Effects of MicroRNAs in Valvular Heart Diseases: From Molecular Pathways to Clinical Effects and Therapeutical Strategies

Micro-RNAs have been recently investigated in preclinical and clinical research as regulators of valvulopathies pathogenesis, diagnostic biomarkers and therapeutical targets. Evidences from in-vivo and in-vitro studies demonstrated stimulatory or inhibitory roles in mitral valve prolapse, aortic leaflet fusion and calcification pathways, specifically osteoblastic differentiation and transcription factors modulation. Tissue expression assessment and comparison between physiological and pathological phenotypes or different disease entities, including mitral valve prolapse and mitral chordae tendineae rupture, emerged as the best strategies to address mi-RNAs over or under-representation. In this review we discuss the fundamental intracellular homeostatic and cardiogenetic pathways regulated by mi-RNAs leading to defects in mitral and aortic valves, congenital heart diseases and the possible therapeutical strategies targeting them. Mi-RNAs inhibitors comprise antisense by the experimental of a high phosphate found low levels of miR-133b and miR-211 and high levels of miR-29b. The correlated with overexpression of osteogenic RUNX2, the

the mitral orifice, causing regurgitation. In addition, annular dilation may develop over time, leading to further advancement in the severity of mitral regurgitation.
A careful evaluation of MVP is achieved with transthoracic echocardiography which serves to determine the mechanism and severity of mitral regurgitation. Although the information provided by echo 2D and 3D yields comprehensive morphological and functional assessment [22][23][24], the lack of effective medical therapy in slowing the progression of these lesions making surgery the only viable treatment option [25][26][27]. Recently a new impetus in mitral valve repair has been afforded by the use of the transcatheter approach, although this requires more solid validation of results for wider application [28][29][30].
The landmark study of Mayeux [1] has highlighted the emergence of biomarkers as an important tool for a better diagnosis, thus, shifting paradigms in clinical practice progression by increasing the possibility of obtaining a definitive prognosis in specific pathological conditions. However, there is currently no likelihood of using specific circulating biomarkers for the identification of mitral valve prolapse in clinical practice.
Regarding MVP, several studies have looked for circumstantial evidence between the presence of biomarkers and the valvular degenerative process. The studies by Songia et al [10,11] investigated the possible association between osteoprotegerin and mitral valve degeneration in Barlow syndrome without recording a specific correlation. The study by Tan et al, which evaluated the presence of biomarkers through proteomics, was in the same direction. The authors reported higher levels of haptoglobin, platelet basic protein, and complement component C4b in individuals with degenerative mitral valve prolapse [12]. The results were not conclusive for a specific correlation between biomarkers and disease development.

Emerging differences in mi-RNAs as biomarkers in stenotic or insufficient valvulopathy
MicroRNAs (miRNAs) have emerged as circulating biomarkers encoding a new category of potential molecules to study. Almost 20 years ago, a study of the eukaryotic genome reported 20-30 nucleotide RNA molecules emerging with the function of playing a critical regulatory role in the expression and function of eukaryotic genomes. Carthew et al [31] investigated the role of two leading classes of these small RNAs with specific regulatory functions leading to direct implications in the fundamental processes of biology, as well as in the aetiology and treatment of the disease. The authors identified short interfering RNAs (siRNAs) and microRNAs (miRNAs) a pivotal role in intervening on both somatic and germ lines alongside a wide range of eukaryotic species. The specific target of siRNA and miRNA were the sentinels of the genome in defence of invasive nucleic acids, through the ability to negatively regulate gene expression in the post-transcriptional phase by precluding protein translation or supporting RNA (mRNA) degradation [31]. MicroRNAs (miRNAs) were shown to modulate gene expression and were altered in the aortic valve leaflets in patients with aortic valve stenosis compared to aortic insufficiency occurring in the landmark paper from the University of California 10 years ago [5].The reduced levels of miRNA-26a, RNA-30b, and miRNA-195 that have been reported in stenotic valves were almost certainly responsible for its higher risk of developing valvular leaflet fusion due to accelerated calcium accumulation compared to regurgitant aortic valves without morphological fusion of valve leaflets. These miRNAs were involved in the biological processes that modulate calcification-related genes in vitro [5].
Several studies have recorded a substantially inferior level of mi-RNAs that were associated with the pivotal function of post-transcriptional modulators of gene expression in aortic valve stenosis. The expression of miRNA-141 is implicated as a regulator of the levels of bone morphogenetic protein 2 (BMP-2), whereby unrestrained activity led to calcification of the aortic valve mediated by a stimulation of osteogenesis. miRNA141 was markedly attenuated in patients with aortic stenosis associated with the bicuspid aorta valve [6]. Yanagawa et al proposed the new key role of miRNA-141 in the modulation of aortic valve calcification disorders, highlighting the strategic therapeutic target that emerged in the assessment of progressive calcification in stenotic aortic valve disease [6]. The peculiar morphologic features of the stenotic aortic valve may probably be explained by the inferior expression of miRNA-30b which is a known repressor of bone morphogenetic protein 2-mediated osteogenesis.
Zhang et al demonstrated the role of miRNA-30b in reducing osteoblast differentiation activity induced by bone morphogenetic protein 2. The latter was implicated in promoting calcific aortic valve disease. The expression of miRNA-30b was effective in reducing the risk of human aortic valve calcification and apoptosis through direct targeting of Runx2, Smad1, and caspase-3   The levels of miR-486 were increased, inducing the   myofibroblastic transition with an upregulation of the expression of Runx2 and Osx, and   synergized with the deficiency of miR-204, which lead to high cellular and valvular pro-osteogenic activity. This study demonstrated the existence of a sophisticated modulation of the epigenetic mechanism that supported pro-osteogenic valve activity, paving the way for the therapeutic potential for the prevention of the progression of calcified aortic valve disease.
Scientific evidence supports the differential expression of miRNAs in the bicuspid aortic valve, with an evident calcific-degenerative focus that differentiates the bicuspid morphology from the differential modulation of the tricuspid aortic valve [9].

Diagnostic and prognostic relevance of mi-RNAs in mitral valve diseases
In recent years, microRNAs have represented an emerging class of widely laboured circulating biomarkers in various pathological states including degenerative mitral valve disease [10][11][12].
Regarding MVP, we note only a small number of circulating microRNAs have been studied in degenerative disorders of the mitral valve, with the findings limited to the experimental animal models. Hulanicka et al [13] evaluated plasma for miRNAs as potential biomarkers of chronic myxomatous mitral valve disease (MMVD) in Dachshunds. The authors focused the study on the expression of 9 miRNAs already discovered and that were involved in cardiovascular pathologies. The plasma levels of two out of nine miRNAs were significantly downregulated with the use of real-time PCR method, so as to record clear involvement of them in dogs developing endocardiosis.
According to the American College of Veterinary Internal Medicine (ACVIM), the authors compared the plasma levels of miRNAs in three groups of dogs. They recorded that miR-30b expression differed between dogs of the ACVIM group in stage B (asymptomatic n = 8) and those that were included in the unaffected stage A group (control N = 8). The expression of mi-133b differed in the ACVIM stage C group, in which mild to moderate heart failure occurred, compared to that of stage A group of dogs. 5 miRNAs (miR-125, miR-126, miR-21, miR-29b, and miR-30b) showed a downregulation trend only in the ACVIM stage C group recording nonsignificance for the expression of these classes of miRNA. Regarding the levels of miR-423, an equivalence was noted between healthy and sick dogs. The expression of miR-208a and 208b was not detected. The plasma level of miR-30b could be correlated as a potential biomarker of ACVIM stage B heart failure in Dachshunds who developed endocardiosis while miR-133b expression could be correlated as a potential biomarker of ACVIM stage C. It should be noted that the lack of expression or notable change in expression in 7 miRNAs which were potential biomarkers for the development of heart disease in humans highlight the lack of transferability from animal models to clinical applicability.
A second study [14] reported the miRNA expression profile in dogs suffering from MMVD (myxomatous mitral valve disease). Li et al quantified 277 miRNAs using RT-qPCR and comparing three groups of 6 dogs. The first group enrolled asymptomatic animals with no disease (ACVIM stage A control group). The second group included dogs exhibiting MMVD with mild to moderate enlargement of the heart chambers (ACVIM Stage B1 / B2). The third group included animals with MMVD and congestive heart failure (ACVIM Phase C / D). For eleven miRNAs, the study results showed a different expression with a False Discovery Rate <0.05. Dogs enrolled in group B who had stage B1 / B2 disease or those included in group C who had stage C / D disease recorded four upregulated miRNAs. These two groups included three cfa-let-7 / cfa-miR-98 family members with upregulation, while seven others were downregulated when compared to the stage A control group. The evidence suggested significant differences in the expression of six of the 11 miRNAs when comparing animals belonging to stage C / D and those that were included in stage B1 / B2. In addition, the changes in miRNA expression were greater with the increasing severity of MMVD. The significance of this study relates to the fact that these miRNAs can be candidates biomarkers, providing further insights into specific genetic regulation pathways in MMVD that developed in dogs ( The major concern with the use of animal models to evaluate the expression of miRNA is the increased risk of discrepancy with human pathoanatomic conditions. Bulent et al [15]  Songia et al [32] performed the first study using human plasma from patients with degenerative mitral valve prolapse and noted a strong correlation between several circulating miRNAs and mitral valves with myxomatous prolapse. Some of the tested miRNAs were also overlapping with those from Bulent et al [15] and a similarity emerged: lower levels of miRNA 223-3p was found in both patients suffering from MVP and patients who developed MCTR (Table 2).

140-3p
150-5p  [15]and Songia et al [32]. Patients affected by MTCR were found to have lower levels of miRNA 150-5p with respect to controls while miRNA-223-3p was found to be lower than controls, compared to patients affected by MVP.
The authors working on circulating biomarkers have provided valuable information on the etiology of degeneration and prolapse of MV alongside the possibility of stratifying patients affected by the disease.
Deroyer et al [33] investigated the role of apolipoprotein-A1 in fibroelastic disorders of MV revealing that the biomarker was indicated as an independent predictor of MR gravity.
In another study, Tan et al [12] performed an analysis using proteomic evaluation on two pooled Although the report [32] claimed that specific circulating biomarkers could be interpreted as molecular signatures, the study has some critical points. The study lacks numerically validated data in the different miRNA expressions between patients who had fibroelastic degenerative disease of MV and those who instead presented a myxomatous degeneration typically related to Barlow syndrome. In addition, the cohort of patients studied concerns those with degenerative prolapse of the MV with severe MR and eligible for surgical treatment. In fact, the study did not report any evaluation of the plasma levels of miRNAs capable of identifying a larger cohort of individuals with a prolapse of the MV coexisting with a mild or moderate mitral insufficiency.
However, given the reported evidence, the future direction postulates that miRNAs identified in plasma could be used in the near future and is an inexpensive screening tool for patients with progressive degenerative mitral valve disease and severe mitral regurgitation.

Complex interplays between mi-RNAs and intracellular osteogenic signals
Calcium phosphate crystals are responsible for abnormal accumulation in either native or prosthetic valves, leading to valvular calcification (VC), loss of elasticity, and ischemic conditions [42]. Intimal and medial layers of major vessels can display calcifications, the former being associated with atherosclerotic phenotypes, the latter with common cardiovascular risk factors including diabetes and osteoporosis. Some authors propose vascular smooth muscle cells (VSMCs) to begin the process by undergoing phenotypical changes to osteoblastic nature and losing contractile markers such as smooth muscle 22 alpha (SM22α) and alpha-smooth muscle actin (α-SMA) [43].
Several coding genes have been studied and linked to the specific development of calcific valves. Osteopontin (OPN), osteocalcin (OC), bone morphogenetic proteins (BMPs), alkaline phosphatase (Alp), and transcription factor Runx2 were demonstrated to be upregulated in calcific processes [42]. Other described signaling pathways include exosomes cross-talk among the three layers of the vascular wall and Wnt/Beta-catenin, advanced glycation end products (AGEs), and osteoprotegerin/ receptor activator of nuclear factor-kB and its ligand (OPG/RANK/RANKL) interacting between either the intima and media layers or the media and adventitia layers [44,45].
Pro-inflammatory activity has also been related to VC. Tumor necrosis factor-alpha (TNFa), interleukins including IL-1B and IL-6, tumor growth factor-beta 1 (TGFB1), and other cytokines mediate vascular smooth muscle cells transition into osteoblast-like cells [46]. The phenomenon is known to be enhanced by reactive oxygen species (ROS) production and determined by the discovery of cytokines and factors expression in the aortic tissue. A Canadian preclinical study by Agharazii et al [46] demonstrated valvular calcification processes developing from chronic kidney disease, to be increased in rats by cytokines. Interleukin-1β, interleukin-6, and tumor necrosis factor were overexpressed in aortic tissues. Also, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase expression was increased while antioxidant enzymes (SOD1, SOD2, Gpx1, and Prdx1) demonstrated lower levels than normal [46].
MicroRNAs, small non-coding RNAs, and long non-coding RNAs are part of the broader group of non-coding RNAs. MicroRNAs, with the exception of miR-29, have been categorized into either activating or promoting valvular calcification. Some authors claim miR-29a/b repression is a pivotal factor in calcification generation. A preclinical study by Du and colleagues [47] revealed how increased expression of ADAMTS-7 (a disintegrin and metalloproteinase with thrombospondin motifs-7), markedly upregulated in calcifying rat vascular smooth muscle cells, is linked to miR-29 inhibition. Calcification inhibitors miRNAs, which roles were revealed by decreased expression in calcific tissues, have been described by in vitro and in vivo studies, in both animals and humans. MiR-30b, 30c were found to directly inhibit Runx2 factors, such as miR-133a, miR-204, and miR-205 [48][49][50][51].
Recently, Lin and colleagues [52] demonstrated miR-34c/5p to be downregulated in calcific tissues (in vitro high glucose-induced human aorta VSMCs) and inhibiting BMF as the primary target. In fact, miR-34c from the miR-34 family is reported to participate in osteoblast differentiation [53,54]. It targets SATB2, a nuclear matrix protein that inhibits the expression of HOXA2, in turn negatively regulating Runx2 and increases the activity of Runx2 and activator transcription factor 4 (ATF4). Runx2 and ATF4 are sophisticated regulators of osteoblastic differentiation, from osteochondral progenitors. Osterix is another important involved factor [55]. Investigators also claim miR-34c regulates Osteocalcin and other genes whose expression is controlled by Runx2 and SATB2 or ATF4 and SATB2 [53].

Over and underexpression of miRNAs from in vivo animal experimentations
Recently, investigators have provided proof of upregulated levels of miRNAs in calcific vessels. An in vitro demonstration of human and murine aortic tissue and, specifically, of smooth muscle cells expressing miR-29b at increased levels comes from a Japanese investigation [56] and a Spanish preclinical study [57].
Panizo et al [57] induced vessels calcification in rats by using the common experimental method of feeding them a high phosphate diet. They found low levels of miR-133b and miR-211 and high levels of miR-29b. The former correlated with overexpression of osteogenic RUNX2, the previously described factor, while the latter with lower expression of inhibitors of osteoblastic differentiation. The reliability of the study is conferred by in-vitro affirmation of the results: authors analyzed miR-29b, miR-133b, and miR-211 to demonstrate how they regulated the calcification process [57]. MiR-29-mediated elastin down-regulation also promotes osteoblastic differentiation [56]. Calcium deposition in human VSMCs favoured by phosphorus (Ph) was evaluated by Sudo and colleagues [56]  Selected significant results are tabulated in Table 3.

Altered miRNAs expression in congenital valve disorders and cardiogenetic processes
Congenital heart diseases (CHD) comprise a large group of functional and structural disorders, response factor (SRF) and promotes the differentiating process [63].
Several other preclinical studies, conducted on zebrafish ventricles, denoted the role of miR-133 to diminish cardiac regenerating processes [64]. Following resection of zebrafish ventricular apex, a reduced expression of miR-133, coupled with an increased regenerative potential, has led to this concept [64].
Cardiogenesis is also suppressed by the miR-15 family, which, specifically inhibited, has shown to promote myocyte proliferation after myocardial infarction [65]. On the contrary, miR-199 and miR-590 have been found to promote the re-entry of cardiomyocytes in the cell cycle. An interesting therapeutical strategy, supported by preclinical mice studies, would be to inject these molecules into the border zones of infarcted hearts. Positive results and stimulation of cardiomyocytes proliferation have been demonstrated [66,67].
Several mi-RNAs also regulate the signals of insulin-growth factor 1 (IGF-1) in skeletal muscle, contribute to muscle development or atrophy [68]. Several other studies have noted their regenerative role by observing common cardiovascular pathologies and the subsequent structural remodeling [69]. Long non-coding RNAs are implied in hypertension-related vascular remodeling, post-ischemic recovery, and myocardial hypertrophy (Table 4) [69].  Specific miRNAs are also differentially expressed in bicuspid aortic valve (BAV), the most common congenital heart disease. Aortic valve endothelial cells on the ventricular side are frequently exposed to high shear forces while on the aortic side turbulent blood flow and high levels of antioxidant enzymes are present [70].

Mechanism of involvement
Conversely, on the ventricular side, factors inhibiting calcification are more abundant [70,71].
Sabatino and colleagues [9] performed a bioinformatic analysis in order to identify the most commonly regulated miRNAs in normal and stenotic bicuspid aortic valves and compared results with normal and stenotic tricuspid valves with respect to calcium metabolism, blood coagulation, phosphate metabolism, and inflammatory pathways. MiR-133 can be used for guiding the therapeutic management of aortic stenosis, due to its potential role in predicting left ventricular hypertrophy [72][73][74]. Authors found that its levels were differentially expressed in bicuspid versus tricuspid aortic valve and were also correlated with the degree of stenosis, as previously discussed. Investigators claim it will be used as a biomarker as it reflects the degree of myocardial fibrosis [9].
The key factor involved in calcium metabolism and inflammatory pathways was found to be Epidermal growth factor receptor (EGFR). Several miRNAs, also associated with calcification, were associated with stenotic tricuspid aortic valves (TAVs) and BAVs, namely miR-100, −130a, −181a/181b, −199a-5p, −199a-3p, and −214 which have been investigated by other authors to display higher expression levels in VECs of the fibrosa on the aortic side, compared to the ventricular side [75].
MiR-181 is another important mi-RNA. Aortic valve endothelial cells were associated with its increased expression but decreased levels of targets, including SIRT1 and GATA6 that negatively affect vascular SMCs elastin production [76]. Several other studies confirmed that its inhibition increases the expression of elastin and collagen while its stimulation, through direct administration, inhibits atherosclerotic lesion formation [77].

Novel therapeutical strategies: mi-RNAs targeting to suppress or activate them 4.1 Results from in-vivo and in-vitro testing for aortic valvular stenosis
Regulating miRNAs expression is an attractive therapeutical challenge. Valvular calcification is currently not a direct target for pharmacological action. Endothelin receptor antagonists have emerged as possible molecules of interest in preclinical studies [78,79]. Chronic kidney diseaseinduced valvular calcification was demonstrated to be slowed by administration of endothelin type A (ETA) receptor atransentan (10mg/kg/day) which reduced SMC differentiation, calcification, and stiffness [78].
Concerning statin treatment, several studies reported increased rates of calcification [80].
Possible strategies for mi-RNAs overexpression include mi-RNA mimics, mi-RNA expression vectors, and small molecules [59,81]. Instead, negatively regulating mi-RNAs seems to encompass different strategies. Antisense oligonucleotides including locked nucleic acid (LNA)-modified anti-miR, or miRNA sponge vectors can be used to specifically binding to mi-RNAs [59]. Toshima and colleagues [82] demonstrated miR-34a as a potential therapeutic target. Its inhibition in human aortic tissue exhibiting either calcific aortic valve stenosis (CAVS) or aortic regurgitation (AR) attenuated calcification signals in porcine aortic valve interstitial cells (AVICs) compared with miR-control. After performing RNA pull-down assays, miR-34a was demonstrated to directly target Notch1 by binding to Notch1 mRNA 3' untranslated region [82]. Also, miR-34a inhibitor suppressed calcium deposition of aortic valves and cardiac hypertrophy, both mechanisms involved in decreased Runx2 and increased Notch1 expressions [82].
Another possible strategy recently proposed, involves melatonin administration. In vitro studies confirmed melatonin reduces the level of CircRIC3, a circular RNA with procalcific effects. It acts as a miR-204-5p sponge to stimulate and increase expression levels of the procalcification gene dipeptidyl peptidase-4 (DPP4). A preclinical in vivo study [83] involving high cholesterol diet (HCD)-treated ApoE -/mice with aortic valve calcification demonstrated that the intragastric administration of melatonin for 24 weeks improved aortic valvular parameters. It reduced thickness and calcium deposition in the leaflets and ameliorated echocardiographic markers, namely transvalvular peak jet velocity and aortic valve area [83]. At the molecular level, it decreased Runx2, osteocalcin, and osterix factors which are involved in osteogenic differentiation, as we discussed above (Figure 2). The authors also demonstrated melatonin caused in vitro suppression of calcification in human valvular interstitial cells (hIVICs) [83].

Technical concerns on stability and efficacy of mi-RNAs as therapeutical targets
The primary concerns in utilizing mi-RNAs as therapeutic targets, either to positively or to negatively affect them, arise from the need of achieving stability and resistance to degradation enzymes. We named expression vectors, antisense nucleotides (ASOs), small molecules and miR-mimics as novel approaches under current experimentation. Expression vectors are also defined mi-RNAs sponges and constitute artificial binding sites for mi-RNAs to reduce their effect on mRNAs [84,85]. Oligonucleotides also bind mi-RNAs but are regarded as anti-miR for their sequence complementarity. With this strategy, they relieve mRNA targets from degradation or transcriptional blockage. Small molecules serve as translational regulators instead but specific targets have not been revealed yet.
A possible strategy to improve stability is modification with 2-O-methyl (2 -OMe) [86]. This can be then further stabilized with sulfur atoms in place of non-bridging oxygen atoms in the phosphate backbone. Serum nucleases, deputed to degradation of mi-RNAs, would find difficult to cleave phosphorothioate bonds, given that they normally cleave phosphate bond [86,87]. The adding of a 3' cholesterol tail is another approach to ameliorate stability and efficacy.

Disadvantages in pharmacokinetics and proposed mechanisms for delivery vehicles
In vitro studies conducted on oligonucleotides had limited pharmacological effects due to unfavourable kinetic characteristics, notably poor tissue distribution and fast excretion. Thus, appropriate delivery systems have been developed, functioning as carriers for in vivo molecular directing [84]. A good delivery system should achieve the following features: evading the immune system response, avoiding nuclear degradation, directed to target cells, and releasing the content for incorporation into RNA processing machinery [89][90][91][92]. The main combination strategies include polymers, lipids, conjugation, antibodies, nanoparticles, and microbubbles [84]. In particular, nanoparticles can deliver anti-miRNAs and small molecules with a greater degree of multifunctionality [93]. The advantages of nanoparticles include large surface-to-volume ratios, hence, controlling their surface properties is crucial [94]. Surface charges also appear fundamental: macrophage scavenging is increased when the charge increases in number (either positive or negative) [93,95,96]. So, minimizing interactions to non-target sites via, as an instance, steric stabilization, would prevent nanoparticles from directing the molecules to undesired locations, and evading the immune system.
Local mi-RNA delivery results have been published and discussed in an Israeli study for metastatic breast cancer prevention by miR-96 and miR-182 treatment [105]. In vivo, local targeting was achieved by coating breast tumour cells with adhesive hydrogel scaffold covered in nanoparticles carrying the miRNAs of interest [105]. Nanoparticle stability achieved with hydrogel was also described for drug delivery in several other studies [106-108].

Conclusions: clinical relevance of preclinical studies and future directions
An Italian group discussed the potential use of mi-RNAs for mitral valve diseases [109]. Matrix metalloproteinase (MMP) with their genetic variants and mi-RNAs appear as predictive diagnostic and prognostic biomarkers but, most promisingly, as potential targets for personalized treatments [109] (figure 3). At present, no clinical data on therapeutic approaches targeting mi-RNAs are available. A preclinical study [110] investigated the use of recombinant adeno-associated virus (AAV-miR155-inhibitor) to inhibit the expression of miR-155-5p for valvular damage caused by rheumatic heart disease (RHD) in rat models. The different method, comprising an adenoviral delivery, enabled promising results to be demonstrated. Valvular expression of miR-155-5p was increased while SOCS1/STAT3 signaling was activated in the study rat population. The authors utilized dual-luciferase assays to display targeting by miR-155-5p of S1PR1 and SOCS1.
Inhibition of valvular miR-155-5p prevented activation of the SOCS1/STAT3 signal pathway and this resulted in suppression of valvular inflammation, detected as decreased tissue levels of Il-6 and Il-17 and fibrosis, both in valves and rats serum.
In conclusion, despite the current limitations of mi-RNAs molecular interactions and pathophysiological mechanisms to the preclinical scenario (both in vitro and in vivo), realising the possible therapeutic effects is fast becoming possible. We auspicate that in the coming years, further emphasis will be placed on these fascinating molecular approaches.
Funding: This research received no external funding.

Conflicts of Interest:
The authors declare no conflict of interest MIAT: myocardial infarction-associated transcript.