1、Opportunities for microRNAs in the Crowded Field of Cardiovascular BiomarkersPerry V.Halushka1,2,Andrew J.Goodwin3,and Marc K.Halushka41Department of Pharmacology,South Carolina Clinical and Translational Research Institute,Medical University of South Carolina,Charleston,South Carolina 29425,USA2Dep
2、artment of Medicine,South Carolina Clinical and Translational Research Institute,Medical University of South Carolina,Charleston,South Carolina 29425,USA3Division of Pulmonary,Critical Care,Allergy,and Sleep Medicine,Medical University of South Carolina,Charleston,South Carolina 29425,USA4Department
3、 of Pathology,Johns Hopkins University School of Medicine,Baltimore,Maryland 21205,USAAbstractCardiovascular diseases exist across all developed countries societies.Biomarkers that can predict or diagnose diseases early in their pathogeneses can reduce their morbidity and mortality in afflicted indi
4、viduals.microRNAs are small regulatory RNAs that modulate translation and have been identified as potential fluid-based biomarkers across numerous maladies.We describe the current state of cardiovascular disease biomarkers across a range of diseases,including myocardial infarction,acute coronary syn
5、drome,myocarditis,hypertension,heart failure,heart transplantation,aortic stenosis,diabetic cardiomyopathy,atrial fibrillation,and sepsis.We present the current understanding of microRNAs as possible biomarkers in these categories and where their best opportunities exist to enter clinical practice.K
6、eywordsmicroRNAs;biomarkers;cardiovascular disease;hypertension;sepsis;aortic stenosisINTRODUCTIONIn the field of cardiovascular disease,biomarkers are essential tools in a clinicians armamentarium.The best of our biomarkers can aid in diagnosing an acute myocardial infarction(AMI),indicate long-sta
7、nding heart failure,and predict the rejection of a transplanted heart.However,not all biomarkers are robust,and many fields within cardiovascular disease management await useful biomarkers to aid in diagnosis and assessing prognosis.Across all of these scenarios there is the opportunity for better D
8、ISCLOSURE STATEMENTThe authors are not aware of any affiliations,memberships,funding,or financial holdings that might be perceived as affecting the objectivity of this review.HHS Public AccessAuthor manuscriptAnnu Rev Pathol.Author manuscript;available in PMC 2020 January 24.Published in final edite
9、d form as:Annu Rev Pathol.2019 January 24;14:211238.doi:10.1146/annurev-pathmechdis-012418-012827.Author ManuscriptAuthor ManuscriptAuthor ManuscriptAuthor Manuscriptbiomarkers.It is with this appreciation that we critically evaluate microRNAs(miRNAs)as a new category of biomarkers for cardiovascula
10、r diseases.In this review,we analyze what the opportunities are for using miRNA biomarkers across particular disease states,specifically comparing miRNA biomarkers to what is currently used for the disease when appropriate.We also provide context for the intricacies of miRNA biomarker studies so eac
11、h reader has the tools to critically evaluate any of those described herein or forthcoming miRNA studies for their potential clinical use in cardiovascular disease.A PRIMER ON miRNAS AND BIOMARKERS IN CARDIOVASCULAR DISEASEA Brief Description of miRNA BiogenesismiRNAs are short(1822 nucleotide)regul
12、atory RNAs that bind mRNAs and decrease protein translation.miRNAs are generally cotranscribed with neighboring genes or co transcribed within a cluster of miRNAs(a polycistronic cluster).These primary miRNAs(pri-miRNAs)are then processed by Drosha into pre-miRNAs and cleaved by Dicer into mature mi
13、RNAs.One half of a mature miRNA(either the 5p or 3p sequence)is loaded into Argonaute 2(Ago2)as part of RISC(the RNA-induced silencing complex).Although complicated,controversial,and with many exceptions to the rule,a six base pair seed sequence at the 5 end binds to a complementary region along the
14、 3 untranslated region of an mRNA,thus enabling specific regulation of scores of mRNAs by a single miRNA(1,2).This entire mechanism has been extensively described and reviewed elsewhere(35).The Strict Nomenclature of miRNAsmiRNAs have a strict naming convention(6,7).A miRNA name begins with a three-
15、letter code for the species(e.g.,hsa for Homo sapiens and cel for Caenorhabditis elegans).This is followed by miR and then a number of some sequential order.If miRNAs have the same seed sequence,indicating a shared family function,there may be a letter,in alphabetical order,after the number.Examples
16、 include hsa-miR-181a,hsa-miR-181b,and hsa-miR-181c.Each processed miRNA has two strands:a dominant strand(more abundant and loaded in RISC)and a passenger strand(degraded,less abundant),one from each of the 5p and 3p ends.These were denoted as the s(sense)and as(antisense)or*strands(star,denoting p
17、assenger)in earlier literature.A complete miRNA name would be hsa-miR-181a-3p or hsa-miR-181a-5p.The one exception to this naming convention is the let-7 family of miRNAs,which kept their original names.Once a species is established,the species code is not commented on further in most manuscripts.Eq
18、ually,if the 5p or 3p designation is not stated,it is generally assumed that the dominant strand is being referenced.The True Number of Human miRNAs is UnknownThe number of human miRNAs is controversial.miRBase(http:/www.mirbase.org/)has been considered the central repository of all known miRNAs.Ver
19、sion 21 of miRBase listed 2,588 human miRNAs,a relatively stable number compared with prior versions of the database(6).However,since that version appeared,with the explosion in small RNA sequencing(RNA-seq)and generic novel miRNA discovery tools,thousands of additional Halushka et al.Page 2Annu Rev
20、 Pathol.Author manuscript;available in PMC 2020 January 24.Author ManuscriptAuthor ManuscriptAuthor ManuscriptAuthor Manuscripthuman miRNAs have been proposed(811).Most of these have been collected into the new miRCarta database(https:/mircarta.cs.uni-saarland.de/)that adds an additional 12,857 huma
21、n miRNA precursors to our collection(12).Are all of these additional sequences truly in the miRNA oeuvre?Many groups suggest otherwise,indicating that miRBase is rife with non-miRNA species,and these newly minted novel miRNAs are mostly dead on arrival(1317).In response to these concerns,MiRGeneDB(h
22、ttps:/mircarta.cs.uni-saarland.de/)was developed as a hand-curated classification of miRNAs based on strict criteria,including genetic conservation and canonical development(14,18).This database lists only 586 human miRNA genes.Clearly,the collection of small RNAexpressed species requires greater cl
23、arity and perhaps additional nomenclature to describe miRNA-like small RNAs(17).Regardless,for the purpose of utilizing miRNAs as biomarkers,any sequence that gives a consistent signal indicating the presence of disease can be useful.Many miRNAs are Expressed in a Cell TypeSpecific FashionA factor i
24、n understanding the function and biomarker utility of miRNAs is the appreciation of the cells in which they are expressed.If a miRNA is expressed in a cardiomyocyte,it may have more relevance as a cardiovascular biomarker than one that also is expressed in a noncardiovascular cell(e.g.,a hepatocyte)
25、.Early expression localization studies were performed in tissue(1921).These limited our understanding of the contribution of the various cell types that are expressed across a variety of tissuessuch as endothelial cells,red blood cells,and fibroblaststo the changing miRNA levels in diseased tissue.T
26、his led to problems that plague the miRNA literature(7,22,23).Better characterization of miRNAs at the cellular level has been slow to develop.We have been actively engaged in the realization of miRNA cellular localization(24,25),culminating in a cellular microRNAome that is based on miRNA data from
27、 46 primary cell types and 42 cancer or immortalized cell lines(8).This work was complemented by two other papers in 2017 describing miRNA expression in many additional cell types(9,26).These combined data are now accessible as a University of California Santa Cruz genome browser barChart that shows
28、 miRNA expression levels across 78 primary cell types and 51 cancer or immortalized cells(Figure 1)(17).For the first time,any investigator can quickly determine the cellular location of their miRNA of interest to determine its relevance to disease(Table 1).This information is vitally important to m
29、iRNA biomarker studies as seen below.MyomiRsBetween organ-specific data and cell-specific data,it has emerged that a group of miRNAs is found either exclusively or more abundantly in myocytes.These are termed myomiRs.As described in Table 2,some of these miRNAs are found within myosin heavy chain ge
30、nes(27).Most of these myomiRs are also expressed in skeletal muscle,with miR-208 being the most specific of the myomiRs for cardiac muscle.This group of miRNAs is particularly important in cardiovascular disease.Because of their exclusivity to muscle cells,they should be found only in blood-based fl
31、uids(plasma and serum)if they are spilled from injured cells or perhaps be present if muscle cells use these miRNAs to signal to other cells(28).Of this group,miR-1(miR-11 and miR-12 have identical sequences from different loci)has mainly been described in cardiac and skeletal muscle cells(8).Howeve
32、r,it is also found in low levels in many organs(17,29),suggesting that some noncardiac-and skeletal muscle Halushka et al.Page 3Annu Rev Pathol.Author manuscript;available in PMC 2020 January 24.Author ManuscriptAuthor ManuscriptAuthor ManuscriptAuthor Manuscriptcell expresses miR-1 in vivo.MyomiRs
33、are an important group of miRNAs in cardiovascular biomarker studies,as discussed below.BiomarkersBiomarkers may be biochemical,molecular,histologic,radiographic,or physiologic.They may be obtained from whole blood,serum,plasma,body fluids,or tissues.For the purposes of this review,we discuss curren
34、t biochemical and molecular biomarkers and newer,potential miRNA biomarkers.Biomarkers have multiple uses.They can be indicators of a pathologic or physiologic process;they can be used to monitor the progress of or provide the prognosis for a disease;they can be used to monitor the efficacy or toxic
35、ity of a therapeutic agent;or they may represent a therapeutic target(30).They may also be used to stratify patients for the purposes of a clinical trial.Thus,their usefulness has been well established.However,they should be used only once they have been well validated for their intended purpose.The
36、y may also undergo considerable improvement in terms of sensitivity or specificity,or both.An excellent example of this is the refinement of the use of blood measurements of cardiac-specific troponin as an indicator of myocardial damage(31).CURRENT BIOMARKERS OF CARDIOVASCULAR DISEASEMyocardial Infa
37、rctionOf all the cardiovascular diseases,the diagnosis of an AMI,usually as the result of an acute coronary artery event,is of the most societal importance.Fortunately,robust science has produced biomarkers that rapidly detect an AMI.The measurement of cardiac troponins either Troponin I(cTnI)or Tro
38、ponin T(cTnT)has emerged as our gold standard diagnostic test.Cardiac troponins are cardiomyocyte-specific proteins that are spilled into the circulation when a cardiomyocyte dies(32).They can be measured rapidly in clinical chemistry laboratories and as point-of-care testing(33).American Heart Asso
39、ciation guidelines recommend taking two measurements,one at first assessment and a second 36 hours later(34).A test is considered positive if at least one value is 99th percentile of the upper reference limit.With the advent of a new high-sensitivity test(hs-cTnT),this biomarker is firmly establishe
40、d as a key diagnostic tool in confirming a myocardial event.However,troponins can be elevated in non-AMI settings,including myocarditis and end-stage renal disease,although they are generally at lower levels than following an AMI and have different kinetics over time due to their sustained release(a
41、lso known as troponin leak)or accumulation.Any cardiac injury can cause a release of troponins,with the extent of the injury correlating roughly with the level of the troponin.Thus,it is a sensitive but not specific test for an AMI,and opportunities may exist to find biomarkers with more discriminat
42、ion.HypertensionHypertension,or high blood pressure,is a polygenic and multifactorial disease,and its course can be altered by many environmental factors.Hypertension is injurious to the bodys organs and is associated with increased incidences of stroke,myocardial infarction,and aortic aneurysm,amon
43、g other problems.The diagnosis is easily made with the use of a Halushka et al.Page 4Annu Rev Pathol.Author manuscript;available in PMC 2020 January 24.Author ManuscriptAuthor ManuscriptAuthor ManuscriptAuthor Manuscriptsphygmomanometer to measure blood pressure,and the criteria for the diagnosis an
44、d treatment are well established(35,36).Biomarkers beyond those obtained by the sphygmomanometer are clearly not needed for diagnosing hypertension;however,they may be of great value in helping to(a)stratify a patient for a therapeutic approach,(b)serve as prognostic indicators of hypertension-relat
45、ed sequelae or comorbidities,and(c)indicate a specific etiology of hypertension.It is in these roles that miRNA biomarkers may hold value.Rejection of Orthotopic Heart TransplantThe gold standard for diagnosing heart transplant rejection is a pathologists review of endomyocardial biopsy material(37,
46、38).However,simpler blood-based methods have emerged.A gene expression profiling method called AlloMap(CareDx,Brisbane,CA)is used clinically to rule out cardiac rejection in patients(39).A second method,still in development,is a cell-free DNA assay that can detect increased levels of DNA from the do
47、nor heart in a patients blood,indicating heart injury(40).A third method utilizes brain natriuretic peptide(BNP)levels as indicators of rejection(41).The pros and cons of these methods as biomarkers are discussed in greater detail elsewhere,but no current blood-based test is useful at distinguishing
48、 between cellular and antibody-mediated rejection,among other limitations(42).MyocarditisMyocarditis can be difficult to diagnose,and having specific biomarkers could greatly improve our diagnostic abilities.Currently,endomyocardial biopsy remains the gold standard,as for heart transplantation(43).S
49、ince this is an invasive procedure and not widely offered,other methods are used as well.Even if myocarditis is present,the heterogeneous nature of the disease results in only 50%of cases being diagnosed positively by biopsy(44).Other biomarker modalities that can be used include imaging studies and
50、 measurement of levels of troponin,BNP,and N-terminal pro-B-type natriuretic peptide(NT-proBNP).These tools have diagnostic overlap with myocardial infarction and other causes of heart failure,so there is not yet a robust biomarker specific to myocarditis.Diabetic CardiomyopathyDiabetic cardiomyopat
