**Non-Coding RNAs: The "Dark Matter" of Cardiovascular Pathophysiology**

**Claudio Iaconetti, Clarice Gareri, Alberto Polimeni and Ciro Indolfi \***

Division of Cardiology, Magna Graecia University, URT Consiglio Nazionale delle Ricerche (CNR), Catanzaro 88100, Italy; E-Mails: iaconetticlaudio@unicz.it (C.I.); c.gareri@unicz.it (C.G.); polimeni@unicz.it (A.P.)

**\*** Author to whom correspondence should be addressed; E-Mail: indolfi@unicz.it; Tel.: +39-961-3694231; Fax: +39-961-6394073.

*Received: 4 July 2013; in revised form: 12 September 2013 / Accepted: 16 September 2013 / Published: 9 October 2013*

**Abstract:** Large-scale analyses of mammalian transcriptomes have identified a significant number of different RNA molecules that are not translated into protein. In fact, the use of new sequencing technologies has identified that most of the genome is transcribed, producing a heterogeneous population of RNAs which do not encode for proteins (ncRNAs). Emerging data suggest that these transcripts influence the development of cardiovascular disease. The best characterized non-coding RNA family is represented by short highly conserved RNA molecules, termed microRNAs (miRNAs), which mediate a process of mRNA silencing through transcript degradation or translational repression. These microRNAs (miRNAs) are expressed in cardiovascular tissues and play key roles in many cardiovascular pathologies, such as coronary artery disease (CAD) and heart failure (HF). Potential links between other ncRNAs, like long non-coding RNA, and cardiovascular disease are intriguing but the functions of these transcripts are largely unknown. Thus, the functional characterization of ncRNAs is essential to improve the overall understanding of cellular processes involved in cardiovascular diseases in order to define new therapeutic strategies. This review outlines the current knowledge of the different ncRNA classes and summarizes their role in cardiovascular development and disease.

**Keywords:** non-coding RNA; microRNA; long non-coding RNA; vascular development; vascular disease; heart pathophysiology

#### **1. Introduction**

Many studies have recently focused on understanding RNA metabolism and its implication in development and disease processes. Genomic tiling arrays and RNA-Sequencing have showed that the human genome is dynamically transcribed and leads to the production of a complex world of RNA molecules of which only a small fraction is translated into proteins [1]. In fact, application of high-throughput sequencing technologies in the analysis of mammalian transcriptomes, revealed a wide spectrum of RNA molecules that do not encode protein, termed non-coding RNAs (ncRNAs) [2]. For many years the role of these molecules remained unknown, so ncRNAs were called the "Dark Matter" of biology. To date many studies have been carried out on these molecules, especially on microRNAs, partially clarifying their roles. However many mechanisms and functions of different classes of ncRNA still remain unknown. Emerging evidence indicates that the non-coding portion of the genome is critical in the regulation of multiple biological processes, such as differentiation, development, post-transcriptional regulation of gene expression and epigenetic regulation [3–5]. Recently, many classes of ncRNA have been described to be associated with human disease [6]. Cardiovascular disease is a major cause of mortality and hospitalization worldwide [7], and the work of multiple research groups has been devoted to determine the molecular mechanism underlying heart and vascular disease. Recent studies indicate that altered ncRNA expression and function have been strongly implicated in cardiovascular disease such as myocardial infarction, cardiac hypertrophy and coronary artery disease [8–10]. The transcriptome of a cell contains different types of ncRNA that can be divided into two principal classes (Table 1): structural and regulatory ncRNAs. Structural ncRNAs include RNA molecules that are usually constitutively expressed such as ribosomal and transfer RNAs. Regulatory ncRNAs can be classified into three major classes based on transcript size: small (small ncRNAs), medium and long non-coding RNAs (lncRNAs) [6]. The most studied class of small ncRNAs in cardiovascular research is the microRNAs (miRNAs). MiRNAs are endogenous, single-stranded molecules consisting of approximately 20–22 nucleotides that regulate their target genes by reducing mRNA stability and/or translation [11]. Changes in microRNA expression lead to changes in gene function. This dysregulation of miRNA expression appears to play a significant role in the onset and progression of cardiovascular diseases [12]. Despite the progress in defining the role of microRNAs in cardiac and vascular biology, the complex network of ncRNAs and their interaction with different states of cardiovascular development and disease is still unknown. This is related to the multiple diversity of biogenesis, expression and functional properties of different classes of ncRNAs. Among these, the long non-coding RNAs (lncRNAs) are apparently the most numerous and functionally different [13]. LncRNAs are broadly classified as transcripts longer than 200 nucleotides and some of them are preferentially expressed in specific tissues [14]. Thus it is becoming increasingly clear that lncRNAs can regulate numerous molecular mechanisms. Recently, lncRNAs have emerged as new players in cardiovascular development and disease demonstrating potential roles in different cellular processes [15,16]. However, the characteristics and functions of the overwhelming majority of these lncRNAs are currently unknown. Accordingly, the functional characterization of lncRNAs is essential to advance our comprehensive understanding of cellular processes underlying cardiovascular development and disease. In the present review, emerging roles of ncRNAs in cardiovascular pathophysiology are discussed. Particular focus will be on the evaluation of biological roles of microRNAs and lncRNAs in vascular as well as cardiac disorders. Moreover, the focus of this review is to provide an overview of the current state of knowledge of molecular processes implicated in differentiation and cardiovascular development, which are related to the function of ncRNAs.


**Table 1.** Classes of non-coding RNAs (ncRNAs).

### **2. An Overview on the Main Methods to Analyze the ncRNAs Expression**

Each ncRNA has expression levels that are tissue- or stage-specific. In recent years several methods have been developed to study ncRNA expression. A common approach is Real-time PCR, which is employed mainly to analyze microRNA expression levels but can be used also for studies on long ncRNA [17]. Also many approaches based on immunoprecipitation assays have been developed in recent years (e.g., RNA immunoprecipitation or RIP, Cross-linking and immunoprecipitation or CLIP, RNA-chromatin immunoprecipitation or RNA-ChIP) [18–20]. RNA-IP was developed to identify ncRNAs, especially ncRNA, that interact with a specific protein. The basic principal behind all immunoprecipitation approaches is the same. Using a specific antibody it is possible to isolate a ncRNA-protein complex, then a cDNA library is constructed and the ncRNA is sequenced. Unfortunately, for any of these immunoprecipitation-based approaches the results are influenced by the specificity and affinity of the antibodies. Moreover, these methods (Real-time PCR or IP) allow evaluation of the expression of a few specific molecules but do not permit the discovery of new ncRNAs or provide an overview of all ncRNAs. Recently, advances in technology enabled the development of new genome-wide screening methods to study ncRNAs and their targets. Among these the most commonly used are microarray analysis and RNA sequencing. These technologies are very accurate and permit large-scale analysis of ncRNAs. In particular, the microarray [21–23] approach offers various platforms allowing the study of microRNAs and mRNAs targets, although to date there are only a few chips to analyze long non-codingRNA. Analysis with traditional microarrays is limited to detecting the presence or the absence of known ncRNAs and it is incapable of identifying new molecules or revealing different splicing variants. To get around this problem a new approach has been defined: tiling arrays. Unlike traditional microarrays, these platforms permit identification of new ncRNAs in a selected DNA region without prior knowledge of their precise location. For instance, Rinn *et al*. used this approach to study lncRNAs expressed in the region of HOX genes in humans [24]. The RNA sequencing or RNAseq [25] refers to the use of high-throughput sequencing technologies to get information about a sample's RNA content. This approach permits information to be obtained on differential expression of the interest gene, microRNA or long ncRNA. RNAseq is very sensitive in detecting less-abundant transcript and it can reveal alternatively spliced isoforms. Moreover, sequencing the entire transcriptome has been widely used to discover new non coding molecules. However, given the time and the cost related to the downstream analysis of the data generated by RNA sequencing, microarrays remain the first choice in many applications.

#### **3. Functions of Non Coding RNAs**

Although the function of most lncRNAs remains unknown, it has become clear that these molecules are intimately involved in many biological processes. LncRNAs can regulate gene expression programs through a variety of mechanisms, such as epigenetic modifications of DNA, alternative splicing, post-transcriptional gene regulation and mRNA stability and translation [5,26,27]. Given their established roles in transcriptional regulation, lncRNAs play a key role in several cellular events including proliferation, migration, apoptosis and development [21,28]. LncRNAs are now known to regulate the expression of protein-coding genes: they can positively or negatively control the expression of their target genes. Several lncRNAs are involved with *in cis* inactivation of larger genomic regions by epigenetic mechanisms. Kcnq1ot1 is a regulatory non coding antisense RNA that regulates epigenetic gene silencing in an imprinted gene cluster *in cis* [29]. This lncRNA specifically interacts with nearby genes in embryonic tissues causing transcriptional gene silencing. More recently, it was found that lncRNAs can act *in cis* to regulate expression of neighboring genes during cardiomyocyte differentiation [30]. Notably, many lncRNAs are now known to regulate the expression of genes by a *trans* mechanism. One example of a lncRNA that acts *in trans* is AK143260, termed Braveheart (Bvht) that specifically promotes activation of a core gene regulatory network to direct cardiovascular lineage commitment [15]. So far, several other functions have been attributed to lncRNAs. These molecules can act as scaffolds bringing together multiple proteins to form ribonucleoprotein complexes. For example, Miao-Chih Tsai *et al*. showed that a long non coding transcript, termed HOTAIR (HOX Antisense Intergenic RNA), acts as a scaffold for Polycomb Repressive Complex 2 (PRC2) and LSD1/CoREST/REST complex [31]. In addition to their role in chromatin regulation, lncRNAs can also function as molecular "decoys" of transcription factors and other regulatory proteins. PANDA (P21 associated ncRNA DNA damage activated) is an example of a lncRNA with decoy functionality. In fact, PANDA interacts with the transcription factor NF-YA to limit expression of pro-apoptotic genes [32]. Finally, the presence of a complex network of interactions between lncRNAs and miRNAs is becoming increasingly clear. In fact, lncRNAs may exert their biological activity through their ability to act as endogenous decoys for miRNAs. For example, a muscle-specific long noncoding RNA, linc-MD1, could interact with two specific miRNAs, miR-133 and miR-135, and promote muscle differentiation by acting as a competing endogenous RNA (ceRNA) in mouse and human myoblasts [33]. Another lncRNA which has been identified in association with microRNAs is the pseudogene PTENP1 [34]. Similar to Linc-MD1, PTENP1 mRNA acts as a decoy for miRNAs that directly target the tumor suppressor protein PTEN. Accordingly, PTENP1 reduces down-regulation of PTEN messenger RNA. Recent reports also show that stable circular lncRNAs (circRNAs) can act as molecular decoys of microRNAs [35,36]. Taken together, these observations suggest that lncRNAs could have profound effects on several molecular mechanisms. Nevertheless, lncRNAs are poorly conserved among species resulting in an additional degree of complexity in the definition of their functions. Despite rapid progress in lncRNA discovery, evidence of physiologic function for lncRNAs remains poor and further investigation is necessary.

**Figure 1.** Role of non-coding RNAs in Vascular Development and Disease.
