- Email: [email protected]
The role of microRNAs in platelet biology during storage
ARTICLE IN PRESS Transfusion and Apheresis Science ■■ (2016) ■■–■■
Contents lists available at ScienceDirect
Transfusion and Apheresis Science j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / t r a n s c i
Review
The role of microRNAs in platelet biology during storage Yuzhong Yan a,b,1, Jingjun Zhang a,1, Qi Zhang a, Yanping Chen b, Xinfang Zhu a, Rong Xia a,* a b
Department of Transfusion Medicine, Shanghai Huashan Hospital, Fudan University, Shanghai, China Department of Clinical Laboratory, Shanghai Pudong Hospital, Fudan University, Shanghai, China
A R T I C L E
I N F O
Keywords: microRNA mRNA Platelet storage lesion
A B S T R A C T
Platelet storage lesions seriously affect the quality of stored platelets, even causing them to be ineffective in vivo after transfusion. Past research have been focused on what mechanism(s) cause the formation of storage lesions. One proposed mechanism is microRNAs (miRNAs)-based molecular regulation of the platelet mRNAs that are relevant to the storage lesion. Platelets continue to translate proteins from mRNA while in a storage environment. A strong correlation exists between the platelet transcriptome and its subsequent proteomic profile, which supports de novo platelet translational capabilities. Thus, miRNA may play a crucial role in platelet biology during storage conditions. Importantly, this suggests the exciting possibility of post-transcriptional regulation of gene expression in platelets that are in storage. Given this, the differential profiling of miRNAs could be a useful tool in identifying changes to ex vivo stored platelets. Any identified miRNAs could then be considered as potential markers to assess the viability of platelet concentrates. The present review summarizes the current experimental and clinical evidence that clarifies the role miRNAs play during platelet ex vivo storage. © 2016 Elsevier Ltd. All rights reserved.
Contents 1. 2. 3. 4. 5.
Introduction .............................................................................................................................................................................................................................. miRNA–targeted mRNA platelet interactions during storage .................................................................................................................................. Differential platelet miRNA profiling during storage .................................................................................................................................................. miRNAs as potential markers for the assessment of platelet concentrate viability ........................................................................................ Conclusion ................................................................................................................................................................................................................................. Funding ....................................................................................................................................................................................................................................... References ..................................................................................................................................................................................................................................
1. Introduction Platelets are anucleated cells, but they contain numerous functional organelles such as the endoplasmic reticulum, Golgi apparatus, and mitochondria. They have numerous
* Corresponding author. Department of Transfusion Medicine, Huashan Hospital, Fudan University, Shanghai 200040, China. Fax: 86 021 62482324. E-mail address: [email protected] (R. Xia). 1 These authors contribute equally to this work.
1 2 2 3 3 4 4
metabolic activities. As small, enucleate cellular fragments, platelets have a wide array of surface receptors and adhesion molecules in addition to numerous granules that have been generated from megakaryocytes. Although in vivo platelets have a natural life span of 8–12 days, in vitro storage conditions cause them to undergo morphological and physiological changes that are collectively known as the platelet storage lesion. Those that are collected for therapeutic or prophylactic transfusion are currently limited to 5–7 days of room temperature storage [1].
http://dx.doi.org/10.1016/j.transci.2016.10.010 1473-0502/© 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Yuzhong Yan, et al., The role of microRNAs in platelet biology during storage, Transfusion and Apheresis Science (2016), doi: 10.1016/j.transci.2016.10.010
ARTICLE IN PRESS 2
Y. Yan et al. / Transfusion and Apheresis Science ■■ (2016) ■■–■■
Platelet storage lesions seriously affect the quality of stored platelets, even resulting in their ineffectiveness in vivo after transfusion. For many years, research have focused on the mechanisms underlying the development of storage lesions [2,3]. One proposed mechanism is that of microRNAs (miRNAs)-based molecular regulation of platelet mRNAs relevant to storage lesions. In ex vivo storage, platelets use their inherent post-transcriptional, regulatory machinery to regulate important physiological processes. Research on miRNA– targeted mRNA interactions would open a new dimension into the experimental biology of stored platelets. Crucially, this could facilitate enhancements to the quality and shelf-life of ex vivo-stored platelets. Despite numerous research into miRNAs over the past decades, surprisingly little is known about how they function in platelets subjected to storage conditions. This is important because platelets continue to translate proteins from mRNA under blood bank storage conditions [4]. Given the lack of information, it is possible that miRNA plays a critical part in platelet biology during storage. This review summarizes the current experimental and clinical evidence concerned with the role of miRNAs during platelet ex vivo storage. 2. miRNA–targeted mRNA platelet interactions during storage Mature platelets receive and maintain from megakaryocytes many components necessary to gene regulation (e.g. mRNAs, pre-miRNAs, Dicer, and Ago2). Platelets being held in storage conditions not only continue to translate inherited mRNAs into proteins [5], but also to process premiRNAs into mature miRNAs [4]. There is a strong correlation between the platelet transcriptome and its proteomic profile, which supports that de novo platelet translational capabilities exist. Moreover, it suggests the exciting possibility of post-transcriptional regulation of gene expression within platelets during storage conditions. In humans, miRNAs are predicted to regulate ~60% of protein-coding genes [6] and are believed to be involved in most – if not all – physiological and pathological processes. As such, one could anticipate that microRNAs would play a significant role in both health and disease [7]. To this end, serial analysis of gene expression (SAGE) data have revealed that the average length of 3’UTR of platelet transcripts (1047 nt) is much longer than that of nucleated cells (492 nt). Since miRNA-mediated gene regulation depends on binding site availability and occupancy at target mRNAs 3’UTR, a longer one not only suggests more robust posttranscriptional regulation, but also mRNA stability via other mechanisms [8]. Based on the SAGE analysis, it was concluded that miRNAs can target multiple mRNAs, with most mRNAs being targeted by multiple miRNAs. Target prediction is a critical aspect of miRNA research, since targets dictate how miRNAs function. Each type of miRNA binds to several different mRNAs, thus creating a complex, interactive network. To this end, Landry et al. have reported that platelet mRNAs might be decorated at multiple sites by Ago2–miRNA complexes. This would result in comparatively stronger mRNA repression than in other cells [9]. The abundance of plate-
let miRNAs relative to mRNAs suggests that the normal ratio seen in nucleated cells of the hematopoietic system is significantly altered in platelets. Furthermore, they suggest an important role for miRNAs in modulating mRNA translation in platelets, unveiling a link between microRNA profiles and platelet reactivity. miRNAs continue to exert activity in platelets undergoing storage conditions. Expression of miRNA–mRNA pairs associated with platelet phenotypes could be altered during platelet storage. Identification of miRNA:mRNA pairs has led to the discovery of novel regulators of platelet function. Reducing miRNAs in platelets by eliminating miRNA processing machinery results in platelets with altered levels of mRNA important to normal platelet functioning [10]. Researchers have found that there is increasing synthesis of integrin, a protein that functions as a fibrinogen receptor involved in platelet aggregation, during platelet preservation and storage [4,11]. Full-length mRNA for αIIbβ3 was present throughout a 10-day storage period and translation of the message into protein was demonstrated by incorporation of [35S]-methionine into sequence-confirmed immunoprecipitated αIIbβ3 protein. To this end, a select number of miRNAs, which regulate integrin subunit expression, have been previously documented [10,12]. Finally, additional expressions of miRNA–mRNA pairs were found to correlate with platelet aggregation [13]. Recently, Dahiya et al. profiled both miRNAs and mRNAs in ex vivo stored platelets at different time points to examine storage-associated changes in mRNA levels. They then correlated them with differentially expressed miRNAs [14]. However, the exact contribution of each of miRNAs – or the contribution of each distinct miRNA–mRNA pair – remains to be determined. 3. Differential platelet miRNA profiling during storage Existing pre-miRNAs undergo cleavage to form mature miRNAs, with these liberated miRNAs participating further in the regulation of specific mRNAs. Dahiya et al. suggested that differential miRNA profiling could be a useful tool in identifying changes that occur to platelets during ex vivo storage [14]. Reports examining storage conditions have also indicated that platelet miRNA profiles at different time intervals are different from those at rest [15–17]. These studies revealed a link between microRNA profiles and subsequent platelet reactivity, thereby suggesting the important role played by post-transcriptional regulation during storage. Differences in platelet miRNA profiles may result from various physiological and pathological conditions. This might be the result of their partial biogenesis from pre-miRNAs. In addition to mature miRNAs, the detection of both premiRNA transcripts and the presence of functional core components of the miRNA effector complex have suggested that partial biogenesis of mature miRNA from premiRNA templates takes place directly within platelets [9]. A possible explanation for such differential miRNA expression could be that platelets simultaneously contain (1) pre-miRNA that are processed to mature miRNAs and (2) miRNA-degrading enzymes and/or an miRNA partitioning mechanism that promotes the maturation of miRNA. Since anucleated platelets are unable to synthesize new miRNAs,
Please cite this article in press as: Yuzhong Yan, et al., The role of microRNAs in platelet biology during storage, Transfusion and Apheresis Science (2016), doi: 10.1016/j.transci.2016.10.010
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it is expected that the number of miRNAs would decrease during storage conditions. However, Pontes et al. found that the number of miRNAs increased by the seventh day of storage [16]. One hypothesis for this finding is that the pre-miRNAs of mature miRNAs charged with regulating senescence were cleaved after the fifth day of storage by RNA editing enzymes. Thus, the number of identified miRNAs would increase [18]. Another hypothesis posits that small regulatory RNAs could be cleaved, resulting in inhibition of stress-induced protein translation [19]. Platelet microparticles are rich in a variety of effector proteins and also contain RNA. These microparticles have been regarded as delivery shuttles and an important part of cell– cell communication [20,21]. During blood processing and storage, platelet microparticle release is triggered by stimuli like shear stresses, activation, generation of platelet agonists, and the ongoing blood cell apoptosis. Decreases to platelet miRNA was due to their activation and release of miRNA-rich microparticles [20,22]. In vitro studies [22,23] have shown that platelets treated for pathogen reduction results in a decrease in miRNA content from the activated platelets via microparticles. The idea was confirmed by an increased amount of microparticular miR-223 in the supernatant of treated platelets. Microparticle miRNA profiles could also differ from source cells, indicating a selective packaging of miRNAs from cells to microparticles [20]. Thus, platelet miRNA profiles may be modified as a consequence of the activation-induced release of miRNA-containing microparticles. Platelet miRNAs bear signs of post-transcriptional modifications, which are expected to influence miRNA biogenesis and stability. For instance, Jones et al. found that posttranscriptional modification of miRNA transcripts by terminal modifications affected their ability to efficiently silence mRNA targets [24]. In vitro uridylation and adenylation assays have shown the capability of platelets to uridylate singlestranded miRNAs, which correlated with platelet expression of the uridyltransferase enzyme TUT-4 [7]. Although these terminal modifications are likely to occur during miRNA biogenesis, the molecular mechanisms underlying the differential, polar modifications that occur to stored platelet miRNAs remains unclear. 4. miRNAs as potential markers for the assessment of platelet concentrate viability The storage period for platelet concentrates depends on local legislation and the additive used in the concentrate solution. Despite these possible differences, all unused platelet concentrate bags are discarded after 5–7 days of storage in a blood bank. However, many of these bags may contain functional platelets since the kinetics of cellular aging are influenced by age, type of nutrition, and exposure to environmental agents of the blood donors [25,26]. As such, there are currently a few methods available to allow for a more selective assessment of viable platelet concentrates. The first report [15] published regarding this issue used membrane arrays and found that a few platelet miRNAs of the 52 selected apoptosis-associated miRNAs were differentially expressed during the various time points of platelet storage. This suggested that this subgroup of differentially
3
expressed platelet miRNAs had potential as storage biomarkers for platelet quality. Furthermore, this work suggested that two miRNAs – let-7b and miR-16—showed increasing levels during storage. In comparison, two others – miR-7 and miR-145—showed decreasing levels. This work was the first to investigate the use of miRNAs as platelet markers for viability and opened the question to further research. To this end, Yu et al. measured miRNA levels in platelets at 1, 3, and 5 days after apheresis and found ten had altered expression levels [17]. Of these, miR-326 was found to increase over time in addition to down-regulating the expression of the anti-apoptotic gene Bcl-xL. Ultimately, this resulted in platelet apoptosis [27]. Consistent with the above results, the increase of another miRNA – miR-16—during platelet storage targeted genes involved in apoptosis. This provided further support for the idea that platelet apoptosis is regulated by miRNAs. Deep sequencing of platelets stored for 1 to 5 days revealed a gradual loss of the most abundant miRNAs and suggested the use of miRNA profiles as biomarkers for stored platelet quality [16]. For example, Pontes et al. found that the inverse expression between miR-127 and miR-320a depended on storage time. Moreover, this relationship could allow for the identification of platelet concentrate bags that still exhibited physiologically normal (not activated) platelets. This would be the case even after long storage periods (more than 5 days) and would allow their use for blood transfusions. A bioinformatics analysis showed that these miRNAs regulated several genes related to mitochondrial energy metabolism. Dahiya et al. found that miR-570 interacted with mitochondrial ATPase subunit g (ATP5L) encoding mRNA in stored platelets. Thus, miR-570 could also serve as a biomarker for stored platelet quality and viability [28] . Finally, Osman et al. reported that miRNA content was altered in stored platelets that had been treated for pathogen reduction [23]. Moreover, it coincided with platelet activation and the impairment of some aspects of platelet function. These results could also provide at least a partial explanation for the platelet losses that occur secondarily to pathogen reduction treatment [23,29,30]. Deregulation of these platelet mRNAs could also be consecutive to alterations in specific miRNA levels [31], thus resulting in impaired protein synthesis during blood bank storage [5]. Taken together, past work has shown that miRNA regulation is not only associated with changes to relevant gene and protein expression, but that these changes also come with functional consequences. These selected miRNAs could be considered as potential markers to assess platelet concentrate viability.
5. Conclusion The discovery of miRNAs revealed a previously unknown layer of regulation for several biological processes important to platelet storage. Research into the regulatory network of platelet miRNA will continue to unravel many poorly understood events that occur during platelet storage (e.g. platelet storage lesions). This continued work will provide
Please cite this article in press as: Yuzhong Yan, et al., The role of microRNAs in platelet biology during storage, Transfusion and Apheresis Science (2016), doi: 10.1016/j.transci.2016.10.010
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information aimed at better understanding the regulatory mechanisms behind platelet apoptosis. With accumulating evidence for the crucial role noncoding RNAs play in biology, it is likely that miRNAs are important regulators of platelet physiology. Differential miRNA expression during platelet storage shows the promise of using miRNAs as biomarkers for platelets undergoing storage. Such studies should help facilitate improvements to platelet quality during storage, enhancements to their storage shelf-life, as well as better patient outcomes after transfusion. Funding This study was funded by National Natural Science Foundation of China (81470351), funding in the 4th round of Shanghai Municipal Key Disciplines of Public Health on Transfusion Medicine (15GWZK0501), and Shanghai Municipal Commission of Health and Family Planning (201440056). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. References [1] Rijkers M, van der Meer PF, Bontekoe IJ, Daal BB, de Korte D, Leebeek FW, et al. Evaluation of the role of the GPIb-IX-V receptor complex in development of the platelet storage lesion. Vox Sang 2016;111(3): 247–56. [2] Cauwenberghs S, van Pampus E, Curvers J, Akkerman JW, Heemskerk JW. Hemostatic and signaling functions of transfused platelets. Transfus Med Rev 2007;21(4):287–94. [3] Prochazkova R, Andrys C, Hubackova L, Krejsek J. Markers of platelet activation and apoptosis in platelet concentrates collected by apheresis. Transfus Apher Sci 2007;37(2):115–23. [4] Thon JN, Devine DV. Translation of glycoprotein IIIa in stored blood platelets. Transfusion 2007;47(12):2260–70. [5] Edelstein LC, Bray PF. MicroRNAs in platelet production and activation. Blood 2011;117(20):5289–96. [6] Bartel DP. MicroRNAs: target recognition and regulatory functions. Cell 2009;136(2):215–33. [7] Ple H, Landry P, Benham A, Coarfa C, Gunaratne PH, Provost P. The repertoire and features of human platelet microRNAs. PLoS ONE 2012;7(12):e50746. [8] Dittrich M, Birschmann I, Pfrang J, Herterich S, Smolenski A, Walter U, et al. Analysis of SAGE data in human platelets: features of the transcriptome in an anucleate cell. Thromb Haemost 2006;95(4): 643–51. [9] Landry P, Plante I, Ouellet DL, Perron MP, Rousseau G, Provost P. Existence of a microRNA pathway in anucleate platelets. Nat Struct Mol Biol 2009;16(9):961–6. [10] Rowley JW, Chappaz S, Corduan A, Chong MM, Campbell R, Khoury A, et al. Dicer1-mediated miRNA processing shapes the mRNA profile and function of murine platelets. Blood 2016;127(14):1743–51.
[11] Weyrich AS, Schwertz H, Kraiss LW, Zimmerman GA. Protein synthesis by platelets: historical and new perspectives. J Thromb Haemost 2009;7(2):241–6. [12] Chen W, Harbeck MC, Zhang W, Jacobson JR. MicroRNA regulation of integrins. Transl Res 2013;162(3):133–43. [13] Nagalla S, Shaw C, Kong X, Kondkar AA, Edelstein LC, Ma L, et al. Platelet microRNA-mRNA coexpression profiles correlate with platelet reactivity. Blood 2011;117(19):5189–97. [14] Dahiya N, Sarachana T, Vu L, Becker KG, Wood WH 3rd, Zhang Y, et al. Platelet MicroRNAs: an overview. Transfus Med Rev 2015;29(4):215– 9. [15] Kannan M, Mohan KV, Kulkarni S, Atreya C. Membrane array-based differential profiling of platelets during storage for 52 miRNAs associated with apoptosis. Transfusion 2009;49(7):1443–50. [16] Pontes TB, Moreira-Nunes Cde F, Maues JH, Lamarao LM, de Lemos JA, Montenegro RC, et al. The miRNA profile of platelets stored in a blood bank and its relation to cellular damage from storage. PLoS ONE 2015;10(6):e0129399. [17] Yu S, Deng G, Qian D, Xie Z, Sun H, Huang D, et al. Detection of apoptosis-associated microRNA in human apheresis platelets during storage by quantitative real-time polymerase chain reaction analysis. Blood Transfus 2014;12(4):541–7. [18] Abdelmohsen K, Srikantan S, Kang MJ, Gorospe M. Regulation of senescence by microRNA biogenesis factors. Ageing Res Rev 2012; 11(4):491–500. [19] Kato M, Slack FJ. Ageing and the small, non-coding RNA world. Ageing Res Rev 2013;12(1):429–35. [20] Diehl P, Fricke A, Sander L, Stamm J, Bassler N, Htun N, et al. Microparticles: major transport vehicles for distinct microRNAs in circulation. Cardiovasc Res 2012;93(4):633–44. [21] Hunter MP, Ismail N, Zhang X, Aguda BD, Lee EJ, Yu L, et al. Detection of microRNA expression in human peripheral blood microvesicles. PLoS ONE 2008;3(11):e3694. [22] Mundt JM, Rouse L, Van den Bossche J, Goodrich RP. Chemical and biological mechanisms of pathogen reduction technologies. Photochem Photobiol 2014;90(5):957–64. [23] Osman A, Hitzler WE, Meyer CU, Landry P, Corduan A, Laffont B, et al. Effects of pathogen reduction systems on platelet microRNAs, mRNAs, activation, and function. Platelets 2015;26(2):154–63. [24] Jones MR, Quinton LJ, Blahna MT, Neilson JR, Fu S, Ivanov AR, et al. Zcchc11-dependent uridylation of microRNA directs cytokine expression. Nat Cell Biol 2009;11(9):1157–63. [25] Pope CA 3rd, Hansen ML, Long RW, Nielsen KR, Eatough NL, Wilson WE, et al. Ambient particulate air pollution, heart rate variability, and blood markers of inflammation in a panel of elderly subjects. Environ Health Perspect 2004;112(3):339–45. [26] Leytin V. Apoptosis in the anucleate platelet. Blood Rev 2012;26(2): 51–63. [27] Yu S, Huang H, Deng G, Xie Z, Ye Y, Guo R, et al. miR-326 targets antiapoptotic Bcl-xL and mediates apoptosis in human platelets. PLoS ONE 2015;10(4):e0122784. [28] Dahiya N, Sarachana T, Kulkarni S, Wood Iii WH, Zhang Y, Becker KG, et al. miR-570 interacts with mitochondrial ATPase subunit g (ATP5L) encoding mRNA in stored platelets. Platelets 2016;1–8. [29] Butler C, Doree C, Estcourt LJ, Trivella M, Hopewell S, Brunskill SJ, et al. Pathogen-reduced platelets for the prevention of bleeding. Cochrane Database Syst Rev 2013;(3):CD009072. [30] Johnson L, Loh YS, Kwok M, Marks DC. In vitro assessment of buffycoat derived platelet components suspended in SSP+ treated with the INTERCEPT Blood system. Transfus Med 2013;23(2):121–9. [31] Ple H, Maltais M, Corduan A, Rousseau G, Madore F, Provost P. Alteration of the platelet transcriptome in chronic kidney disease. Thromb Haemost 2012;108(4):605–15.
Please cite this article in press as: Yuzhong Yan, et al., The role of microRNAs in platelet biology during storage, Transfusion and Apheresis Science (2016), doi: 10.1016/j.transci.2016.10.010
Contents lists available at ScienceDirect
Transfusion and Apheresis Science j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / t r a n s c i
Review
The role of microRNAs in platelet biology during storage Yuzhong Yan a,b,1, Jingjun Zhang a,1, Qi Zhang a, Yanping Chen b, Xinfang Zhu a, Rong Xia a,* a b
Department of Transfusion Medicine, Shanghai Huashan Hospital, Fudan University, Shanghai, China Department of Clinical Laboratory, Shanghai Pudong Hospital, Fudan University, Shanghai, China
A R T I C L E
I N F O
Keywords: microRNA mRNA Platelet storage lesion
A B S T R A C T
Platelet storage lesions seriously affect the quality of stored platelets, even causing them to be ineffective in vivo after transfusion. Past research have been focused on what mechanism(s) cause the formation of storage lesions. One proposed mechanism is microRNAs (miRNAs)-based molecular regulation of the platelet mRNAs that are relevant to the storage lesion. Platelets continue to translate proteins from mRNA while in a storage environment. A strong correlation exists between the platelet transcriptome and its subsequent proteomic profile, which supports de novo platelet translational capabilities. Thus, miRNA may play a crucial role in platelet biology during storage conditions. Importantly, this suggests the exciting possibility of post-transcriptional regulation of gene expression in platelets that are in storage. Given this, the differential profiling of miRNAs could be a useful tool in identifying changes to ex vivo stored platelets. Any identified miRNAs could then be considered as potential markers to assess the viability of platelet concentrates. The present review summarizes the current experimental and clinical evidence that clarifies the role miRNAs play during platelet ex vivo storage. © 2016 Elsevier Ltd. All rights reserved.
Contents 1. 2. 3. 4. 5.
Introduction .............................................................................................................................................................................................................................. miRNA–targeted mRNA platelet interactions during storage .................................................................................................................................. Differential platelet miRNA profiling during storage .................................................................................................................................................. miRNAs as potential markers for the assessment of platelet concentrate viability ........................................................................................ Conclusion ................................................................................................................................................................................................................................. Funding ....................................................................................................................................................................................................................................... References ..................................................................................................................................................................................................................................
1. Introduction Platelets are anucleated cells, but they contain numerous functional organelles such as the endoplasmic reticulum, Golgi apparatus, and mitochondria. They have numerous
* Corresponding author. Department of Transfusion Medicine, Huashan Hospital, Fudan University, Shanghai 200040, China. Fax: 86 021 62482324. E-mail address: [email protected] (R. Xia). 1 These authors contribute equally to this work.
1 2 2 3 3 4 4
metabolic activities. As small, enucleate cellular fragments, platelets have a wide array of surface receptors and adhesion molecules in addition to numerous granules that have been generated from megakaryocytes. Although in vivo platelets have a natural life span of 8–12 days, in vitro storage conditions cause them to undergo morphological and physiological changes that are collectively known as the platelet storage lesion. Those that are collected for therapeutic or prophylactic transfusion are currently limited to 5–7 days of room temperature storage [1].
http://dx.doi.org/10.1016/j.transci.2016.10.010 1473-0502/© 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Yuzhong Yan, et al., The role of microRNAs in platelet biology during storage, Transfusion and Apheresis Science (2016), doi: 10.1016/j.transci.2016.10.010
ARTICLE IN PRESS 2
Y. Yan et al. / Transfusion and Apheresis Science ■■ (2016) ■■–■■
Platelet storage lesions seriously affect the quality of stored platelets, even resulting in their ineffectiveness in vivo after transfusion. For many years, research have focused on the mechanisms underlying the development of storage lesions [2,3]. One proposed mechanism is that of microRNAs (miRNAs)-based molecular regulation of platelet mRNAs relevant to storage lesions. In ex vivo storage, platelets use their inherent post-transcriptional, regulatory machinery to regulate important physiological processes. Research on miRNA– targeted mRNA interactions would open a new dimension into the experimental biology of stored platelets. Crucially, this could facilitate enhancements to the quality and shelf-life of ex vivo-stored platelets. Despite numerous research into miRNAs over the past decades, surprisingly little is known about how they function in platelets subjected to storage conditions. This is important because platelets continue to translate proteins from mRNA under blood bank storage conditions [4]. Given the lack of information, it is possible that miRNA plays a critical part in platelet biology during storage. This review summarizes the current experimental and clinical evidence concerned with the role of miRNAs during platelet ex vivo storage. 2. miRNA–targeted mRNA platelet interactions during storage Mature platelets receive and maintain from megakaryocytes many components necessary to gene regulation (e.g. mRNAs, pre-miRNAs, Dicer, and Ago2). Platelets being held in storage conditions not only continue to translate inherited mRNAs into proteins [5], but also to process premiRNAs into mature miRNAs [4]. There is a strong correlation between the platelet transcriptome and its proteomic profile, which supports that de novo platelet translational capabilities exist. Moreover, it suggests the exciting possibility of post-transcriptional regulation of gene expression within platelets during storage conditions. In humans, miRNAs are predicted to regulate ~60% of protein-coding genes [6] and are believed to be involved in most – if not all – physiological and pathological processes. As such, one could anticipate that microRNAs would play a significant role in both health and disease [7]. To this end, serial analysis of gene expression (SAGE) data have revealed that the average length of 3’UTR of platelet transcripts (1047 nt) is much longer than that of nucleated cells (492 nt). Since miRNA-mediated gene regulation depends on binding site availability and occupancy at target mRNAs 3’UTR, a longer one not only suggests more robust posttranscriptional regulation, but also mRNA stability via other mechanisms [8]. Based on the SAGE analysis, it was concluded that miRNAs can target multiple mRNAs, with most mRNAs being targeted by multiple miRNAs. Target prediction is a critical aspect of miRNA research, since targets dictate how miRNAs function. Each type of miRNA binds to several different mRNAs, thus creating a complex, interactive network. To this end, Landry et al. have reported that platelet mRNAs might be decorated at multiple sites by Ago2–miRNA complexes. This would result in comparatively stronger mRNA repression than in other cells [9]. The abundance of plate-
let miRNAs relative to mRNAs suggests that the normal ratio seen in nucleated cells of the hematopoietic system is significantly altered in platelets. Furthermore, they suggest an important role for miRNAs in modulating mRNA translation in platelets, unveiling a link between microRNA profiles and platelet reactivity. miRNAs continue to exert activity in platelets undergoing storage conditions. Expression of miRNA–mRNA pairs associated with platelet phenotypes could be altered during platelet storage. Identification of miRNA:mRNA pairs has led to the discovery of novel regulators of platelet function. Reducing miRNAs in platelets by eliminating miRNA processing machinery results in platelets with altered levels of mRNA important to normal platelet functioning [10]. Researchers have found that there is increasing synthesis of integrin, a protein that functions as a fibrinogen receptor involved in platelet aggregation, during platelet preservation and storage [4,11]. Full-length mRNA for αIIbβ3 was present throughout a 10-day storage period and translation of the message into protein was demonstrated by incorporation of [35S]-methionine into sequence-confirmed immunoprecipitated αIIbβ3 protein. To this end, a select number of miRNAs, which regulate integrin subunit expression, have been previously documented [10,12]. Finally, additional expressions of miRNA–mRNA pairs were found to correlate with platelet aggregation [13]. Recently, Dahiya et al. profiled both miRNAs and mRNAs in ex vivo stored platelets at different time points to examine storage-associated changes in mRNA levels. They then correlated them with differentially expressed miRNAs [14]. However, the exact contribution of each of miRNAs – or the contribution of each distinct miRNA–mRNA pair – remains to be determined. 3. Differential platelet miRNA profiling during storage Existing pre-miRNAs undergo cleavage to form mature miRNAs, with these liberated miRNAs participating further in the regulation of specific mRNAs. Dahiya et al. suggested that differential miRNA profiling could be a useful tool in identifying changes that occur to platelets during ex vivo storage [14]. Reports examining storage conditions have also indicated that platelet miRNA profiles at different time intervals are different from those at rest [15–17]. These studies revealed a link between microRNA profiles and subsequent platelet reactivity, thereby suggesting the important role played by post-transcriptional regulation during storage. Differences in platelet miRNA profiles may result from various physiological and pathological conditions. This might be the result of their partial biogenesis from pre-miRNAs. In addition to mature miRNAs, the detection of both premiRNA transcripts and the presence of functional core components of the miRNA effector complex have suggested that partial biogenesis of mature miRNA from premiRNA templates takes place directly within platelets [9]. A possible explanation for such differential miRNA expression could be that platelets simultaneously contain (1) pre-miRNA that are processed to mature miRNAs and (2) miRNA-degrading enzymes and/or an miRNA partitioning mechanism that promotes the maturation of miRNA. Since anucleated platelets are unable to synthesize new miRNAs,
Please cite this article in press as: Yuzhong Yan, et al., The role of microRNAs in platelet biology during storage, Transfusion and Apheresis Science (2016), doi: 10.1016/j.transci.2016.10.010
ARTICLE IN PRESS Y. Yan et al. / Transfusion and Apheresis Science ■■ (2016) ■■–■■
it is expected that the number of miRNAs would decrease during storage conditions. However, Pontes et al. found that the number of miRNAs increased by the seventh day of storage [16]. One hypothesis for this finding is that the pre-miRNAs of mature miRNAs charged with regulating senescence were cleaved after the fifth day of storage by RNA editing enzymes. Thus, the number of identified miRNAs would increase [18]. Another hypothesis posits that small regulatory RNAs could be cleaved, resulting in inhibition of stress-induced protein translation [19]. Platelet microparticles are rich in a variety of effector proteins and also contain RNA. These microparticles have been regarded as delivery shuttles and an important part of cell– cell communication [20,21]. During blood processing and storage, platelet microparticle release is triggered by stimuli like shear stresses, activation, generation of platelet agonists, and the ongoing blood cell apoptosis. Decreases to platelet miRNA was due to their activation and release of miRNA-rich microparticles [20,22]. In vitro studies [22,23] have shown that platelets treated for pathogen reduction results in a decrease in miRNA content from the activated platelets via microparticles. The idea was confirmed by an increased amount of microparticular miR-223 in the supernatant of treated platelets. Microparticle miRNA profiles could also differ from source cells, indicating a selective packaging of miRNAs from cells to microparticles [20]. Thus, platelet miRNA profiles may be modified as a consequence of the activation-induced release of miRNA-containing microparticles. Platelet miRNAs bear signs of post-transcriptional modifications, which are expected to influence miRNA biogenesis and stability. For instance, Jones et al. found that posttranscriptional modification of miRNA transcripts by terminal modifications affected their ability to efficiently silence mRNA targets [24]. In vitro uridylation and adenylation assays have shown the capability of platelets to uridylate singlestranded miRNAs, which correlated with platelet expression of the uridyltransferase enzyme TUT-4 [7]. Although these terminal modifications are likely to occur during miRNA biogenesis, the molecular mechanisms underlying the differential, polar modifications that occur to stored platelet miRNAs remains unclear. 4. miRNAs as potential markers for the assessment of platelet concentrate viability The storage period for platelet concentrates depends on local legislation and the additive used in the concentrate solution. Despite these possible differences, all unused platelet concentrate bags are discarded after 5–7 days of storage in a blood bank. However, many of these bags may contain functional platelets since the kinetics of cellular aging are influenced by age, type of nutrition, and exposure to environmental agents of the blood donors [25,26]. As such, there are currently a few methods available to allow for a more selective assessment of viable platelet concentrates. The first report [15] published regarding this issue used membrane arrays and found that a few platelet miRNAs of the 52 selected apoptosis-associated miRNAs were differentially expressed during the various time points of platelet storage. This suggested that this subgroup of differentially
3
expressed platelet miRNAs had potential as storage biomarkers for platelet quality. Furthermore, this work suggested that two miRNAs – let-7b and miR-16—showed increasing levels during storage. In comparison, two others – miR-7 and miR-145—showed decreasing levels. This work was the first to investigate the use of miRNAs as platelet markers for viability and opened the question to further research. To this end, Yu et al. measured miRNA levels in platelets at 1, 3, and 5 days after apheresis and found ten had altered expression levels [17]. Of these, miR-326 was found to increase over time in addition to down-regulating the expression of the anti-apoptotic gene Bcl-xL. Ultimately, this resulted in platelet apoptosis [27]. Consistent with the above results, the increase of another miRNA – miR-16—during platelet storage targeted genes involved in apoptosis. This provided further support for the idea that platelet apoptosis is regulated by miRNAs. Deep sequencing of platelets stored for 1 to 5 days revealed a gradual loss of the most abundant miRNAs and suggested the use of miRNA profiles as biomarkers for stored platelet quality [16]. For example, Pontes et al. found that the inverse expression between miR-127 and miR-320a depended on storage time. Moreover, this relationship could allow for the identification of platelet concentrate bags that still exhibited physiologically normal (not activated) platelets. This would be the case even after long storage periods (more than 5 days) and would allow their use for blood transfusions. A bioinformatics analysis showed that these miRNAs regulated several genes related to mitochondrial energy metabolism. Dahiya et al. found that miR-570 interacted with mitochondrial ATPase subunit g (ATP5L) encoding mRNA in stored platelets. Thus, miR-570 could also serve as a biomarker for stored platelet quality and viability [28] . Finally, Osman et al. reported that miRNA content was altered in stored platelets that had been treated for pathogen reduction [23]. Moreover, it coincided with platelet activation and the impairment of some aspects of platelet function. These results could also provide at least a partial explanation for the platelet losses that occur secondarily to pathogen reduction treatment [23,29,30]. Deregulation of these platelet mRNAs could also be consecutive to alterations in specific miRNA levels [31], thus resulting in impaired protein synthesis during blood bank storage [5]. Taken together, past work has shown that miRNA regulation is not only associated with changes to relevant gene and protein expression, but that these changes also come with functional consequences. These selected miRNAs could be considered as potential markers to assess platelet concentrate viability.
5. Conclusion The discovery of miRNAs revealed a previously unknown layer of regulation for several biological processes important to platelet storage. Research into the regulatory network of platelet miRNA will continue to unravel many poorly understood events that occur during platelet storage (e.g. platelet storage lesions). This continued work will provide
Please cite this article in press as: Yuzhong Yan, et al., The role of microRNAs in platelet biology during storage, Transfusion and Apheresis Science (2016), doi: 10.1016/j.transci.2016.10.010
ARTICLE IN PRESS Y. Yan et al. / Transfusion and Apheresis Science ■■ (2016) ■■–■■
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information aimed at better understanding the regulatory mechanisms behind platelet apoptosis. With accumulating evidence for the crucial role noncoding RNAs play in biology, it is likely that miRNAs are important regulators of platelet physiology. Differential miRNA expression during platelet storage shows the promise of using miRNAs as biomarkers for platelets undergoing storage. Such studies should help facilitate improvements to platelet quality during storage, enhancements to their storage shelf-life, as well as better patient outcomes after transfusion. Funding This study was funded by National Natural Science Foundation of China (81470351), funding in the 4th round of Shanghai Municipal Key Disciplines of Public Health on Transfusion Medicine (15GWZK0501), and Shanghai Municipal Commission of Health and Family Planning (201440056). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. References [1] Rijkers M, van der Meer PF, Bontekoe IJ, Daal BB, de Korte D, Leebeek FW, et al. Evaluation of the role of the GPIb-IX-V receptor complex in development of the platelet storage lesion. Vox Sang 2016;111(3): 247–56. [2] Cauwenberghs S, van Pampus E, Curvers J, Akkerman JW, Heemskerk JW. Hemostatic and signaling functions of transfused platelets. Transfus Med Rev 2007;21(4):287–94. [3] Prochazkova R, Andrys C, Hubackova L, Krejsek J. Markers of platelet activation and apoptosis in platelet concentrates collected by apheresis. Transfus Apher Sci 2007;37(2):115–23. [4] Thon JN, Devine DV. Translation of glycoprotein IIIa in stored blood platelets. Transfusion 2007;47(12):2260–70. [5] Edelstein LC, Bray PF. MicroRNAs in platelet production and activation. Blood 2011;117(20):5289–96. [6] Bartel DP. MicroRNAs: target recognition and regulatory functions. Cell 2009;136(2):215–33. [7] Ple H, Landry P, Benham A, Coarfa C, Gunaratne PH, Provost P. The repertoire and features of human platelet microRNAs. PLoS ONE 2012;7(12):e50746. [8] Dittrich M, Birschmann I, Pfrang J, Herterich S, Smolenski A, Walter U, et al. Analysis of SAGE data in human platelets: features of the transcriptome in an anucleate cell. Thromb Haemost 2006;95(4): 643–51. [9] Landry P, Plante I, Ouellet DL, Perron MP, Rousseau G, Provost P. Existence of a microRNA pathway in anucleate platelets. Nat Struct Mol Biol 2009;16(9):961–6. [10] Rowley JW, Chappaz S, Corduan A, Chong MM, Campbell R, Khoury A, et al. Dicer1-mediated miRNA processing shapes the mRNA profile and function of murine platelets. Blood 2016;127(14):1743–51.
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Please cite this article in press as: Yuzhong Yan, et al., The role of microRNAs in platelet biology during storage, Transfusion and Apheresis Science (2016), doi: 10.1016/j.transci.2016.10.010