affects the pathological mechanisms of CVDs.NcRNAs that regulate ubiquitination or are regulated by the UPS are involved in the execution of target protein stability.

FIGURE 2
FIGURE 2Crosstalk of ncRNAs and UPS.

FIGURE 3
FIGURE 3NcRNAs and ubiquitination in cardiac diseases.The crosstalk of ncRNAs and ubiquitination regulates cardiac diseases, such as MI/R, MI, myocardial.NcRNAs and ubiquitination are involved in cardiac diseases by regulating target protein expression.

FIGURE 4
FIGURE 4    NcRNAs and ubiquitination in other cardiac diseases.The crosstalk of ncRNAs and ubiquitination regulates cardiac diseases, such as hypertrophy, myocarditis, heart failure, heart aging, myocardial fibrosis and calcific AV disease.NcRNAs and ubiquitination are involved in cardiac diseases by regulating target protein expression.

FIGURE 5
FIGURE 5    NcRNAs and ubiquitination in vascular diseases.The crosstalk of ncRNAs and ubiquitination regulates vascular diseases, such as endothelial cell inflammatory injury, VSMC proliferation and migration, atherosclerosis, stroke, and hypertension.NcRNAs and ubiquitination are involved in vascular diseases by regulating target protein expression.

TABLE 1 ncRNAs
as diagnostic indicators for CVDs.

TABLE 2
ncRNAs as therapeutic targets for CVDs.