The Expression Profile of MicroRNAs in Small and Large Abdominal Aortic Aneurysms

Background Abdominal aortic aneurysms (AAA) are relatively frequent in elderly population, and their ruptures are related with high mortality rate. There are no actually used laboratory markers predicting the AAA development, course, and rupture. MicroRNAs are small noncoding molecules involved in posttranscriptional gene expression regulation, influencing processes on cell and tissue levels, and are actually in focus due to their potential to become diagnostic or prognostic markers in various diseases. Methods Tissue samples of AAA patients and healthy controls were collected, from which miRNA was isolated. Microarray including the complete panel of 2549 miRNAs was used to find expression miRNA profiles that were analysed in three subgroups: small (N = 10) and large (N = 6) aneurysms and healthy controls (N = 5). Fold changes between expression in aneurysms and normal tissue were calculated including corresponding p values, adjusted to multiple comparisons. Results Six miRNAs were found to be significantly dysregulated in small aneurysms (miR-7158-5p, miR-658, miR-517-5p, miR-122-5p, miR-326, and miR-3180) and 162 in large aneurysms, in comparison with the healthy control. Ten miRNAs in large aneurysms with more than two-fold significant change in expression were identified: miR-23a-3p, miR-24-3p, miR-27a-3p, miR-27b-3p, miR-30d-5p, miR-193a-3p, miR-203a-3p, miR-365a-3p, miR-4291, and miR-3663-3p and all, but the last one was downregulated in aneurysmal walls. Conclusion We confirmed some previously identified miRNAs (miR-23/27/24 family, miR-193a, and miR-30) as associated with AAA pathogenesis. We have found other, yet in AAA unidentified miRNAs (miR-203a, miR-3663, miR-365a, and miR-4291) for further analyses, to investigate more closely their possible role in pathogenesis of aneurysms. If their role in AAA development is proved significant in future, they can become potential markers or treatment targets.


Introduction
Abdominal aortic aneurysm (AAA), the enlargement of abdominal aorta to a diameter of at least 3 cm, is a common disease in the western part of the world, mainly in developed countries. e most common occurrence is in men over 65 years, where the prevalence is around 4-7% [1]. However, a significant percentage of the deaths caused by AAA rupture are also in women [2]. Due to high mortality rate when AAA ruptures (about 70-90%), it poses a serious health and public social problem in a lot of countries [3]. Although our understanding of this issue and its mechanisms is getting better, its complete aetiology is still unclear and we are still missing the crucial trigger in majority of AAAs. ere are actually no prognostic laboratory markers used in clinical practice predicting the AAA behaviour. e identification of molecules involved in the deregulation of gene expression in the process of pathogenesis of AAA could be one option. Published data showed dysregulated expression of micro-RNAs in AAA tissue, and presumed the microRNA can play pivotal role in AAA development [4][5][6]. e positive family AAA history increases the risk of developing the same condition in relatives and indicates substantial portion of the genetic component. e heritability of AAA is over 0.7, and first-degree relatives of a patient with AAA have a 2-fold higher risk of developing an aneurysm as well [7]. Gene variants, found in genome-wide association studies (GWAS) associated with increased AAA risk, are usually not located in the coding regions and/or do not necessarily represent the causal ones. ese gene variants may rather be in linkage disequilibrium with the causal alleles contributing to AAA formation, being located close to them [8]. Up to day, some risk loci on a few chromosomes were identified, but these explain only a small proportion of the heritability of AAA [7]. Positions of these genetic variants in noncoding regions suggest that they probably more often influence gene regulation than the protein structure [8]. e regulation of gene expression can be influenced in many ways including microRNAs (miRNAs). miRNAs are short noncoding RNAs, first found and described at the very end of the last century, that are recently in focus of research due to their potential to become useful diagnostic and/or prognostic markers in a large variety of diseases. miRNAs are transcribed as long primary transcripts (pri-miRNAs) that are partly processed in the nucleus (pre-miRNAs) and finally in the cytoplasm. Mature miRNAs, about 20 nucleotides in length, associate with Argonaute family protein members and form RISCs (RNAinduced silencing complexes). RISCs interact with proteincoding mRNAs and inhibit their translation, or destabilize their molecules that are degraded and so decrease the levels of proteins coded by target mRNAs. On the other hand, in some cases miRNAs can also activate translation of target genes [9]. One miRNA can interact with many mRNAs, and, vice versa, one particular mRNA can be regulated by many different miRNAs. miRNAs can regulate gene expression either in the cell in which they were synthetized, or in other neighbouring or more distant cells, as they can be exported into circulation in the form of membrane-bound vesicles (exosomes and microvesicles), or in association with protein complexes [10].
A few thousands of miRNAs have been identified, over 2500 in the human genome. Some miRNAs, or significant changes in their levels, are associated with particular diseases, their stages, or acute events, and so became potential diagnostic or prognostic markers, for example, increased levels of miR-208a, miR-499, miR-1, and miR-133 in plasma are associated with myocardial damage [10]. e levels of miRNAs can be measured either in cells/tissue, or in circulation (serum, plasma, and blood), or other body fluids.
In this study, we aimed to evaluate the changes in 2533 miRNAs expression in small and large aneurysms compared to the normal vessel wall.

Patients.
Our study included ten patients with small AAA (maximum diameter ≤ 5 cm), six patients with large AAA (>5 cm), and finally five "healthy controls" who were cadaveric organ donors. Only patients indicated for the AAA resection were included, either due to the size of AAA, or symptoms present. Patients with oncological diagnoses, autoimmune diseases, and mycotic or inflammatory aneurysms were excluded. Control aortic tissue samples were from the dead brain and heart-beating donors (without warm ischemia or any other tissue injury) and were collected at the same time as the kidneys were harvested and immediately frozen. All patients with AAA have signed an informed consent; cadaveric donors have been used based on the principle of presumed consent (approved by the local Ethical committee; decision from the 12 th of August 2014).

Tissue Samples.
Tissue samples were collected within the aortic surgery. AAA samples during the open aneurysm resection, approximately 1 cm 3 tissue sample from the anterior aneurysmal wall; control group samples from cadaveric donors, from the same region of aorta, were collected. Samples were immediately washed with physiological saline solution, cleaned from intraluminal thrombus, frozen with liquid nitrogen, and stored in the freezer (− 80 degrees of centigrade).

miRNA Isolation.
Sample aliquots (approximately 0.125 cm 3 ) were frozen in liquid nitrogen and grinded to prepare homogeneous powder that was transferred into 1 ml of chilled TRI Reagent ® RT (Molecular Research Center, Inc., Cincinnati, USA). Total RNA was isolated according to manufacturer's protocol (Manual Part Number: G4170-90011, Version 3.1.1, August 2015). Isolated RNA was dissolved in RNAse/DNAse-free water ( ermo Fisher Scientific Inc., Waltham, USA), and its concentration and purity were assessed spectrophotometrically using the Infinite M200 instrument (Tecan Trading AG, Männedorf, Switzerland). e RNA sample was stored at − 80°C until further use.
2.4. miRNA Array. RNA samples were prepared for hybridization using miRNA Complete Labelling and Hybridization Kit (Agilent, Santa Clara, USA), following manufacturer's protocol, without optional spike-in control and purification of labelled RNA. Samples were randomized and hybridized for 20 hours on SurePrint G3 Unrestricted miRNA arrays with 8 × 60 K fields and Amadid 070156 (Agilent, Santa Clara, USA).

Data Analysis.
After hybridization, microarrays were scanned using the Agilent G 4900DA scanner, and the resulting image was processed using Agilent Feature Extractor software version 11.5.1.1. Resulting data were processed using R version 3.5.1 with limma package version 3.36.3. e weights for imported probes showing saturation, nonuniformity, or significantly higher signal over background were set to zero. e background was then corrected using the normexp method with offset set to 50. e "cyclicloess" with "affy" method was used for normalization to allow comparison of samples between arrays. Probes annotated with systematic name of detected miRNA were considered as within array replicates and combined using "avereps function" from limma package version 3.36.3 to obtain the expression of particular miRNA. Fold changes between aneurysms and normal tissue were then calculated as well as corresponding p values. Benjamini & Hochberg method was used for adjustment of p values to multiple comparisons.

Baseline Characteristics of Patients.
Samples of 21 patients were analysed. Six were obtained from large aneurysms, ten from small aneurysms, and five from healthy controls. e baseline characteristics of patients are given in Table 1.

miRNAs Dysregulated in Small
AAA. miRNAs expression in small and large aneurysm tissue was compared with healthy tissue. In small aneurysms, six miRNAs were detected with significantly different expression in comparison with the healthy aortic wall, including miR-7158-5p, miR-658, miR-517-5p, miR-122-5p, miR-326, and miR-3180 ( Figure 1(a)). All of them were also significantly changed in large aneurysms. All but miR-326 were downregulated in the AAA tissue. In both large and small aneurysms the difference between the aneurysmal and healthy aortic wall was less than two-fold, and both had the same direction. Interestingly, fold changes in small and large aneurysms against healthy tissue were significantly correlated (Pearson's coefficient � 0.9521; p value � 0.0034).

Discussion
Based on miRNAs expression profiles, we have proved our assumption that some miRNAs are differently expressed in small and/or large AAA as compared to healthy tissue. In this analysis, we found six miRNAs dysregulated in small aneurysms in comparison with healthy controls (miR-7158-5p, miR-658, miR-517-5p, miR-122-5p, miR-3180 downregulated, and miR-326 upregulated). Twenty-seven times more miRNAs were found to be differentially expressed in large aneurysms, suggesting increasing miRNA levels dysregulation related to the disease progression. Despite higher number of small aneurysms than the large ones (10 vs. 6), we were unable to detect any other significantly changed miRNAs in comparison with healthy tissue, when correction for multiple comparison was applied.
is suggests that small aneurysms are relatively close to the normal aortic wall regarding miRNAs expression.
is miR-24 also affects smooth muscle cells proliferation, function, and apoptosis and inhibits proliferation, migration, and sprouting of human umbilical venous endothelial cells (HUVECs) [14]. Derived from the abovementioned facts, decreased levels of miR-23 attenuate its proangiogenic effect and downregulation of miR-27 and miR-24 results in the cell growth, angiogenesis, and loss of contractile phenotype. Maegdefessel et al. studied miR-24 in aneurysmal tissue and identified it as a key regulator of vascular inflammation and AAA pathology [15]. miR-24 was found to be colocalized with activated macrophages in aneurysmal aortic mouse tissue and visualized its expression in the aneurysm intimal-medial region. miR-24 downregulation was proinflammatory in macrophages [10]. e authors found miR-24 downregulated in both animal model and human AAA tissue (− 1.9 ± 0.09-fold) versus control, and they found no significant differences between the small (52-67 mm; n � 12) and large AAA (69-115 mm, n � 10) but suggested a trend towards lower miR-24 expression with larger AAA. is downregulation is in good agreement with our findings in large aneurysms, as our subgroup of "large" AAA (diameter over 50 mm) involves both large and small Maegdefessel's groups. In addition, miR-27b was found to be downregulated in AAA in their study [15], which is in line with our results. Furthermore, levels of both miR-24 and miR-23b were significantly lower also in the intracranial aneurysmal tissue than in the healthy controls [16]. Our findings support the hypothesis that the downregulation of miRNA-23/27/24 family dysregulates angiogenesis, smooths muscle cells proliferation, function, and apoptosis, and together with their proinflammatory effect can contribute to the AAA development/progression.
In the cardiovascular system, the downregulation of miR-30 family contributes to endoplasmic reticulum (ER) stress and is associated with the upregulation of glucoseregulated protein 78 (GRP78) [19]. ER stress plays fundamental roles in the development and progression of various cardiovascular diseases such as ischemic heart diseases, heart failure, atherosclerosis, hypertension, and stroke [19]. Artificial  knockdown of miR-30 triggered the phenotypic ER stress with significant GRP78/ATF6/CHOP/caspase-12 upregulations and cell death in rat cells [19]. In patients with acute coronary syndrome were found higher levels of circulating miR-30d-5p, in comparison with healthy controls (p < 0.001). e authors found this miRNA suitable as a diagnostic predictor of myocardial infarction [20]. In this context, the downregulation of miR-30 family members in our AAA samples could indicate an increased ER stress, dysregulation of endothelial cell growth, and increased cell death.

miR-203a.
Levels of miR-203a were lower in aneurysmal tissue. is miRNA is dysregulated in chronic inflammatory diseases that suggest its involvement in immune-mediated diseases [26]. miR-203 is highly expressed in skin (and keratinocytes) and oesophagus and overexpressed in patients with psoriasis in comparison with healthy controls [27]. One of its targets is suppressor of cytokine signalling-3 (SOCS-3), negative regulator of the STAT3 pathway that is activated by inflammatory cytokines. SOCS-3 is involved in cell growth, differentiation, and survival and in the regulation of immunity [27]. SOCS-3, a target of miR-203a, was shown to play a critical role in AAA development and dissection [28,29]. e long-term suppression of SOCS-3 by increased levels of miR-203 may lead to stronger or prolonged inflammation [26]. As the inflammation is one of the key players in aneurysmal pathology [18], dysregulation of miRNAs influencing this process could contribute to the aneurysm formation.

miR-3663 and miR-4291.
e expression of miR-3663 was upregulated, and the expression of miR-4291 was downregulated in the analysed aneurysmal tissue. Little is known about miR-3663. miR-3663-3p was identified as downregulated in tissue samples of patients with degenerative aortic stenosis, and miR-193-3p as overexpressed in comparison with heathy subjects [30]. is information is just opposite to our findings in aortic dilated tissue of AAA patients. We have not found any published specific functions associated with hsa-miR-4291. e majority of miRNAs, we found dysregulated in the tissue of AAA patients, are potentially related to the aneurysmal pathology, being either involved in endothelial cell or smooth muscle cell metabolism and survival, or angiogenesis, or inflammation, or atherosclerosis ( Table 3). Some of them (miR-24, miR-27b, and miR-193a) were identified in previously published literature as downregulated in aneurysmal tissue, or in plasma of patients with aortic aneurysms [15,22,23], what is in line with our findings. In addition to these three miRNAs, we identified miR-23a, miR-27a, miR-30d, miR-365a, miR-203a, and miR-4291 to be downregulated and miR-3663 upregulated.
In our analysis, a complete panel of 2549 miRNAs was tested. We found 6 miRNAs dysregulated in small and 162 miRNAs in large aneurysms in comparison with healthy controls. e number of samples in particular groups was limited, and therefore microarray results were not verified using qPCR. To compensate this main limitation of this study, the Benjamini and Hochberg method Table 3: miRNAs with more than two-fold significant change in expression in large aneurysm tissue, vascular biology processes in which they are involved, and their target genes.

Conclusion
In our analysis, we tested the complete panel of 2549 miRNAs and we confirmed some previously identified miRNAs (miR-23/27/24 family, miR-193a, and miR-30) as associated with AAA pathogenesis. In addition, we identified other potential miRNA candidates not yet found dysregulated in AAA tissue (miR-203a, miR-3663, miR-365a, and miR-4291) for further analyses, to investigate more closely their possible role in pathogenesis of aneurysms. If their role in AAA development is proved significant in future, they can become potential markers or treatment targets.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.