MicroRNA Expression Signature in Human Calcific Aortic Valve Disease

Altered microRNA (miRNA, miR) expression has been related to many disease processes; however, the miRNA expression signature in calcific aortic valve disease (CAVD) is unclear. In this study, microarrays were used to determine the miRNA expression signature of tissue samples from healthy individuals (n = 4) and patients with CAVD (n = 4). TargetScan, PITA, and microRNAorg 3-way databases were used to predict the potential target genes. DIANA-miRPath was used to incorporate the aberrant miRNAs into gene pathways. miRNA microarrays identified 92 differentially expressed miRNAs in CAVD tissues. The principal component analysis (PCA) of these samples and the unsupervised hierarchical clustering analysis based on the 92 aberrantly expressed miRNAs noted that miRNA expression could be categorized into two well-defined clusters that corresponded to healthy control and CAVD. Bioinformatic analysis showed the miRNA targets and potential molecular pathways. Collectively, our study reported the miRNA expression signature in CAVD and may provide potential therapeutic targets for CAVD.


Background
Valve diseases continue to occur in many patients with significant morbidity and mortality. The age-adjusted prevalence of moderate or severe valve diseases was estimated at 2.5% [1]. Calcific aortic valve disease (CAVD) is the most common valve heart disease in the elderly and a leading cause of aortic stenosis [2]. In developing countries, CAVD represents a major cause for surgical valve replacement [3]. As a result of rising life expectancy and ageing populations, the burden of CAVD will significantly increase in the near future.
While CAVD was originally thought to be a degenerative process with passive deposition of calcium phosphate in the valve occurring with age, it now appears to be a complex and actively regulated progress mediated by inflammation, cell apoptosis, lipid deposition, renin-angiotensin system activation, extracellular matrix remodeling, and bone formation [4][5][6]. To better monitor progression of CAVD and identify the most appropriate instances for surgical intervention, biomarkers can be serially monitored. Such biomarkers would represent objective laboratory measurements, as older patients with CAVD might have atypical symptoms associated with comorbidities such as pulmonary disease or orthopaedic disabilities [7,8].
MicroRNAs (miRNAs, miRs) are endogenous, small, single-stranded, 21-25 nucleotide noncoding RNAs, regulating target gene expressions by hybridizing to messenger RNAs (mRNAs). An individual miRNA is able to target tens to hundreds of genes while a single gene can also be targeted by lots of miRNAs [9]. Since miRNAs play critical roles in many physiological processes, increasing reports indicate that a distinct pattern of altered miRNA expressions may be linked to specific disease processes [10][11][12][13][14][15]. We previously reported the miRNA expression signature in degenerative aortic stenosis [14]. In this study, we explored the miRNA expression signature in CAVD.

Tissue Sample Collection and RNA Isolation.
This study was officially approved by the Ethics Committees of the First Affiliated Hospital of Nanjing Medical University and conformed to the principles outlined in the Declaration of Helsinki. All written informed consent was obtained 2 BioMed Research International from patients, and parents where applicable. Tissue samples from four healthy subjects were collected from prospective multiorgan donors in cases because of technical reasons that prevented transplantation, while stenotic aortic valve samples were obtained from four patients who underwent surgical valve replacement for aortic stenosis. All samples were examined by gross examination, and microscopic examination of hematoxylin and eosin-stained cryosections was conducted to confirm the presence/absence of CAVD. Tissue samples harvested from subject donors were snap-frozen in liquid nitrogen, and RNA was then isolated using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions.

miRNA Microarray Analysis.
Total RNAs were isolated from heart tissues using mirVana6 RNA Isolation Kit, quantified by NanoDrop ND-2100 (Thermo Scientific), and controlled for RNA integrity using Agilent Bioanalyzer 2100 (Agilent Technologies) according to the manufacturer's instructions. miRNA profiling was performed with OE Biotech's (Shanghai, China) miRNA microarray service. The arrays from the control group are the same as we previously used [14].

Bioinformatic Analysis.
TargetScan, PITA, and microR-NAorg 3-way databases were used to identify potential human miRNA target genes and a Venn diagram was made to provide relations among the 3 databases. DIANA tool miRPath v2.0, a web-based analysis tool, was used for pathway enrichment analysis for the miRNA set identified [16]. DIANA tool miRPath assigns Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway with the significance level determined by the number of target genes affected by the identified microRNAs.

Statistical
Analysis. Independent Student's -test was used to determine whether there were any significant differences between the miRNA expression profiles between two groups. values less than 0.05 ( < 0.05) were considered to be statistically significant. Significant data were further analyzed by clustering, and the expression profiles were visualized with GeneSpring 10.0 (Agilent Technology).

Principal Component Analysis of miRNA Expression Profiles.
Principal component analysis (PCA) is a mathematical algorithm that reduces the dimensionality of the data while retaining most of the variation in the data set [17]. Dots in two colors separated in two axes based on the differences of the data, suggesting that samples in this study were prepared appropriately and could be grouped as CAVD or healthy control ( Figure 1).

Unsupervised Hierarchical Cluster Analysis of miRNA
Microarray Data. miRNA arrays identified 92 miRNAs with a statistically significant differential expression of 2.0-fold or greater in CAVD samples relative to normal controls. Fifty-three miRNAs were underexpressed and 39 were overexpressed in aortic tissue from CAVD patients ( Table 1). Unsupervised hierarchic clustering of the two groups was performed on the 92 differently expressed miRNAs and displayed as heatmap ( Figure 2).

Target Genes Analysis.
MicroRNAorg, TargetScan, and PITA were used to predict the targets of differentially expressed miRNAs in CAVD samples. A Venn diagram was made to highlight the relations among the three databases.
There are 8717 genes overlapping by all three sets, which are most likely to be targets of miRNAs in patients with CAVD ( Figure 3).

Discussion
miRNAs have been shown to be critical regulators in cardiovascular diseases [18][19][20][21][22][23][24][25]. However, there are no reports revealing distinct miRNA expression signatures in the CAVD patients and healthy controls. In this study, we identified global changes in the miRNA expression profile in CAVD and healthy control. Calcific aortic valve stenosis is characterized by lipid accumulation, inflammation, formation  [4,26]. Previous studies have shown that miRNAs play crucial roles in those processes such as angiogenesis, fibrogenesis, proliferation, and apoptosis [9]. miR-126 is one of the most abundantly expressed microR-NAs in endothelial cells (ECs) [27]. Upregulation of miR-126 increases EC survival, decreases EC apoptosis, and prevents reactive oxygen species (ROS) mediated endothelial damage  Specific types of cancers and infections were not included. [28]. Our findings of decreased miR-126 in CAVD may suggest a detrimental effect in human calcific aortic valve. The differentially expressed miRNAs identified in the current study also included many profibrotic miRNAs such as miR-21 and miR-125b that might contribute to CAVD by promoting fibrosis. Several expression profiling studies identify that increased level of miR-21-5p in cardiac fibroblasts promotes cardiac fibrosis via its target genes: phosphatase and tensin homolog (PTEN) [29] and Sprouty-1 (Spry1) [30]. Additionally, miR-125b is a novel regulator of cardiac fibrogenesis, proliferation, and fibroblast-to-myofibroblast transition. Nagpal et al. demonstrated the upregulation of miR-125b in fibrotic human heart and murine models of cardiac fibrosis [31].
The abnormal expression of miR-21-5p was found in many cardiovascular diseases [33]. Programmed cell death 4 (PDCD4) is identified as a direct target gene of miR-21-5p. It has been reported that miR-21-5p prevented cardiomyocyte apoptosis in ischaemia/reperfusion heart model through PDCD4 repression [34]. Furthermore, miR-21/PDCD4 pathway was proved to be involved in cardiac valvulogenesis by regulating endothelial cell migration [35]. In our work, miR-21-5p was upregulated in calcific aortic valve which indicates −3.00 −2.00 −1.00 0.00 1.00 2.00 3.00 Con-1 Con-3 Con-2 Con-4 CAVD-2 CAVD-1 CAVD-3 CAVD-4 that miR-21-5p might take part in CAVD. However, the effects and mechanisms of miR-21-5p on calcific aortic valve are still to be investigated in further studies. A comprehensive knowledge of miRNA expression is essential to improve our understanding of this disease. This study provides the first evidence that there exists a distinct miRNA expression signature in individuals with CAVD, as compared to healthy controls. There are 92 differently expressed miRNAs in the CAVD patients compared with healthy controls by miRNA arrays. PCA and unsupervised hierarchical clustering with these miRNAs demonstrates that this profile could accurately classify the samples according to their disease status. Moreover, bioinformatic tools indicate that the differential expression of miRNAs could be linked to several targets and pathways.
As a limitation of our study, the exact pathways by which dysregulated miRNAs cause CAVD in human remain elusive. Further studies are required to fully characterize the function of candidate miRNAs.

Conclusions
Taken together, the current study provides insight into the importance of microRNA expression signature in CAVD. A deeper understanding of the molecular alternations in CAVD may provide potential targets for future clinical applications.

Conflicts of Interest
The authors have declared that no conflicts of interest exist.