Blood Cell MicroRNAs: What Are They and What Future DoThey Hold?

Patricia Ryan and Chintamani Atreya

The advent of blood component storage revolutionizedhealth care by allowing for a managed supply oftransfusion quality blood products. During storage,blood components undergo a series of physiologicalchanges that affect the product quality, whichultimately can interfere with the safety and efficacyof such products after transfusion. Despite continuousimprovements in blood component quality and safety,it is still desirable to have in vitro standard markers ofmeasurable characteristics that predict blood compo-

Transfusion Medicine Reviews, Vol 25, No 3 (July), 2011: pp 247-25

nent safety and efficacy in vivo following theirtransfusion. Over the last decade, research on thefeasibility of using microRNAs as biomarkers forvarious clinical manifestations and cellular pathologieshas exploded. Here, we review the literature on bloodcell microRNAs and discuss the potential of thesemolecules to act as measurable characteristics (pro-duct biomarkers) for stored blood component qualityand safety.Published by Elsevier Inc.

icroRNAs (miRNAs) ARE A class of small, sion can lead to disease and cellular pathologies,

From the Section of Cell Biology, Laboratory of CellularHematology, Division of Hematology, Office of Blood Researchand Review, Center for Biologics Evaluation and Research, USFood and Drug Administration.

PR is a recipient of a postdoctoral fellowship at the Center forBiologics Evaluation and Research administered by the OakRidge Institute for Science and Education through an intra-agency agreement between the US Department of Energy and theUS Food and Drug Administration. The authors thank Drs Kethaand Rao, Center for Biologics Evaluation and Research, Foodand Drug Administration, for their review of this manuscript.

Address reprint requests to Chintamani Atreya, Bldg 29A,Room 2C-11, NIH campus, 8800 Rockville Pike, Bethesda, MD20892.E-mail: Chintamani.Atreya@fda.hhs.gov0887-7963/$ - see front matterPublished by Elsevier Inc.doi:10.1016/j.tmrv.2011.01.005

M endogenous, evolutionarily conserved, non-coding RNAs that regulate gene expression and playa role in diverse cellular processes, includingproliferation, differentiation, and cell death.1 As anabundant class of regulatory molecules, there havebeen more than 1000 distinct miRNAs identified inthe human genome to date and thousands morepredicted.2 A singlemiRNA can regulate expressionof multiple genes, and the expression of a singlegene may be regulated by several distinct miRNAs,creating complicated regulatory networks.3,4 It isestimated that roughly 60% of human protein-coding genes are regulated by miRNAs.5

Precursor miRNAs (pri-miRNAs) are transcribedby RNA polymerase II from independent transcrip-tion units or represent the introns of protein-codinggenes. Precursor miRNAs fold into hairpins, whichare processed by a microprocessor complex con-taining the RNaseIII enzyme Drosha to produceanother precursor molecule (pre-miRNA) roughly60 nucleotides (nt) in length.6-8 The pre-miRNA istransported to the cytoplasm where Dicer processesit to a duplex miRNA of approximately 20 nt.9 Inthe cytoplasm, these miRNA duplexes are unwoundand the guide strand is incorporated into the RNAinduced silencing complex (RISC), which containsan Argonaute (Ago) family member (proteins thatbind to miRNAs) and TAR RNA binding protein(TRBP).10,11 After incorporating into RISC, themiRNAs mediate posttranscriptional repression ofgene expression by pairing to complementarysequences of messenger RNA (mRNA) targets,resulting in transcript destabilization, translationalrepression, or both (Fig 1).

MicroRNAs play a crucial role in gene regulation;therefore, while altered or defective miRNA expres-

miRNA expression may also be dysregulated as aconsequence of disease. The idea of miRNAsserving as biomarkers of a specific disease, or of acellular state, has recently emerged as a newinvestigational field of study. Currently, efforts areunder way to evaluate miRNAs as potential clinicalbiomarkers for Alzheimer's disease, Huntington'sdisease, kidney disease, and arthritis.12-15 Micro-RNAs have also been exploited as clinical biomar-kers in cancer biology. Initially, miRNAs werestudied for their possible role in tumorigenesis andtheir potential as therapeutic targets. Subsequently,several independent investigations of miRNA ex-pression indicated that miRNAs are dysregulated innearly all tumors evaluated, and various studiesusing serum or tissue samples further indicated thatthese specimens display unique miRNA expressionprofiles relative to healthy subjects.16 Since then,efforts to use differential miRNA profiling of serum

1 247

Fig 1. Model of miRNA pathway in megakaryocyte-erythrocyte progenitor cells (MEPs) and anucleate blood cells. In the nucleus oMEPs, miRNA genes are transcribed by RNA polymerase II into long primary miRNA transcripts (60-100 nt) that fold into stem loopstructures called pri-miRNAs. Subsequently, pri-miRNAs are processed into pre-miRNAs (~60 nt) by the microprocessor complex thacontains the enzyme Drosha (class 2 RNase III enzyme).6-8 Pre-miRNAs are transported to the cytoplasmwhere they are further processedinto small (~22 nt) miRNAs by the Dicer enzyme (RNase III family endoribonuclease).9 A miRNA guide strand is then loaded by Dicer ontoRISC, which contains an Ago family member (proteins that bind to miRNAs) and TRBP.10,11 As part of miRISC, miRNAs base-pair withtarget mRNA and induce either translational repression (due to imperfect base-pairing) or degradation (due to perfect base-pairing).39,4

Endonucleolytic cleavage activity of Ago2 targets mRNAs that possess a perfect complementarity binding site for miRNA. Anucleate bloodcells receive pre-miRNAs fromMEPS. Dicer, TRBP, and Ago2, which are the functional components of the miRNA pathway, were recentlyidentified in platelets.28

248 PATRICIA RYAN AND CHINTAMANI ATREYA

f

t

0

249MicroRNAs IN STORED BLOOD CELLS

or tissue to classify cancer type, predict prognosis,and diagnose disease have taken a prominent rolein the field of cancer diagnostics. For instance, apanel of 6 miRNAs has been linked to short- orlong-term survival in pancreatic adenocarcinoma.In addition, several studies have demonstrated thatmiRNA-21 is overexpressed in several types ofcancer and that this overexpression often correlateswith drug resistance.17-19

The rapid progression from identifying miRNAinvolvement in cancer biology to developingtranslational applications for these small RNAmolecules in cancer diagnostics suggests that thescope of usingmiRNAs as clinical biomarkers couldbe extended to evaluate the measurable quality andsafety characteristics of cellular therapeutics, as wellas their stability during storage. MicroRNAs arerelatively stable molecules and easily quantified,and recent technical developments have allowedresearchers to measure the absolute and relativelevels of many cellular miRNAs under both healthand disease conditions.

The potential advantages of using miRNAs inclinical and cellular therapeutic settings are man-ifold. For example, in the area of ex vivo storedblood cells, especially concentrated platelets andpacked red blood cells (RBCs), there is no singlemeasurable quality characteristic (“gold standard”)of the product that predicts its in vivo survival andfunction following transfusion. Current practice ofquality assurance for these cellular productsincludes a battery of in vitro tests, followed by invivo assessment of radiolabeled autologous cells inhealthy volunteers.20 Although this comprehensiveapproach no doubt has provided reliable qualityassurance for stored blood cells, it still is worthsearching for simpler and possibly better alter-natives by exploiting the wealth of bioinformaticsavailable, and adapting some of the emergingmolecular tools such as miRNA profiling fromother fields of science to the field of transfusionmedicine. Here we review available literature onblood cell miRNA studies and discuss thefeasibility of using miRNA profiling as a measur-able characteristic of quality for stored cells intransfusion medicine.

STORED RED BLOOD CELLS AND PLATELETS

More than 10 million units of platelets and 14million units of RBCs or whole blood are transfusedannually in the United States.21 Red blood cells play

a key role in tissue and organ oxygenation and arethe most widely transfused blood componentworldwide.22 One of the current practices for RBCstorage includes collection of whole blood intoanticoagulant solutions, concentrating RBCs bycentrifugation, removal of platelet-rich plasma,and storage of the final product at 4°C ± 2° for upto 42 days. During storage, RBCs undergo a seriesof molecular and physiological changes, collective-ly referred to as storage lesions, which could lead tocell death and ultimately affect the quality of storedRBCs and reduce their shelf-life.23 Efforts are thusunder way to invent new ways to increase the shelf-life of RBC units.

Platelets are routinely used in clinical settings totreat bleeding arising from surgery, trauma, orvarious bleeding disorders. Currently, platelets arecollected and stored at 22°C to 24°C for up to 5days with continuous gentle agitation. As is the casewith RBCs, a series of morphological and molec-ular changes occur during storage (platelet storagelesions) that affect platelet viability and functionafter transfusion.23

POTENTIAL FOR miRNA AS BIOMARKERS FORSTORED BLOOD CELL QUALITY

Although a wealth of information about miRNAexists for many cell types, the role of miRNA interminally differentiated, anucleate blood cells suchas platelets and erythrocytes is only beginning to beunraveled. Historically, these cells were consideredrelatively inert owing to the lack of a nucleus and denovo transcription. However, recent studies havedemonstrated that miRNAs may play a role inregulating gene expression in these cells.

Released into the bloodstream from megaka-ryocytes, platelets are highly specialized anucleatecells. Despite lacking genomic DNA, it isestimated that platelets contain up to 32% of themRNA transcripts for protein-coding genes, andstudies indicate platelet ribosomes are capable ofde novo protein synthesis.24-27 A recent study hasindicated that platelets not only contain anappreciable amount of mRNA and miRNA, butalso host a functional miRNA pathway (Fig 1).28

Preliminary evidence indicates that miRNA-96may play a role in modulating platelet reactivityby regulating levels of VAMP8 mRNA, andmiRNA-96 was shown to be elevated in storedplatelets.29,30

250 PATRICIA RYAN AND CHINTAMANI ATREYA

During erythropoiesis, erythroid cells shed manystructures that are present in typical mammaliancells, including the nucleus and endoplasmicreticulum, and undergo significant mRNA reduc-tion to yield highly specialized RBCs.31 Neverthe-less, RBCs maintain a diverse population ofmiRNAs at levels similar to those of nucleatedcells.25,32,33 The purpose of these miRNAs in RBCbiology, if any, remains to be defined. The presenceof miRNA in such abundance is consistent with arole for miRNAs in these cells, and based on whatwe know now about miRNAs in platelets, it iscertain that efforts to establish evidence for theexistence of a miRNA pathway (or lack there of) inRBCs are under way. Presumably, any residualtranslation that occurs in these cells would beregulated by these miRNAs.Given the existence of miRNA and mRNAs in

stored blood cells and the fact that blood cells doundergo morphological and molecular changesduring storage (storage lesions), it is reasonable topostulate that miRNAs provide translational regu-lation of existing mRNAs in these cells. Thesedecisional steps within the stored cell will ulti-mately determine the fate of the cell, that is,survival during storage by resisting the onset ofphysiological changes (storage lesions) or ulti-mately undergoing cell death. If this hypothesis iscorrect, then the miRNA and mRNA expressionprofiles should be altered during storage in bothRBC and platelets. Indeed, 2 separate studies usinga panel of selected apoptosis-related miRNAsdemonstrated that miRNA expression signatureschange over time ex vivo in both platelets andRBCs.29,34 In stored platelets, differential profilingdemonstrated a time-dependent detection of 4 of the52 miRNAs studied, as measured over a period of 9days. Two of these miRNAs, let-7b and miRNA-16, demonstrated an increasing trend, whereasanother 2 miRNAs, miRNA-7 and miRNA-145,exhibited decreased levels during storage.29 Ana-lyzing the same set of 52 miRNAs in RBC, 4miRNAs displayed a time-dependent increasingtrend up to day 20 during storage. The 4 miRNAsare miRNA-96, miRNA-150, miRNA-196a,miRNA-197.34 Given the recent discovery of aprogrammed cell death pathway in RBCs,35 and themiRNA biogenesis pathway in platelets,28 themiRNA trends observed in both types of storedblood cells may be indicative of a relationshipbetween miRNA expression signatures and storage

lesions. Although these 2 recent studies provided aproof-of-concept that miRNAs can be profiled instored blood cells, these results are only apromising first step toward undertaking globalmiRNA profiling to identify a correlative trend ofmiRNAs that could serve as surrogates of measur-able quality characteristics for these cells.

FUTURE PROSPECTS FOR miRNAs IN STOREDBLOOD CELLS

Further studies designed to assess whether adirect relationship between these miRNAs andtheir predicted apoptosis-related targets (mRNAs)exists will also provide evidence of an interactionthat is relevant to storage lesions in stored bloodcells. Given the association of these miRNAs withapoptosis, it would seem reasonable to predictthat they will serve as appropriate markers forstorage lesions. However, storage lesion is acollective description for a diverse set ofmolecular and physiological changes, and estab-lishing a comprehensive miRNA profile mightreveal multiple miRNAs indicative of varioustypes of storage lesions.

Storage lesions are but one issue affecting theefficacy of stored blood cells after transfusion.Some of the examples taken from other areas ofresearch—plasma miRNA-122 levels as a bio-marker of hepatic diseases,36 induction of miRNA-155 expression in T cells by Helicobacter pylori,37

and the observed correlation between miRNAexpression levels and clinical parameters associat-ed with chronic hepatitis C viral infection inhumans38—all suggest that if changes in miRNAprofiles are proven consistent with the cellularstatus of stored blood cells, then these small RNAsmight one day play a role in predicting bloodproduct safety and efficacy. For example, corre-lating and validating miRNA profiles of ex vivostored blood components associated with specificimpurities, contamination, and activation of plate-lets as well as with a host of other adventitiousfactors affecting these products would provide newopportunities in ensuring blood component safety.Additional work on miRNA expression profilingwill no doubt provide a wealth of informationabout the role of these regulatory molecules instored blood cells, and perhaps provide anindication of stored blood cell quality that couldbe useful in both clinical and point-of-carelaboratory settings.

251MicroRNAs IN STORED BLOOD CELLS

REFERENCES

1. Bartel DP: MicroRNAs: Genomics, biogenesis, mecha-nism, and function. Cell 116:281-297, 2004

2. Griffiths-Jones S, Saini HK, vanDS, et al: miRBase: Toolsfor microRNA genomics. Nucleic Acids Res 36:D154-D158,2008 (Database issue)

3. Calin GA, Cimmino A, Fabbri M, et al: MiR-15a andmiR-16-1 cluster functions in human leukemia. Proc Natl AcadSci U S A 105:5166-5171, 2008

4. Vatolin S, NavaratneK,Weil RJ: A novelmethod to detectfunctional microRNA targets. J Mol Biol 358:983-996, 2006

5. Friedman RC, Farh KK, Burge CB, et al: Mostmammalian mRNAs are conserved targets of microRNAs.Genome Res 19:92-105

6. Denli AM, Tops BB, Plasterk RH, et al: Processing ofprimary microRNAs by the microprocessor complex. Nature432:231-235, 2004

7. Gregory RI, Yan KP, Amuthan G, et al: The micropro-cessor complex mediates the genesis of microRNAs. Nature432:235-240, 2004

8. Lee Y, Ahn C, Han J, et al: The nuclear RNase III Droshainitiates microRNA processing. Nature 425:415-419, 2003

9. Provost P, Dishart D, Doucet J, et al: Ribonucleaseactivity and RNA binding of recombinant human Dicer. EMBO J21:5864-5874, 2002

10. Chendrimada TP, Gregory RI, Kumaraswamy E, et al:TRBP recruits the Dicer complex to Ago2 for microRNAprocessing and gene silencing. Nature 436:740-744, 2005

11. Liu J, Carmell MA, Rivas FV, et al: Argonaute2 is thecatalytic engine of mammalian RNAi. Science 305:1437-1441,2004

12. Cogswell JP, Ward J, Taylor IA, et al: Identification ofmiRNA changes in Alzheimer's disease brain and CSF yieldsputative biomarkers and insights into disease pathways. J Alzhei-mersDis14:27-41,2008

13. DeSE, Ferretti E,GulinoA:MicroRNAs as biomarkers forCNS cancer and other disorders. Brain Res 1338:100-111, 2010

14. Li JY, Yong TY, Michael MZ, et al: Review: The role ofmicroRNAs in kidney disease. Nephrology (Carlton) 15:599-608, 2010

15. Buckland J: Biomarkers: Micrornas under the spotlight ininflammatory arthritis. Nat Rev Rheumatol 6:436

16. Croce CM: Causes and consequences of microRNAdysregulation in cancer. Nat Rev Genet 10:704-714, 2009

17. Bloomston M, Frankel WL, Petrocca F, et al: MicroRNAexpression patterns to differentiate pancreatic adenocarcinomafrom normal pancreas and chronic pancreatitis. JAMA 297:1901-1908, 2007

18. Chan SH, Wu CW, Li AF, et al: miR-21 microRNAexpression in human gastric carcinomas and its clinicalassociation. Anticancer Res 28:907-911, 2008

19. Lawrie CH, Gal S, Dunlop HM, et al: Detection ofelevated levels of tumour-associated microRNAs in serum ofpatients with diffuse large B-cell lymphoma. Br J Haematol141:672-675, 2008

20. Kulkarni S, Kannan M, Atreya CD: Omic approaches toquality biomarkers for stored platelets: Arewe there yet? TransfusMed Rev 24:211-217, 2010

21. US Department of Health and Human Services. The 2007NationwideBlood Collection andUtilization SurveyReport. 2007.

22. D'Alessandro A, Liumbruno G, Grazzini G, et al: Redblood cell storage: The story so far. Blood Transfus 8:82-88,2010

23. Solheim BG, Flesland O, Seghatchian J, et al: Clinicalimplications of red blood cell and platelet storage lesions: Anoverview. Transfus Apher Sci 31:185-189, 2004

24. Gnatenko DV, Dunn JJ, McCorkle SR, et al: Transcriptprofiling of human platelets using microarray and serial analysisof gene expression. Blood 101:2285-2293, 2003

25. Hamilton AJ: MicroRNA in erythrocytes. Biochem SocTrans 38(Pt 1):229-231, 2010

26. Warshaw AL, Laster L, Shulman NR: Protein synthesisby human platelets. J Biol Chem 242:2094-2097, 1967

27. Weyrich AS, Schwertz H, Kraiss LW, et al: Proteinsynthesis by platelets: Historical and new perspectives. J ThrombHaemost 7:241-246, 2009

28. Landry P, Plante I, Ouellet DL, et al: Existence of amicroRNA pathway in anucleate platelets. Nat Struct Mol Biol16:961-966, 2009

29. Kannan M, Mohan KV, Kulkarni S, et al: Membranearray-based differential profiling of platelets during storage for52 miRNAs associated with apoptosis. Transfusion 49:1443-1450, 2009

30. Kondkar AA, Bray MS, Leal SM, et al: VAMP8/endobrevin is overexpressed in hyperreactive human platelets:Suggested role for platelet microRNA. J Thromb Haemost8:369-378, 2010

31. Peter KS: Red blood cells. Int J Biochem Cell Biol34:1513-1518, 2002

32. Chen SY, Wang Y, Telen MJ, et al: The genomic analysisof erythrocyte microRNA expression in sickle cell diseases.PLoS One e2360:3

33. Rathjen T, Nicol C, McConkey G, et al: Analysis of shortRNAs in the malaria parasite and its red blood cell host. FEBSLett 580:5185-5188, 2006

34. Kannan M, Atreya C: Differential profiling of human redblood cells during storage for 52 selected microRNAs.Transfusion 50:1581-1588, 2010

35. Bratosin D, Estaquier J, Petit F, et al: Programmed celldeath in mature erythrocytes: A model for investigating deatheffector pathways operating in the absence of mitochondria.Cell Death Differ 8:1143-1156, 2001

36. Zhang Y, Jia Y, Zheng R, et al: PlasmaMicroRNA-122 asa biomarker for viral-, alcohol-, and chemical-related hepaticdiseases. Clin Chem 56:1820-1838, 2010

37. Fehri LF, Rechner C, Janssen S, et al: Helicobacterpylori-induced modification of the histone H3 phosphorylationstatus in gastric epithelial cells reflects its impact on cell cycleregulation. Epigenetics 4:577-586, 2009

38. Marquez RT, Bandyopadhyay S, Wendlandt EB, et al:Correlation between microRNA expression levels and clinicalparameters associated with chronic hepatitis C viral infection inhumans. Lab Invest 90:1727-1736, 2010

39. Krol J, Loedige I, Filipowicz W: The widespreadregulation of microRNA biogenesis, function and decay. NatRev Genet 11:597-610, 2010

40. Wang B, Li S, Qi HH, et al: Distinct passenger strand andmRNA cleavage activities of human Argonaute proteins. NatStruct Mol Biol 16:1259-1266, 2009