MicroRNA as predictive and informative diagnostic tools and promising drug targets to monitor chronic cardiac disorders and to manage the patients and persons-at-risks

Ivkin Dmitriy Yu.; Lisitskiy Dmitriy S.; Chistyakova Elizaveta Yu.; Galagudza Mikhail M.; Zakharov Evgeniy A.; Karpov Andrey A; Okovity Sergey V.; Suchkov Sergey V.; Tyukavin Alexnder I.

ОРИГИНАЛЬНЫЕ СТАТЬИ | ORIGINAL PAPERS

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МИКРОРНК КАК ПРЕДИКТИВНый ДИАГНОСТИЧЕСКИй ИНСТРУМЕНТ

и многообещающая мишень для лекарственных средств

в лечении хронических сердечных расстройств

для использования у пациентов и лиц из группы риска

© Дмитрий Юрьевич Ивкин1, 3, Дмитрий Сергеевич Лисицкий1, 3 4, Елизавета Юрьевна Чистякова1, Михаил Михайлович Галагудза3, Евгений Александрович Захаров1, 3, Андрей Александрович Карпов2, 3, Сергей Владимирович Оковитый1, Сергей Владимирович Сучков5, Александр Иванович Тюкавин1, 3

1 ГБОУ ВПО «Санкт-Петербургская государственная химико-фармацевтическая академия» Минздрава России. 197376, Россия, Санкт-Петербург, ул. проф. Попова, 14

2 ФГБОУ ВО «Первый Санкт-Петербургский государственный медицинский университет имени академика И.П. Павлова» Минздрава России. 197022, Россия, г. Санкт-Петербург, ул. Льва Толстого, д. 6-8

3 ФГБУ «СЗФМИЦ им. В.А. Алмазова» Минздрава России. 197341, Санкт-Петербург, ул. Аккуратова, д. 2

4 Федеральное государственное бюджетное учреждение науки «Институт токсикологии Федерального медико-биологического агентства». 192019, Россия, г. Санкт-Петербург, Бехтерева ул., д. 1.

5 ФГБОУ ВО Первый МГМУ им. И.М. Сеченова Минздрава России. 194223, г. Санкт-Петербург, пр. Тореза, д. 44.

Контактная информация: Дмитрий Юрьевич Ивкин — к.б.н., доцент, доцент кафедры фармакологии и клинической фармакологии ГБОУ ВПО СПХФА, с.н.с. НИЛ системного кровообращения ИЭМ СЗФМИЦ им. В.А. Алмазова. E-mail: [email protected]

РЕЗЮМЕ. МикроРНК относятся к классу небольших некодирующих РНК, которые играют важную роль в регуляции экспрессии генов. При помощи микроРНК можно вызвать разнообразные эффекты в тканях и органах. Исследования последних лет показали, что разные формы микроРНК, возможно, могут быть использованы в лечении и при диагностике различных заболеваний. Широко обсуждаются принципы применения микроРНК в таргетной терапии. Другой важный аспект их применения включает методы доставки в клетку как аналогов, так и ингибиторов эндогенных микроРНК. Настоящий обзор фокусируется на роли микроРНК в патогенезе разных типов патологии. Показаны перспективы и способы возможного применения разнообразных микроРНК в качестве диагностического или фармакологического агента в особенности в отношении лечения заболеваний сердечно-сосудистой системы.

КЛЮЧЕВЫЕ СЛОВА: микроРНК; сердечно-сосудистые заболевания; таргетная терапия; диагностические агенты .

MICRORNA AS PREDICTIVE AND INFORMATIVE DIAGNOSTIC TOOLS AND PROMISING DRuG TARGETS TO MONITOR CHRONIC CARDIAC DISORDERS AND TO MANAGE THE PATIENTS AND PERSONS-AT-RISKS

© Dmitriy Yu. Ivkin1, 3, Dmitriy S. Lisitskiy1, 3, 4, Elizaveta Yu. Chistyakova1, Mikhail M. Galagudza3, Evgeniy A. Zakharov1, 3, Andrey A. Karpov2, 3, Sergey V. Okovity1, Sergey V. Suchkov5, Alexnder I. Tyukavin1, 3

1 Saint-Petersburg Chemical Pharmaceutical Academy. Prof. Popova str. 14. Saint-Petersburg, Russia, 197376

2 First Pavlov State Medical University of St. Petersburg. L'va Tolstogo str. 6/8, Saint Petersburg, Russia, 197022

3 Federal Almazov Medical Research Centre. 2 Akkuratova str., Saint-Petersburg, Russia, 197341

4 Federal State Scientific Institution «Institute of Toxicology» Federal Medico-Biological Agency. Bechtereva str. 1, Saint-Petersburg, Russia, 192019

5 I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry of the Russian Academy of Sciences. Thorez Ave., 44, Saint-Petersburg, Russia, 194223.

Contact Information: Dmitry Yu. Ivkin — PhD, Professor, Department of Pharmacology and Clinical Pharmacology SPCPA. Junior Researcher at the Laboratory of systemic circulation of Federal Almazov Medical Research Centre. E-mail: [email protected]

ABSTRACT: MicroRNAs are a class of small noncoding RNAs that are important regulators of gene expression. Different effects in organs and tissues can be obtained using miRNAs. Recent studies have shown that different miRNA forms can be possibly used in therapy as well as in diagnosis of various diseases. The principles of application of miRNA in target therapy are now widely discussed. Another important question includes methods of delivery of inhibitors and analogs of endogenous miRNAs in cell. This review focuses on the role of miRNAs in the pathogenesis of various types of pathology. Prospects and possible ways of applying different miRNAs as diagnostic and pharmacological agents are shown, particularly relevant in the treatment of diseases of the cardiovascular system.

KEY WORDS: microRNA; cardiovascular disease; target therapy; diagnostic agents.

introduction

MicroRNAs is a class of small non-coding single-stranded endogenous RNA molecules (containing about 20-30 nucleotides) which are important components of the programmable regulation of the metabolic processes of eukaryotic cells [58, 61]. MiRNA functions in biological regulation of gene expression by modulation of the translation process (inhibition or stimulation) and ultimately leads to a decrease of the protein product of the gene [34].

The first miRNA lin-4, which leads to a violation of metamorphosis of nematode Caenorhabditis elegans, was discovered in 1993 by scientists at Harvard University under the guidance of V. Ambros [16]. The second miRNA let-7 with the opposite effect of metamorphosis process stimulation was discovered 7 years later. Ever since it was discovered more than a thousand microRNAs, including human miRNAs (Tab. 1).

MICRORNA BIOSYNTHESIS

MiRNA molecules form hairpin structures which are processed to form small RNAs (containing 21-23 nucleotides) that block the synthesis of certain proteins on the RNA level, mainly through the inhibition of translation of sequences homologous sequences of messenger RNA (mRNA) [3] (Fig. 1).

On the one hand, each microRNA can potentially serve as a regulator of expression of several hundreds of mRNA, on the other hand, the expression of a single gene can be controlled by several miRNAs. Thus, microRNAs represent a new level of coordinated gene expression, supporting the action of protein

transcription factors, and the regulation by microRNAs is fast and reversible [30].

It was shown that miRNAs are involved in the regulation of important cellular processes such as differentiation, proliferation, apoptosis and response to stress. Often, several components of regulatory pathways responsible for homeostasis of a cell are controlled by the particular microRNA, thus disturbance of expression of microRNAs leads to dysregulation of the whole signal network and cell dysfunction. It is found that the dysfunction of certain microRNAs is associated with malignant transformation of cells, appearance of neurological diseases and various cardiovascular diseases [15, 52].

Numerous recent studies produce evidence of potentially important role that miRNAs may play in the pathogenesis of heart failure and myocardial hypertrophy [45, 55]. Differential expression of several miRNAs was observed in normal tissue compared to cardiomyopathy [42], and changes in the expression of miRNAs — after unloading of ventricles of heart by means of mechanical auxiliary devices [36, 50].

Since each miRNA may be associated with a large number of unidirectional processes the potential controllability of such signals is likely for pathological phenotype reversibility [56]. In fact, they may represent potential targets for therapy in order to delay or reverse the development of cardiac remodeling or fibrosis [43].

The possibility of different miRNA forms application by expression or knock-down for the therapy of different types of pathology as well as in the diagnosis of diseases is now widely discussed [31, 32].

Table 1

Databases

miRanda PicTar-4way Pic Tar-5way Target ScanS

Number of miRNAs 470 179 131 139

Number of target genes 15 274 9152 3455 7709

Number of binding sites 284714 154 894 28 870 22 837

Average number of target genes per miRNA 32,5 51,1 26,4 55,5

Average number of binding sites per miRNA 606 865 220 164

Average number of binding sites per gene 18,6 16,9 8,3 3,0

Interspecies conservation 2 species 4 species 5 species 5 species

Statistics for target prediction methods in humans [47]

target mRNA

Fig. 1. Biosynthesis of microRNA (dimming marks noncanonical pathways) (S. deRosa, 2014)

DIAGNOSIS OF CARDIOVASCuLAR DISEASES

Early life-time diagnosis of ischemic myocardial injury is carried out using biochemical tests with determination of troponin, myoglobin, and CPK-MB.

Most often in clinical practice and preclinical trials of pharmacological agents the diagnosis of myocardial injury is carried out using cardiac troponin I and T, structural proteins of cardiomyocytes which were discovered in the second half of the 20th century; moreover, determination of the former is considered to be more sensitive. The advantages of troponin test include the possibility of differentiation of unstable angina and myocardial infarction without ST-segment elevation. The disadvantages of troponin determination include low sensitivity in the first 6 hours after acute coronary syndrome (latency), possibility of false-positive result because of insufficient specificity of the method. Troponin levels may increase in case of tachyarrhythmia, bradyarrhythmia, hypertensive crisis, severe pulmonary hypertension, myocarditis, aortic aneurysm, myocardial contusion, as well as stroke, hypothyroidism, kidney failure, toxic effects, burns, sepsis and other critical conditions.

Specificity of the CPK-MB test is significantly reduced in case of skeletal muscle damage. Concentration of myoglobin which is the earliest but non-specific marker of myocardial necrosis increases rapidly and also rapidly decreases due to intense renal excretion. Evaluation microRNA can be a promising alternative to the triad of «myoglobin — troponin — CPK-MB».

DIAGNOSTIC POTENTIAL OF MICRORNAS

MicroRNAs are important physiological regulators of post-transcriptional gene expression. MicroRNAs not only reside in the cytoplasm but are also stably present in several extracellular compartments, including the circulation. MicroRNAs are molecules increasingly investigated for both diagnostic and therapeutic strategies and are being now considered to be potential biomarkers of cardiovascular diseases [39]. Those molecules are demonstrated to be present in blood of humans and have been increasingly suggested as a novel biomarker for various pathological processes in the heart, including myocardial infarction (MI), myocardial remodeling and progression to heart failure. Their diagnostic value has been examined in numerous studies and animal models with respect to, for instance, coronary artery disease (CAD) and myocardial infarction (MI). And signatures consisting of sets of multiple miRNAs seem to improve the predictive power compared to single miRNAs. For instance, pre-early and even subclinical diagnosis of acute coronary syndrome (ACS), especially non-ST elevated MI, is essential for optimal treatment outcome, and due to the ongoing need for additional identifiers, miRNAs are of special interest as biomarkers for ACS [25].

Moreover, various miRNA combinations have been proven to be useful biomarkers for HF, and also in the differentiation of

HFpEF from HFrEF. As a whole entity, miRNA biomarkers may support diagnostic, predictive and monitoring strategies in subpopulations of patients with HF.

Meanwhile, whilst the biomarker value of microRNAs for MI or any sorts of heart failure has been extensively studied, less attention has been devoted to their prognostic value after cardiac arrest. Moreover, technological platforms and armamentarium used for miRNA detection still lacks ways of standardization and need to be considered carefully.

MiRNAs can be used as early predictors for diagnosis of cardiovascular diseases that is determined by tissue-specific expression profile and output of nucleotide sequences out of cells into body fluids including blood. As cardiac miRNAs were observed 1, 133a, 133b, 208a, 208b, 499. MicroRNAs 1 and 133 proved to nonspecific, their expression was observed in case of skeletal muscle damage. MicroRNAs 208a, 499 were determined as the myocardium-specific markers [65].

Thereby, advanced method for determination of myocar-dial injury organospecificity is the evaluation of microRNA 208a and 499, or the determination of their pool with the possibility of death risk prediction [11-13]. It is obvious that the definition of miRNAs may be one of the most promising directions in the failure diagnosis of ischemia-reperfusion and this gives promise that the screening of these molecules will be implemented in experimental and clinical practice in the near term.

Methods of miRNA detection include determination in fixed tissues by hybridization and living visualization using oligonucle-otide probes and PCR techniques (Fig. 2) [4, 12].

MIRNAS AS TARGETS IN therapy of cardiovascular DISEASES

At the present time there have been described a number of miRNAs, affecting which we can obtain different effects in organs and tissues of cardiovascular system (apoptotic and an-tiapoptotic, arrhythmogenic and antiarrhythmic, ischemic and anti-ischemic etc.) [20]. Thus, human hsa-microRNA-1 is involved in forming of liability to diseases of cardiovascular system (negatively regulates the expression of genes of calmodulin and Mef2a associated with hypertrophy). As possible mechanisms of its influence are considered: negative regulation of expression of genes CALM and Mef2a associated with hypertrophy; contribution to the re-expression of channel sinus genes HCN2 and HCN4; regulation of arrhythmogenic potential by influence on GJA1 and KCNJ2; posttranscriptional repression of HSP60, HSP70 affecting apoptosis; regulation of content Hand2 during cardiomyogenesis [2, 46].

It has been proved that in addition to the above mentioned genes there are another targets for hsa-microRNA-1: mRNA for genes Hand2, HDAC4, TMSB4X, KCNJ2. Thus, Hand2 gene product is a transcription factor determining the expansion of the cardiomyocytes within the ventricles during the formation of heart, and hsa-miR-1 «titrates» its effect on cardiac critical regulatory proteins that control the balance between differentiation and proliferation during cardiomyogene-

Technology "PCR chip"

9 10 11 12

A 01 02 03 04 05 06 07 08 09 10 11 12

В 13 14 15 16 17 18 19 20 21 22 23 24

С 25 26 27 28 29 30 31 32 33 34 35 36

0 37 38 39 40 41 42 43 44 45 46 47 48

E 49 50 51 52 53 54 55 56 57 58 59 60

F 61 62 63 64 65 66 67 68 69 70 71 72

G 73 74 75 76 77 78 79 80 81 82 83 84

™RTC m ¡RTC PPC PPC

Analysis of expression 84 types of miRNA at one time

C. elegans miR-39 miScript Primer Assay

snoRNA/snRNA miScript PCR Controls

Reverse Positive transcription PCR control control

Scheme of PCR reaction in PCR chip (QIGEN manual)

Fig. 2. using the PCR technique for determination of miRNA

sis [48, 66]. In accordance with modern ideas about the effect of the same microRNA on the level of translation of a number of proteins, it is believed that hsa-microRNA-1 is involved in the regulation of expression of the complex of genes whose products are responsible for the development and functioning of the heart [44].

The regulatory potential of this class of molecules can be substantially higher. According to the information contained in different databases (miRanda [22], TargetScan [24], Pictar [23]) for miRNA hsa-miR-1 potential targets may be hundreds of genes (e. g., in database miRanda 901 genes are considered as targets for hsa-miR-1).

Relatively small sample of these genes is shown in Table. 2. It demonstrates a broad spectrum of physiological processes in which given miRNA can be involved. In database TargetScan 584 genes are indicated as potential targets for hsa-miR-1, which contain 636 conservative and 136 slightly conservative binding sites.

uSE OF MIRNA AS A TARGET FOR THERAPY OF DIFFERENT KINDS OF DISEASES

A study of the functional role of miRNAs in the regulation of cellular processes in conjunction with the analysis of data on changes in levels of microRNAs in the development of the disease allows predicting specific microRNA expression damage of which may be a factor in the development of pathological processes [60].

The potential use of modified synthetic oligonucleotides for the treatment and diagnosis of miRNA-dependent states is intensively studied at the present time. A number of properties of microRNAs make them prospective candidates for targets for new drugs. Firstly, a small size and known conserved miRNA sequence. Secondly, under the control of a particular miRNA is typically several functionally related genes. Therefore, the impact on a particular miRNA leads to coordinated changes in several components of the signal pathway, blocking of compensatory mechanisms and as a result, may have a strong regula-

Table 2

Various potential miRNA hsa-miR-1 targets and their functions («MicroCosm Targets» Database, The European Bioinformatics Institute [21, 22])

Process/function/localization Target genes

Processes

Regulation of growth IHPK2

Metabolism PPIB, SFRS9, GTF2B, MON2, DDX5, SPTLC1, CEBPZ, MGAT4A, ERH, DUPS12, MRPS33, ORC6L, TGM3

Morphogenesis NCL, SLC33A1, GLI3, DLX5

Regulation of physiological processes E2F5

Regulation of physiological processes in cells TRAPPC3, CLTC, MTX1, AP2A1

Response to biotic stimulus TLR3

Cellular interaction PRKCSH, PDGFA

Function

Structural component of ribosomes MRPS33

Transferase activity IHPK2, SPTLC1, MGAT4A, TGM3

Oxidoreductase activity PGD

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Hydroxylase activity DUPS12

Isomerase activity PPIB

Binding of nucleic acids SFRS9, NCL, DMRT2, TLR3, E2F5, CEBPZ, GLI3, ORC6L, DLX5

Binding of ions GTF2B, PRKCSH, RP9

Binding of proteins MTX1, SMARCB1, ERH

Ion transfer MON2

Helicase activity DDX5

GTPase regulatory activity DOCK9

Cofactor of transport activity SLC33A1

Receptor activity NEONO2

Receptor binding PDGFA

Component

Membrane-bound organelles IHPK2 SFRS9, GTF2B, TRAPPC3, DDX5, DMRT2, CEBPZ, DLX5

Cavity of organelles PPIB

Non-membrane bound organelles NCL, SMARCB1, TGM3

Membranes MON2, TLR3, SLC33A1, MTX1, SPTLC1, NEONO2, MGAT4A, ORC6L

Intracellular localization CLTC, E2F5, PRKCSH, AP2A1, GLI3, DUPS12

Extracellular localization PDGFA

RNA-induced complex MRPS33

Receptor complex RP9

tory effect. This is particularly important for improving the methods of therapy of diseases, which pathogenesis is connected with failure of functioning of several signal pathways, not single genes [57].

To modulate the levels of miRNA two alternative strategies can be used depending on whether the increase or decrease in the expression of endogenous miRNA is considered as a causative factor in the development of the disease. Effects of different miRNAs are shown in Table 3.

INHIBITORS OF ENDOGENOuS MICRORNAS

If it is necessary to reduce the level of endogenous microRNA, short single-stranded oligonucleotides complementary to this miRNA can be used. These synthetic molecules are called miRNA antagonists (anti-microRNA). In a cell anti-miRNA interact with complementary endogenous microRNA and damage its repressing function. The exact mechanism of anti-miRNA action of is not completely established [54]. In order to increase the sensitivity and specificity of binding to the tar-

get, as well as resistance to degradation a number of modified nucleotides are incorporated into anti-microRNAs. 2'-O-methyl and 2'-O-methoxyethyl analogs is also used in addition to the above described LNA-nucleotides [9, 10, 29]. Stability of the anti-miRNA can be also increased by introduction of Phospho-rothioate internucleotide groups and efficiency of intracellular delivery improves the conjugation of anti-miRNA with cholesterol [49].

ANALOGS OF ENDOGENOuS MICRORNAS

Normally a number miRNAs inhibit the development of pathological processes. Therefore reducing of miRNA levels, for example, with functions of tumor suppressors, causes the development of the relevant diseases [33]. In such cases, functions of signal pathways can be recovered by increasing of endogenous miRNA level. Synthetic short double-stranded RNAs, designed in such a way that one strand of the duplex is identical to a mature microRNAs can be used for this purpose [62]. Double-stranded structure of analogs of microRNAs is necessary for the recognition by cellular machinery of RNA-dependent regulation of gene expression. Additional chemical modifications of RNA in the content of the synthetic duplex are applied for correct inclusion of strand identical to the mature miRNA into the effector complex [35].

After entering the cell microRNA synthetic analogues are included in the effector complex, functionally replace deregulated endogenous miRNAs and recover the signal pathways functioning normally. Analogs of miRNA do not differ from endogenous molecules, for this reason it is unlikely that their introduction will lead to the non-specific side effects. As the analogs and endogenous miRNAs have the same nucleotide sequence, it can be expected that synthetic analogue will regulate the same set of genes that normally endogenous miRNA. Importantly, the drug based on a synthetic analogue will have an effect on all the endogenous miRNA targets, including those that are still unknown.

METHODS OF DELIVERY OF INHIBITORS AND ANALOGS OF ENDOGENOuS MICRORNAS IN CELL

At the present time, several methods of delivery of agents based on microRNAs are used in experimental animal models and systems of cultivation in vitro.

Viral vectors encoding miRNAs precursors exemplify the analog delivery systems is miRNA. In this case an additional intracel-lular processing of precursor transcript involving proteins Drosha and Dicer is necessary for the maturation of short functional double-stranded RNA [17, 18, 37, 38]. The advantage of this delivery method is a high transfection efficiency and prolonged expression of exogenous miRNA. The complexity of vector constructs manufacture, as well as difficulties associated with immunogenicity and toxicity limits their use [27, 47].

An alternative method is the direct delivery of short single-stranded synthetic miRNA inhibitors or double-stranded analogs of miRNA. In this case, the delivery can be mediated by a carrier

such as liposomes. Particular attention is paid to the development of methods of targeted delivery of microRNAs. For this purpose the surface of carriers are covered with tissue-specific ligands [1, 6, 26]. Additionally high stability of modified synthetic oligonucleotides allows delivering them to the cells without a carrier. Delivery of synthetic oligonucleotides without a carrier can be completed in cell cultures. It has been shown that LNA-modified phosphothio-ate oligonucleotides are efficiently absorbed by cells in vitro and are involved in the specific regulation of genes [53, 59]. Using of this method allows avoiding of possible side effects of transfection reagents on the system under study.

In animal models synthetic oligonucleotides can also be delivered without a carrier. One of the most common ways is a systemic delivery by intravenous or subcutaneous administration of the oligonucleotide solution in physiological buffer. Within a few hours after administration levels of analogs or inhibitors of microRNAs in plasma are reduced through their absorption by cells. In the case of miRNA inhibitors it was shown that after systemic administration these molecules form heteroduplexes with endogenous microRNAs, and thus inhibit their activity [9]. In addition high metabolic stability of synthetic oligonucleotides allows them to be able to function in the tissues for several weeks [14, 28].

The effects, caused by the administration of analogs or inhibitors of some miRNAs are not immediate, that is probably determined by the necessity of initialization or disabling of a number of successive regulatory processes, which are controlled by endogenous miRNAs [40, 41].

conclusion

Thus, the class of microRNAs is one of the most promising for further comprehensive study of oligonucleotides as diagnostic molecules and therapeutic targets. Potential application of miR-NAs in therapy of different diseases is now widely recognized. Target therapy of cardiovascular pathology with use of viral vectors, single-stranded synthetic miRNA inhibitors or double-stranded analogs of miRNA and other delivery technologies is the subject of great interest. Discovering of miRNAs mechanisms of action and obtaining information about their proved efficacy and safety is a major focus of experimental research.

REFERENCES

1. Anand S., Majeti B.K., Acevedo L.M. et al. MicroRNA-132-mediated loss of p120RasGAP activates the endothelium to facilitate pathological angiogenesis. Nat Med. 2010; 16: 909-914.

2. Asada S., Takahashi T., Isodono K. et al. Downregulation of Dicer expression by serum withdrawal sensitizes human endothelial cells to apoptosis. Am. J. Heart. Circ. Phisiol. 2008; 295: 2512-2521.

3. Babushkina N.P., Kucher A.N. Geneticheskaya osnova funktsion-irovaniya malykh regulyatornykh RNK u cheloveka [The genetic basis of functioning of small regulatory RNAs in humans]. Sbornik nauchnyj trudov «Genetika cheloveka i patologija» vyp. 8 [Collection of scientific works Human genetics and pathology, 8th ed.]. Tomsk, 2007: 219-228 (in Russian).

Table 3

The role of microRNAs in the regulation of the cardiovascular system [5, 7, 8, 19, 63, 64]

MiRNA ID Possible target Effect Possible application

1 Cx43 (connexin 43) Selective cardiac overexpression reduces left ventricular hypertrophy and prevents ventricular tachyarrhythmias Myocardial infarction, left ventricular hypertrophy, ventricular tachyarrhythmias

2 Irx5, Kcnd2 Combined loss of function of Irx5 and Irx4 causes abnormal ventricular repolarization and predispose to arrhythmias Reduced heart rate, short interval PR, QRS interval lengthened

7 EGF-R Transcription is activated in senescent cells Recovery of regenerative capacity in chronic lesions in elderly patients

9 PDGFR-ß Reduces paracrine angiogenic potential of cardiomyocytes LV hypertrophy, fibrosis

15 a/16 VEGF, AKT3 Relative inhibition of circulating pro-angiogenic cells, improving of post-ischemic blood flow, recovery of arteriole muscle density Critical ischemia

18/19 TSP, CTGF, or CCN2 Reduces the production of collagen Myocardial infarction

20 a MKK3 Inhibits the expression of MKK3 and VEGF-induced migration of endothelial cells and angiogenesis Reduced angiogenesis in ischemic conditions

21 PDCD4 Inhibition of reverse vascular remodeling activated by balloon injury Restenosis

22 OGN, SIRT-1 Causes aging of cardiac fibroblasts. Promotes hypertrophy LV hypertrophy, myocardial infarction

23 a Determining of level along with the precursor of N-terminal fragment of brain natriuretic peptide can improve the diagnosis of myocardial injury Heart failure

24 eNOS Local delivery improves angiogenesis and increases blood flow in the area of myocardial infarction, causes reduction of infarct size, induces apoptosis in fibroblasts and improves cardiac function Post-MI

26 KIR2.1 Atrial fibrillation activates NFAT, increases its movement into nucleus, where it transcriptionally suppresses the expression of miR-26 genes Atrial fibrillation

27 SEMA6A Angiogenesis, adipogenesis, inflammation, metabolic syndrome, oxidative stress, insulin resistance and type 2 diabetes

30b - Elevated levels can diagnose heart failure in breathless patients Heart failure

33a - - Atherosclerosis and metabolic syndrome

34 SIRT-1 Bcl2 Cdk4 Cyclin D2 Causes aging of endothelial cells. Causes aging and/or destruction of pro-angiogenic cells Heart failure

92a KLF4, MKK4 Its inactivation promots proliferation of endothelium and reduces neointima hyperplasia after vascular injury Angiogenesis and restenosis

98/let-7 Cyclin D2 Its regulation by thioredoxin 1 inhibits cardiac hypertrophy LV hypertrophy

103 - Elevated levels can diagnose heart failure in breathless patients Heart failure

106 - - Atherosclerosis and metabolic syndrome

122 TGF-ß1 Inversely correlated with the content of collagen, promotes the development of myocardial fibrosis and aortic fibrosis Atherosclerosis and metabolic syndrome, inhibited in aortic stenosis

125a-5p ET-1 Reducing confirms the hypothesis of microvessels spasm in Takotsubo cardiomyopathy Takotsubo cardiomyopathy

Продолжение табл. 3

MiRNA ID Possible target Effect Possible application

126 Positive modulation of androgen receptor Tie-1, c-kit, IL-8; CXCL12. VEGF, VEGF receptor — inhibition Spred-1, inhibition — EGFL7 Stimulates angiogenesis, anti-apoptotic Atherosclerosis, myocardial infarction, heart failure

132 CREB, VEGF Promotes angiogenesis, regulates cardiac hypertrophy and autophagy, modulates inflammation Heart failure; myocardial infarction; hypertrophy of the heart; atherosclerosis

133a — Activated by ST-elevated myocardial infarction, controls phenotypic switch off of vascular smooth muscle cells after injury; decreases in case of LVH Restenosis, myocardial infarction, LV hypertrophy

143/145 Actal (pha2), Tnnc2, Tnni2, Mylpf - Aortic aneurysm

146 IRAK, NOX4 Associated with inflammatory and oxidative response in human endothelial cells -

181 а - Negatively correlated with proinflammatory cytokines (IL-6, TNF). Positively correlates with anti-inflammatory cytokines (TGF, IL-10) Myocarditis

199a-3p - Determining of level along with the precursor of N-terminal fragment of brain natriuretic peptide can improve the diagnosis of myocardial injury Heart failure

200 ZEB1 Induced by oxidative stress. Causes aging of endothelial cells in vitro In experiment

204 KK Expression during ischemia and heart failure, protective role due to reducing of congestion in calcium Heart failure, coronary heart disease

208 Myh6, Myh7, Myh7b Its therapeutic inhibition improves cardiac function in heart failure LV hypertrophy, heart failure

214 Calcium channels Expression during ischemia and heart failure, protective role due to reducing of congestion in calcium Coronary heart disease

217 SIRT-1 Causes aging of endothelial cells, reduces the availability of endothelium-derived relaxing factor -

223 IGF1-R Platelets play an important role in the pathogenesis of atherosclerosis and stroke through active interaction with neutrophils, monocytes and vascular endothelial cells. Coming out of the platelets, this microRNA promotes apoptosis of vascular endothelial cells induced by advanced glycation end products. Atherosclerosis, stroke

320 - Elevation in myocardial infarction, proapoptotic factor Myocardial infraction

324-5p - Determining of level along with the precursor of N-terminal fragment of brain natriuretic peptide can improve the diagnosis of myocardial injury Heart failure

328 - - Atrial fibrillation

342-3p - Elevated levels can diagnose heart failure in breathless patients Diagnosis of heart failure

350 MAPK 11/14, MAPK8/9 Are activated in the myocardium in the later stages of aortic stenosis. Inhibition decreases cardiac hypertrophy by reducing the size and cardiomyocyte apoptosis Aortic stenosis, myocardial hypertrophy

370 - - Atherosclerosis and metabolic syndrome

Окончание табл. 3

MiRNA ID Possible target Effect Possible application

467b - Provides protection against atherosclerosis by reducing the level of lipids and inflammatory cytokine production of apolipoprotein E Atherosclerosis

499 MRFs, Eos Cardiac regeneration by regulating cardiac differentiation and proliferation; Genetic reprogramming of the heart; antiapoptotic; regulates stress response genes Myocardial infarction; hypertrophy of the heart; fibrosis and cardiac conduction

590-3p - - Atrial fibrillation

622 - Elevation in cardiovascular disease The diagnostic marker of heart failure with dyspnea

675 - Elevation in cardiovascular disease The diagnostic marker of heart failure with dyspnea

758 - - Atherosclerosis and metabolic syndrome

1248 GAPVD1 Cdk2 Participation in inflammatory pathways (NF-kB) -

1254 - Elevation in cardiovascular disease The diagnostic marker of heart failure with dyspnea

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