Microrna Biomarkers For Peripheral Blood Fractions Identification: Possible Forensic Applications

MARIA TERESA VIANA MACHADO MICRORNA BIOMARKERS FOR PERIPHERAL BLOOD FRACTIONS IDENTIFICATION: possible forensic applications Dissertação de Candidatura ao grau de Mestre em Medicina Legal submetida ao Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto Orientador – Professor Doutor Rui Manuel de Medeiros Melo Silva Categoria – Professor Associado Convidado Afiliação – Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto e Grupo de Oncologia Molecular e Patologia Viral, Centro Investigação do Instituto Português de Oncologia do Porto Co-orientadora – Doutora Ana Luísa Pereira Teixeira Afiliação – Grupo de Oncologia Molecular e Patologia Viral, Centro Investigação do Instituto Português de Oncologia do Porto INFORMAÇÃO TÉCNICA TÍTULO: microRNA biomarkers for peripheral blood fractions identification: possible forensic applications Dissertação de Candidatura ao Grau de Mestre em Medicina Legal, apresentada ao Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto AUTOR: Maria Teresa Viana Machado DATA: Julho de 2015 EDITOR: Maria Teresa Viana Machado MORADA: Rua Maria Lina Alves Maia, n.º 124 LOCALIDADE: Moreira da Maia CÓDIGO POSTAL: 4470-397 Maia CORREIO ELETRÓNICO: [email protected] 1ª EDIÇÃO: Julho de 2015 “Don't be afraid of hard work. Nothing worthwhile comes easily.” – Gertrude B. Elion AGRADECIMENTOS Este projeto foi possível graças ao apoio e dedicação de várias pessoas, pelo que gostaria de agradecer a todos aqueles que contribuíram para a sua realização. Em primeiro lugar agradeço ao meu orientador, Professor Doutor Rui Medeiros, pela oportunidade e privilégio da realização deste projeto, pela integração no grupo de investigação, pela supervisão, orientação, conselhos e sugestões, que foram determinantes para o sucesso do mesmo. À minha co-orientadora, Doutora Ana Luísa Teixeira, o meu sincero agradecimento por tudo: pelo constante apoio, incentivo e motivação; pela confiança depositada em mim; pelo tempo dedicado à minha orientação e ao projeto e, acima de tudo, pela simpatia, dedicação e paciência com que sempre me transmitiu os conhecimentos e que me ajudou a ultrapassar todos os obstáculos. Agradeço ainda à Professora Maria José Pinto da Costa, Diretora do Mestrado em Medicina Legal, pela cordialidade sempre manifestada e pela oportunidade de frequentar este mestrado, o qual foi fundamental para o enriquecimento da minha formação académica e científica. A todo o grupo de Oncologia Molecular e Patologia Viral do Instituto Português de Oncologia do Porto, pela simpatia com que me receberam e pelo permanente apoio. Em especial à Mara Fernandes e à Francisca Dias, pela integração no grupo e no laboratório, por todos os conselhos e por toda a incansável e preciosa ajuda dada ao longo do projeto. À Sarah Silva, pela simpatia com que me apresentou a esta área e pela sempre total disponibilidade demonstrada em me ajudar. Aos meus amigos e colegas de mestrado. À Sílvia Navega, minha colega de licenciatura, de mestrado e de laboratório, pela amizade e companheirismo, essencialmente por me ter acompanhado e aturado em todos os momentos deste ano e por ter tornado todo este percurso mais divertido. Agradeço também a colaboração e a contribuição de todos os dadores de sangue, sem os quais este projeto não teria sido possível. Não posso deixar de agradecer a todos os professores que influenciaram o meu percurso académico e desenvolvimento pessoal, não só pela transmissão de conhecimentos, mas também pela formação e bases que me deram para poder chegar até aqui. Um agradecimento especial à minha família e amigos. Aos meus pais e aos meus avós, pelo apoio incondicional, por acreditarem sempre em mim e nas minhas capacidades, por estarem sempre disponíveis para me ajudarem, pelo incentivo e por todos os sacrifícios que fizeram por mim. Aos meus irmãos, Isabel e Miguel, por me apoiarem à sua maneira, por me “chatearem” menos um bocadinho nesta fase e por me fazerem querer trabalhar mais e melhor para ser um bom exemplo. Aos meus padrinhos e tios, pela confiança transmitida. Aos meus primos, em especial à Ana e à Filipa, por todos os momentos passados e por sempre me fazerem rir. A todos, muito obrigada! Abbreviations microRNA biomarkers for peripheral blood fractions identification: possible forensic applications 7 ABBREVIATIONS A ACC Accuracy AGO Argonaute AKE Ammonium chloride, potassium hydrogen phosphate and EDTA AUC Area Under the Curve C cDNA Complementary Deoxyribonucleic Acid Ct Cycle threshold D DNA Deoxyribonucleic acid E EDTA Ethylenediaminetetraacetic Acid H HDL High Density Lipoprotein M miR microRNA miRNA microRNA mL milliliter mRNA Messenger Ribonucleic Acid MVs Microvesicles N NPV Negative Predictive Value O OR Odds ratio Abbreviations microRNA biomarkers for peripheral blood fractions identification: possible forensic applications 8 P PBS Phosphate Buffered Saline pg picogram PPV Positive Predictive Value or Precision pre-miRNA miRNA precursor pri-miRNA Primitive miRNA PTH Parathyroid Hormone Q qPCR Real-time polymerase chain reaction R RBCs Red Blood Cells RCC Renal Cell Carcinoma RISC RNA induced silencing complex RNA Ribonucleic acid RNase Ribonuclease ROC Receiver Operating Characteristic rpm Revolutions per minute RT-PCR Reverse transcription polymerase chain reaction U L microliter V vs. versus W WBCs White Blood Cells Table of contents microRNA biomarkers for peripheral blood fractions identification: possible forensic applications 9 TABLE OF CONTENTS Page Abstract ………………………………………………………………………………...………... 15 Resumo ………………………………………………………………………………....……….. 19 1. Introduction ………………………………………………………………………………...... 23 1.1. microRNAs …………………………………………………………………………... 23 1.2. Peripheral blood fractions ………………………………………………………….. 25 1.3. Extracellular/circulating microRNAs ……………………………………….. 26 1.4. Origin of circulating plasma and serum microRNAs ……………………... 27 1.4.1. Circulating microRNAs derived from endothelial cells ……………. 29 1.4.2. Circulating microRNAs derived from blood cells …………………... 31 1.4.3. Circulating microRNAs derived from endothelial and blood cells ... 32 1.4.4. Circulating microRNAs with an organ origin ……………………….. 33 1.5. Forensic applications of microRNAs as biomarkers for peripheral blood fractions characterization ……………………………………………………. 36 1.6. Potential microRNA profile for peripheral blood fractions identification ... 38 2. Objectives …………………………………………………………………………………… 41 2.1. Main objective ……………………………………………………………………... 41 2.2. Specific objectives …………………………………………………………….…..... 41 3. Material & Methods …………………………………………………………………….…… 45 3.1. Study population, sample collection and processing …………………………… 45 3.2. microRNA extraction, cDNA synthesis and microRNA relative quantification ………………………………………………………………………… 45 3.3. Statistical Analysis ……………………………………………………………..…… 46 4. Results & Discussion ……………………………………………………………….……… 51 4.1. microRNA detection frequencies ………………………………………………… 51 4.2. microRNA relative quantification in peripheral blood fractions ………………… 52 4.3. microRNA profile according to age and gender ………………………….. 55 4.4. Specificity and sensitivity analysis of miR-24-1-5p as a WBCs biomarker ……………………………………………………………………... 56 5. Conclusion & Future Perspectives .............................................................................. 63 6. References .................................................................................................................. 69 7. Attachments ................................................................................................................ 79 Abstract microRNA biomarkers for peripheral blood fractions identification: possible forensic applications 16 Furthermore, for miR-24-1-5p, the odds ratio (OR), specificity, sensitivity, positive predictive value or precision (PPV), negative predictive value (NPV) and accuracy (ACC) values remained elevated regardless of gender and age sub-divisions. Conclusion: Our results indicate that miR-801, miR-369-3p and miR-16-5p have the potential to be biomarkers for whole peripheral blood identification. However, before its implementation in forensic analysis, further studies with other body fluids are needed, in order to confirm whether or not they can be found in other body fluids. Moreover, miR-165p could also be a biomarker for serum identification and miR-24-1-5p for WBCs identification. Resumo Resumo microRNA biomarkers for peripheral blood fractions identification: possible forensic applications 19 RESUMO Introdução: Os microRNAs (miRNAs) são pequenas moléculas de RNA não codificante, com um comprimento de 18 a 24 nucleótidos, que desempenham um papel regulador em diversos processos celulares. Desde a sua descoberta, os miRNAs têm sido detetados em órgãos, tecidos, células e fluidos corporais, sendo que o seu potencial como biomarcadores moleculares para o diagnóstico de várias condições patológicas, assim como para a identificação forense de fluidos corporais tem sido explorado. No caso particular do sangue periférico, este fluido corporal é normalmente visto como um todo, apesar do facto de apresentar várias diferenças entre as suas frações (plasma, soro e células sanguíneas). Uma destas diferenças consiste no padrão de expressão de miRNAs, uma vez que alguns miRNAs apresentam diferentes níveis e padrões de expressão de acordo com a fração analisada. Desta forma, torna-se relevante o estabelecimento do perfil de miRNAs específico associado às diferentes frações de sangue periférico, o que poderá ser muito útil em Medicina Legal, nomeadamente na análise de provas forenses. Objetivos: Neste estudo avaliou-se o padrão de expressão dos miRNAs miR-801, miR369-3p, miR-16-5p e miR-24-1-5p, nas diferentes frações de sangue periférico (plasma, soro e leucócitos), de modo a identificar miRNAs específicos destas frações. Material & Métodos: As amostras de sangue periférico foram processadas e os miRNAs foram extraídos a partir das amostras de soro, plasma e leucócitos. Após a síntese de DNA complementar, os miRNAs foram analisados por PCR quantitativo em tempo real e os resultados obtidos foram analisados estatisticamente com recurso ao software estatístico IBM®SPSS®Statistics (Versão 22.0). Resultados & Discussão: Verificou-se que os miRNAs analisados se encontravam diferencialmente expressos nas amostras testadas de plasma, soro e leucócitos. Os resultados indicaram que o miR-24-1-5p foi detetado quase exclusivamente em leucócitos, apresentando uma especificidade de 81,82% e uma sensibilidade de 90.91% para a identificação de leucócitos versus soro e plasma. Adicionalmente, o miR-801, o miR-369-3p e o miR-16-5p exibiram níveis de expressão mais elevados no soro, o que leva a concluir que esta é a fração ideal para a análise de miRNAs. Atendendo que estes três miRNAs podem ser encontrados em todas as frações de sangue periférico testadas, podemos considerar que estes poderão ser potenciais biomarcadores para a identificação de sangue total. O miR-16-5p apresentou uma expressão significativamente mais elevada no soro, em comparação com o plasma e os leucócitos, no entanto, a sua utilização como um biomarcador de soro deve ser feita com precaução atendendo à influência do género encontrada no presente estudo. Observamos também que, de um modo geral, os níveis Resumo microRNA biomarkers for peripheral blood fractions identification: possible forensic applications 20 de expressão dos miRNAs testados não exibiram diferenças significativas entre amostras de indivíduos de diferente género ou idade, o que é um fator importante no desenvolvimento de biomarcadores para procedimentos forenses, uma vez que um bom biomarcador deve manter-se inalterado em toda a população, de modo a permitir uma correta identificação da fração de sangue periférico. Quanto ao miR-24-1-5p, verificamos que os valores de razão de possibilidades, especificidade, sensibilidade, valor preditivo positivo ou precisão, valor preditivo negativo e acurácia permaneceram elevados independentemente das sub-divisões de género e idade. Conclusão: O miR-801, o miR-369-3p e o miR-16-5p apresentam um elevado potencial de serem biomarcadores moleculares úteis na identificação de sangue periférico. Porém, antes da sua implementação nas análises e procedimentos forenses, são necessários estudos adicionais que comparem outros fluidos corporais, de modo a confirmar se estes miRNAs podem ou não ser encontrados noutros fluidos. O presente estudo revelou ainda que o miR-16-5p poderá ser um biomarcador para a identificação de soro e o miR-24-15p para identificação de leucócitos. 1. Introduction 1. Introduction microRNA biomarkers for peripheral blood fractions identification: possible forensic applications 23 1. INTRODUCTION The different fractions of peripheral blood (serum, plasma and blood cells), despite being normally seen as a whole, show several differences among them. This is the case of the protein fibrinogen, which can be found in plasma, but is absent in serum [1]. Other variation between the fractions is the microRNA (miRNA) expression levels within the different peripheral blood fractions. In the literature there are some studies that point to the differentiated expression of certain miRNAs in blood fractions, however given the lack of information in this area, a better characterization of the miRNA content is needed [2, 3]. Therefore, it is important to address this subject in order to, not only, establish and characterize the existing differences, but also to standardize the methodologies used. Thus, once the miRNA expression pattern in the peripheral blood fractions is established it will facilitate and allow the implementation of miRNA analysis in forensic procedures. In fact, in comparison to other nucleic acids, such as the deoxyribonucleic acid (DNA) and the messenger ribonucleic acid (mRNA), miRNAs are smaller, what makes them more stable and less prone to degradation [4, 5]. Moreover, these molecules exhibit different expression profiles according to the tissues or body fluid tested [5]. Due to these characteristics, they have a great potential of being reliable and efficient biomarkers for body fluid characterization, as well as for the diagnosis of pathologic conditions. 1.1. microRNAs MiRNAs are short non-coding RNA, with a length of 18 to 24 nucleotides that play a regulative role in several cellular processes. Currently, there are, approximately 1881 known sequences of human miRNAs (miRBase release 21.0) [6, 7]. MiRNAs are transcribed from the genomes of nucleated cells by RNA polymerase II or III and these primary miRNA transcripts (pri-miRNAs) are then modified by the addition of a 5’ cap and a 3’ poly-A tail [4]. Subsequently, the pri-miRNAs are processed first in the nucleus by the ribonuclease (RNase) III enzyme Drosha, being later exported by Exportin 5 to the cytosol, where they are processed by the RNase III enzyme Dicer [4, 8]. This processing of pri-miRNAs originates initially a miRNA precursor (pre-miRNA) with approximately 70 nucleotides of length, and finally a smaller and mature double-stranded miRNA of 18–24 nucleotides [4, 8]. One strand of this mature miRNA is incorporated in the RNA-induced silencing complex (RISC), where it can regulate the expression of target mRNAs [4]. This miRNA can induce post-transcriptional gene silencing, through the 1. Introduction microRNA biomarkers for peripheral blood fractions identification: possible forensic applications 24 guidance of the RISC complex to the target mRNAs, or mRNA cleavage and degradation (Figure 1) [8, 9]. The RISC complex is also responsible for the process RNA interference, in which a double-stranded RNA silences the expression of homologous genes by mRNA cleavage [8, 10-12]. The other strand could be degraded, or prepared for being exported from the cell [4]. Thus, miRNAs could be found not only on the intracellular compartments, but also in the extracellular microenvironment, namely in serum, plasma and urine [13]. However, there is little information concerning the subject of the origin of circulating miRNAs (both in healthy and sick individuals) and what factors may influence the levels of circulating miRNAs [14]. Figure 1 – Biogenesis and post-transcriptional suppression of microRNAs. MiRNAs are transcribed from the genomes of nucleated cells into pri-miRNA transcripts, which are processed by Drosha in the nucleus, originating ~70-nucleotide pre-miRNAs. These pre-miRNAs are transported to the cytoplasm by Exportin 5 and are processed by Dicer into a mature double-stranded miRNA of 18-24 nucleotides. One strand of this miRNA is incorporated into the RNA-induced silencing complex (RISC), which later can induce translational repression or mRNA cleavage. 1. Introduction microRNA biomarkers for peripheral blood fractions identification: possible forensic applications 25 Nevertheless, when compared to DNA and mRNA, the small size of miRNAs makes them more stable and, therefore, less prone to degradation, making them good options for the development of biomarkers. Another factor that plays a key role in their stability is the argonaute (AGO) proteins, which are a catalytic component of the RISC complex [10-12, 15]. The interaction of AGO proteins with mature miRNAs makes them much more stable to the degradation process [6, 11, 16]. In fact, Chen and colleagues demonstrated that serum miRNAs were able to remain stable even after being exposed to severe environmental conditions, like high temperatures, extreme pH and prolonged storage [17]. Another characteristic of miRNAs is the differential expression of these molecules among tissues and fluids [4, 5]. For example, the miR-124 is considered a brain-specific miRNA and miR-122 is highly expressed in liver [3, 4, 18, 19]. Alterations on the normal miRNA expression pattern have also been associated with a diversity of pathologic conditions, such as cancer or cardiac diseases [19-28]. In fact, an ideal biomarker should have a series of characteristics, such as being accessible through non-invasive methods, being inexpensive to quantify, it must be specific to the disease or physiologic condition of interest and translatable from model systems to humans and, in the case of biomarkers for pathologic conditions, it should be a reliable early indicator of disease before the appearance of clinical symptoms [5]. Since miRNAs have these characteristics, they are very promising when it comes to the development of new non-invasive biomarkers [5]. However, some authors have suggested that the concentration for some miRNAs may be affected by gender and age, and due to the fact that an ideal biomarker should maintain unaltered, this information emphasizes the need of further studies, in order to uncover the miRNAs that are stable and that exhibit higher potential of being good biomarkers [11, 29]. 1.2. Peripheral blood fractions The different fractions of peripheral blood, which include plasma, serum and blood cells – leucocytes or white blood cells (WBCs), erythrocytes or red blood cells (RBCs) and platelets – differ from each other based on several factors [1]. Serum and plasma doesn’t contain cells, as opposite to blood cells. Both serum and plasma contain hormones, glucose, electrolytes, antibodies, antigens and other particles. Plasma corresponds to the cell-free supernatant resulting from centrifuged blood collected in the presence of an anticoagulant [30]. On the other hand, serum is the cell-free supernatant resulting from 1. Introduction microRNA biomarkers for peripheral blood fractions identification: possible forensic applications 32 In the same way, miR-150 is present in plasma, serum, WBCs, RBCs and platelets and Pritchard et al. detected that the plasma levels of this lymphoid-enriched miRNA reflected lymphoid counts [14, 29]. Plasma/serum miR-451 and miR-486-5p might also originate in blood cells, since these miRNAs are both common to the cellular and the circulation miRNAs populations, being present in plasma, serum, WBCs, RBCs and platelets, being specially enriched in RBCs [14, 29, 53]. Interestingly, miR-223, miR-150, miR-451 and miR-486-5p are highly expressed in hematologic cells and are absent in endothelial cells and in epithelial cells [46]. The expression of miR-574-3p is detected in plasma, serum, WBCs, RBCs and platelets, being expressed in hematologic cells and not in endothelial cells [29, 46]. Additionally, the plasma levels of this miRNA presented significant positive correlations with myeloid blood cell counts, such as neutrophils and platelets [14]. 1.4.3. Circulating microRNAs derived from endothelial and blood cells Other circulating plasma and serum miRNAs, like miR-126, miR-22, miR-16 (currently referred to as miR-16-5p) or let-7a, which exhibit high expression levels in both blood cells as well as endothelial cells, could be equally secreted by these two types of cells into plasma/serum [5, 27, 29, 41-46, 48-50, 53, 55-59]. MiR-126, which is present in whole blood, as well as in plasma and serum, was reported to be an endothelial cell-restricted miRNA, highly expressed in the endothelium, blood vessels, heart, and lung and is involved in the endothelial cell function, suggesting an endothelial origin [27, 29, 55, 56, 58]. However, this miRNA was also identified in blood cells – WBCs, RBCs and platelets, and, although being present at lower levels in platelets that in endothelial cells, the shedding of platelet microparticles in plasma or serum also represents a major contributor to circulating miR-126 levels [29, 45]. Likewise, miR-22 is present in plasma, serum, WBCs, RBCs and platelets, being also highly expressed in endothelial cells [5, 29, 43]. MiR-16-5p is present in plasma, serum, WBCs, RBCs and platelets and it is particularly abundant in erythrocytes and endothelial cells [29, 59]. Moreover, this miRNA was detected in endothelial cells by six profiling studies [41, 42, 44, 48, 49, 57]. Let-7a, a miRNA present in plasma, serum and blood cells, common to the cellular and the circulation miRNAs populations and detected in endothelial cells by six profiling 1. Introduction microRNA biomarkers for peripheral blood fractions identification: possible forensic applications 33 studies is another candidate of a miRNAs derived both from endothelial and blood cells [29, 41, 42, 44, 49, 50, 53, 57]. Furthermore, McCall et al. observed that miR-22, miR-16-5p and let-7a are common to endothelial, epithelial and hematologic cells, and that miR-126 is expressed by endothelial and hematologic cells, supporting the hypothesis of a shared origin of these miRNAs in plasma and serum [46]. All these circulating plasma/serum miRNA that might have originated from both endothelial and blood cells could be differentiated by those which are derived by only one of these sources based on theirs expression levels in plasma and serum. It would be expected that the miRNAs that are secreted into the plasma/serum by both endothelial and blood cells would have higher levels in these fluids, when compared to the ones that are secreted either from endothelial or blood cells. As a future perspective it would be interesting to further explore this possibility in order to enlighten the miRNA origin in blood plasma and serum. 1.4.4. Circulating microRNAs with an organ origin Due to the fact that some tissue-specific miRNAs have been identified in plasma, and when we consider the close contact between organs and the bloodstream, we can conclude that a miRNA transfer between these organs and the blood plasma/serum can occur [3]. Particularly, in highly vascularized organs, such as kidneys, liver, brain and lungs, this miRNA transfer between organs and blood plasma might represent a more significant contribution for the circulating miRNAs. For this reason, some plasma miRNAs may be organ-derived (Figure 3). Sun et al. detected five miRNAs, including miR-192, miR-194, miR-204, miR-215 and miR-216, which were preferentially expressed in human kidney when compared with other organs/tissues, such as heart, spleen, lung, muscle and prostate [61]. Later, Chandrasekaran and co-workers, profiled the miRNA expression in human organs, including the kidneys, and concluded that these five miRNAs are among the few miRNAs that were found to be renal-specific [60]. Of these five miRNAs, miR-192, miR-194, miR204 and miR-215 can be found in plasma and since they are preferentially highly expressed in the kidney, this organ might secrete them into the bloodstream, and consequently, they may have a kidney origin [5, 29, 60, 61]. Regarding normal human liver tissues, very few circulating plasma miRNAs are reported to be highly expressed in this organ. Perhaps the most well studied miRNA in 1. Introduction microRNA biomarkers for peripheral blood fractions identification: possible forensic applications 34 liver is miR-122, a highly expressed miRNA that represents 70% of the total miRNA pool of this organ [18]. This miRNA is also a liver-specific miRNA that can be detected in plasma, and therefore its origin in plasma might derive from the liver [3, 19, 29, 64]. Likewise, miR-148, a miRNA that can be also found in plasma presents high expression in the adult liver and the results of Barad et al. validated the liver-specific expression of miR148 [5, 62, 63]. In the case of the brain, miR-124 is considered a brain-specific miRNA that have also been detected consistently in blood plasma, and therefore could be brain-derived [3, 19]. Similarly, miR-128 is present in plasma and serum and was also reported to be a brain-specific miRNA [5, 29, 62]. Other similar miRNAs are miR-129-5p, miR-191 and miR-342-3p, since that they can be found in blood plasma and exhibit high expression levels in brain in comparison with other tissues, according to miRNAMap [5, 29, 39]. In the lungs, according to miRNAMap, miR-21, miR-30b, miR-30c-1 and miR146b-5p, are highly expressed in this organ, compared with others organs and tissues [39]. These miRNAs are also present in plasma and serum and based on theirs expression profiles, in the circulation they probably originate in the lungs, as a result of the lung irrigation process [5, 29]. Recent studies have also demonstrated a correlation between some miRNAs and specific pathologic conditions, such as cancer [3, 20, 24, 26, 65, 66]. Furthermore, since tumor cells can secrete miRNAs into the bloodstream, some studies, such as the one performed by Turchinovich et al., have considered the tumor as an organ that contributes to the blood miRNA population [3]. For example, the expression of serum miR-375 was reported to be greater in prostate cancer patients than in healthy individuals, and greater in metastasized cancer than in primary prostate cancer, what leads to believe that in individuals with this pathologic condition, this particularly serum miRNA could be originated in the cancer cells [26]. Moreover, this is corroborated by the study performed by Brase et al., in which was found that miR-375 was highly expressed in serum of prostate cancer patients and the release of this miRNA into the bloodstream is further associated with advanced disease [20]. Another miRNA in a similar situation is miR-195. This miRNA is present in the plasma of healthy individuals and its levels were considerably elevated in samples of whole blood of patients diagnosed with breast cancer from stage I to IV, when compared to age-matched disease-free individuals [5, 24, 26]. In this study the expression of miR-195 in ageand stage-matched tumor tissues was also compared with whole blood samples and the result was that in tumor tissues and circulation, the levels of miR-195 were increased at progressive stages of breast cancer. In other words, this miRNA was more expressed in stage IV than in stage I or II [24]. This 1. Introduction microRNA biomarkers for peripheral blood fractions identification: possible forensic applications 35 positive correlation between tissue and circulating miR-195 suggests that this miRNA could be released into the blood by the tumor cells. MiR-221 is another example of a miRNA associated with cancer, more specifically, with the renal cell carcinoma (RCC) [66]. Teixeira et al. reported that patients with higher plasma levels of this miRNA presented a significantly lower survival rate, when compared to those with lower expression levels, thus, revealing miR-221 as an independent prognosis factor in RCC [66]. Furthermore, the plasma expression levels of miR-221 were higher in patients with advanced disease (metastasis at diagnosis) comparatively with patients with localized disease [66]. MiR-210 and miR-1233 are other miRNAs that are implicated in the RCC, since a recent study (and not yet published) revealed that these miRNAs presented higher expression levels in plasma samples of RCC patients, when compared to healthy individuals, being good candidates for biomarkers of prognosis and aggressiveness in RCC. The expression levels of peripheral whole-blood miR-221 and miR-7 were also reported to be implicated as potential predictive biomarkers of castration-resistant prostate cancer development [65]. Figure 3 – Overview of several circulating plasma/serum miRNAs organ-derived. Adapted from http://upload.wikimedia.org/wikipedia/commons/6/6b/Man_shadow_with_organs.png [71] 1. Introduction microRNA biomarkers for peripheral blood fractions identification: possible forensic applications 36 1.5. Forensic applications of microRNAs as biomarkers for peripheral blood fractions characterization MiRNAs biomarkers for peripheral blood fractions exhibit a wide variety of forensic applications. One of the main aspects in the identification of miRNAs specific to peripheral blood fractions is to characterize the differences/variations existent within blood. This factor should be considered, not only in forensic sciences, but also in all the sciences that use miRNAs, since that in several studies is selected one fraction in particular without taking into account this variation. For this reason, the miRNA differences in the peripheral blood fractions should be analyzed in order to correctly select one of them according to the purpose of the study. Another forensic application of miRNAs biomarkers for peripheral blood fractions is related to body fluid identification. In fact, during a forensic investigation the correct identification of the possible body fluids found is crucial in order to identify possible sources for DNA collection to be later used in the identification of the donor of the biologic material [13]. Within this purpose, over the years, several methods were developed towards the body fluid identification, such as serologic tests [11]. However, these methodologies exhibit low sensitivity and specificity levels [11]. Thus, due to the small size of miRNAs, theirs increased stability and the fact that they show different expression profiles according to the tissue or fluid tested, recently they have been studied, not only as a resource in the diagnostic of different pathologic conditions, but also as an alternative to the conventional methods for the forensic body fluid identification. Although miRNA profiling is not currently a standard technique in forensic investigation, with the improvement of miRNA knowledge and respective methods, it could eventually be implemented in forensic procedures. Furthermore, given the high stability of miRNAs and the fact that these potential biomarkers can be detected even in degraded samples – which are normally not legally allowed – in the future, miRNAs could lead to a change in legislation, allowing the acceptance of these types of samples as forensic evidence. This would represent a major progress and improvement in forensic investigations and evidence analysis. In the literature there are several studies about the use of miRNA biomarkers in the identification of body fluids [5, 13, 56, 72]. Courts and Madea analyzed the presence of miRNAs in blood and saliva samples of five healthy donors and presented two miRNA assays consisting of three differentially expressed miRNAs for the identification of blood (miR-126, miR-150, miR-451) and saliva (miR-200c, miR-203, miR205) [56]. On the other hand, Hanson and co-workers evaluated the miRNA expression 1. Introduction microRNA biomarkers for peripheral blood fractions identification: possible forensic applications 37 profile in dried, forensically relevant biological fluids (such as blood, semen, saliva, vaginal secretions, and menstrual blood) of healthy individuals to identify possible body fluidspecific miRNAs and were able to determine a panel of nine miRNAs that were differentially expressed (miR-451, miR-16, miR-135b, miR-10b, miR-658, miR-205, miR124a, miR-372, and miR-412), allowing the identification of the body fluid origin of forensic biological stains using only 50 pg of total RNA [13]. Other study performed by Weber and co-workers examined the presence of miRNAs in 12 human body fluids of five healthy donors, concluding that the miRNAs tested were present in all fluids analyzed and showed distinct compositions in different fluid types [5]. Zubakov and colleagues screened a set of 718 human miRNA markers in forensically relevant body fluids (saliva, venous blood, menstrual blood, semen and vaginal secretion), finding that two miRNA markers for blood (miR-144 and miR-185) and two for semen (miR-135a and miR-891a) were suggestive to be most useful for body fluid identification in forensic applications [72]. Peripheral blood fractions miRNAs profiles associated with diseases could also play a key role in forensic sciences, namely in the identification of victims and suspects or in the determination of the cause of death. For example, regarding the first case, in an investigation where there is an unidentified corpse, the determination of the best peripheral blood fraction to profile the miRNA expression, and the posterior corresponding analysis could indicate what type of diseases were present and lead to the victim identification, through the consultation of medical records. In a similar situation, in a crime scene, in which a possible blood stain was found, but a DNA comparison to blood samples from suspects can’t be performed, because that stain is not viable for DNA extraction and analysis, considering that miRNAs are much more stable, this same sample might allow a miRNA analysis, which might indicate specific pathologies of the sample donor, which could be crucial information to narrow down the suspects list. Or even, in a forensic investigation, where there isn’t permission for the collection of blood samples from the suspects to compare to a presumable blood stain that was found in the crime scene, in this situation, a miRNA profiling could also uncover eventual pathologies of donor of the crime scene sample. In the second case, when the cause of death is not apparent to the macroscopic/histological level or in sudden death cases, the presence of certain blood fractions miRNAs associated with diseases may indicate more clearly the factors that have caused the death. 1. Introduction microRNA biomarkers for peripheral blood fractions identification: possible forensic applications 38 1.6. Potential microRNA profile for peripheral blood fractions identification Some miRNAs, due to theirs expression patterns in biological samples, have a higher potential of being specific of different fractions of peripheral blood and, therefore, could be used as biomarkers for plasma, serum, and blood cells. According to Weber and co-workers, miR-369-3p and miR-801 are unique to the plasma when compared to other body fluids such as urine, saliva, and seminal fluid [5]. Similarly, in blood cells, Wang and colleagues reported that miR-24-1* (currently referred to as miR-24-1-5p) is present in blood cells (leukocytes, erythrocytes and platelets), being absent in plasma and serum [29]. In the case of serum, it is known that miR-16 (currently referred to as miR-16-5p) is abundant in this fraction [73]. Regarding the fact of the presence of this miRNA in plasma, in the literature there is contradictory information, since Weber and co-workers didn’t detected miR-16-5p in plasma and Blondal and colleagues reported that miR-16-5p is present in plasma [5, 74]. Due to the information previously described, these four miRNAs (miR-369-3p, miR801, miR-24-1-5p and miR-16-5p) exhibit a higher potential of being exclusive of the fractions of peripheral blood, and therefore, were selected for this study. 2. Objectives 2. Objectives microRNA biomarkers for peripheral blood fractions identification: possible forensic applications 41 2. OBJECTIVES 2.1. Main objective The main objective of the present study is the identification of a miRNA profile used for biological identification of the different fractions of peripheral blood, namely, plasma, serum and white blood cells. 2.2. Specific objectives 1) Systematically revision of the literature to define the miRNA profile to be evaluated in biological samples (plasma, serum and white blood cells samples); 2) Detection and determination of the expression levels of miR-801, miR-369-3p, miR-16-5p and miR-24-1-5p in plasma, serum and white blood cells peripheral blood fraction samples; 3) Specificity and sensitivity analysis regarding the miRNA profile used in the identification of the peripheral blood fractions. 4. Results & Discussion 4. Results & Discussion microRNA biomarkers for peripheral blood fractions identification: possible forensic applications 51 4. RESULTS & DISCUSSION 4.1. microRNA detection frequencies Figure 5 displays the miRNA detection frequencies of the four studied miRNAs in serum, plasma and WBCs, i.e. for each peripheral blood fraction, the graphic shows the percentage of cases in which the miRNAs were detected. Through the analysis of this figure, we can conclude that miR-801 and miR-16-5p were both detected in the majority (95%) of the serum, plasma and WBCs samples tested. Moreover, these miRNAs have a more uniform distribution throughout the fractions, when compared to miR-369-3p and mir-24-1-5p, which were the miRNAs that exhibited more variations in the detection frequency in the several peripheral blood fractions. Overall, and based on the detection frequencies, miR-24-1-5p appears to be the miRNA with major differences in the fractions, as it was detected in 91% of the WBCs samples tested and only in 32% of the plasma samples and 5% of the serum samples tested. Figure 5 – miRNA detection frequencies in percentage (%) of miR-801, miR-369-3p, miR-16-5p and miR-241-5p in serum, plasma and white blood cells. 0 10 20 30 40 50 60 70 80 90 100 miR-801 miR-369-3p miR-16-5p miR-24-1-5p miRNA detection frequency (%) Serum Plasma White Blood Cells 4. Results & Discussion microRNA biomarkers for peripheral blood fractions identification: possible forensic applications 52 4.2. microRNA relative quantification in peripheral blood fractions Figure 6 exhibits the results of the miRNA relative quantification across the four miRNAs tested (miR-801, miR-369-3p, miR-16-5p and miR-24-1-5p) in the peripheral blood fractions (serum, plasma and WBCs). The selected miRNAs show different expression patterns according to the peripheral blood fractions tested. MiR-801, miR-3693p and miR-16-5p are expressed in all of the peripheral blood fractions, leading us to consider that these miRNAs could be good biomarkers for whole blood identification. However, these miRNAs should be tested in other body fluids in order to determine theirs effective potential as a blood biomarker. The higher expression levels of these miRNAs are observed in serum, followed by plasma and, lastly, in WBCs. This is a very interesting result, considering that in the majority of studies, plasma is preferentially used instead of serum. Based on our results, serum seems to be the optimal peripheral blood fraction for miRNA analysis, since this fraction exhibits higher miRNA content. Furthermore, within these three miRNAs, miR-16-5p is the one that exhibits higher expression levels throughout the fractions of peripheral blood, thus it might have an important role in a blood-related process. In fact, according to the literature, miR-16-5p contributes to the regulation of mammalian hematopoiesis, through the control of the terminal stages of hematopoietic development [76, 77]. Moreover, a study where the variability of miRNAs in whole blood stored until 12 hours at room temperature was assessed concluded that miR16-5p was able to remain unaltered despite of the room temperature [78]. This stability of miR-16-5p, combined with its expression pattern in peripheral blood fractions, makes this miRNA an ideal biomarker for whole peripheral blood identification. On the other hand, miR-24-1-5p is the miRNA that reveals a more differentiated expression pattern in the peripheral blood fractions. The expression levels of this miRNA were significantly more elevated in plasma in comparison to WBCs (P=0.001) (Figure 6). The mean -ΔCt values of miR-24-1-5p in serum are not represented in Figure 6, as this miRNA was only detected in one of the 22 serum samples tested. The fact that there were detected higher expression levels of miR-24-1-5p in plasma rather than in WBCs might appear to go against what was found through the miRNA detection frequency analysis. However, when interpreting the relative quantification results we must consider that the number of samples used to determine these values was quite different: this miRNA was found only in 7 plasma samples and the mean of the respective quantification is being compared against the relative quantification of 20 WBCs samples. We can conclude that these results related to the expression of miR-24-1-5p can represent an addition to the existent data. To the best of our knowledge, the only study to date that analyzes the 4. Results & Discussion microRNA biomarkers for peripheral blood fractions identification: possible forensic applications 53 expression of this miRNA in the several fractions of peripheral blood was performed by Wang and co-workers, in which the peripheral blood fractions of 10 individuals were tested [29]. This study detected miR-24-1-5p in blood cells (leukocytes, erythrocytes and platelets), and not in plasma or serum [29]. In the present work, we took a step forward and analyzed the miRNA content of plasma, serum and WBCs from 22 individuals. Figure 6 – miRNA relative quantification of miR-801, miR-369-3p, miR-16-5p and miR-24-1-5p in serum, plasma and white blood cells. (mean ± standard error of mean). In Table 2, the fold change in the miRNAs expression in the different peripheral blood fractions allow us to evaluate and compare the differences between the expression levels of each of the selected miRNAs in two types of peripheral blood fractions. Table 2 – Fold change values, and correspondent P values of miR-801, miR-369-3p, miR-16-5p and miR-241-5p in serum, plasma and white blood cells (WBCs). Fold change (2-ΔΔCt) (P values) miR-801 miR-369-3p miR-16-5p miR-24-1-5p Serum vs. WBCs 28.64 (P<0.001) 942.27 (P<0.001) 461.44 (P<0.001) * Plasma vs. WBCs 2.45 (P=0.097) 129.79 (P<0.001) 22.63 (P<0.001) 70.03 (P=0.001) Serum vs. Plasma 11.71 (P<0.001) 7.26 (P=0.109) 20.39 (P<0.001) * *Not calculated, since this miRNA was only detected in one serum sample. -20 -15 -10 -5 0 5 10 15 miR-801 miR-369-3p miR-16-5p miR-24-1-5p miRNA relative quuantification (-ΔCt) Serum Plasma White blood cells 4. Results & Discussion microRNA biomarkers for peripheral blood fractions identification: possible forensic applications 54 The analysis of the miRNA expression levels in serum comparing to WBCs levels reveals that miR-801, miR-369-3p and miR-16-5p are, approximately, 29, 942 and 461 times, respectively more expressed in serum (P<0.001). The comparison of plasma vs. WBCs indicates statistically significant differences in miR-369-3p, miR-16-5p and miR-241-5p expression. These miRNAs are approximately 130, 23 and 70 times more expressed in plasma in comparison to WBCs (P<0.001; P<0.001 and P=0.001, respectively). As it was mentioned early, we need to approach these results with caution and in the light of the miRNA detection frequencies. Thus, for miR-24-1-5p the interpretation may not be as straightforward as one might think, as the 70 fold change value might not be a good reflection of the results, because it is based in the relative quantification of only 7 plasma samples. In order to assess whether or not miR-24-1-5p can be used as a WBCs biomarker, further statistics need to be performed (see point 4.4.). Regarding the serum vs. plasma fold change expression analysis, the values obtained reveal that miR-801, miR-369-3p and miR-16-5p are, respectively, 12, 7 and 20 times more expressed in serum than in plasma (P<0.001; P=0.109 and P<0.001, respectively). The fold change analysis achieved lead us to conclude that miR-16-5p could be a good biomarker for serum identification, as its expression values allow us to distinguish this blood fraction from plasma and WBCs samples. Additionally, as it was mentioned early, this miRNA could also be a good biomarker for whole peripheral blood identification, since it showed high expression levels in all the fractions. In other words, if the screening of miR-16-5p in an unknown fluid indicates its presence, we can propose that the fluid in question is blood. According to our results, for the miRNAs tested we observed that WBCs was the fraction that exhibit lower expression levels (Figure 6, Table 2). This is an interesting result, especially when taking into account that miRNAs are produced in cells and, hence, without any additional knowledge, it would be expected to find higher expression levels in WBCs. This illustrates the fact that miRNAs, despite originating in cells, can and do, indeed, exhibit different expression patterns amongst the fluid or tissue tested, due to the fact that cells are able to export miRNAs into the extracellular environment [3]. In this particular case, we observed higher expression levels in serum, which means that, for these miRNAs, there is a larger quantity of miRNA molecules in serum, in comparison to WBCs and plasma. For miR-24-1-5p, as stated early, the interpretation of the results isn’t as straightforward as it is for the other miRNAs, and, with these results we cannot yet dismiss this miRNA as a WBCs biomarker. 4. Results & Discussion microRNA biomarkers for peripheral blood fractions identification: possible forensic applications 55 4.3. microRNA profile according to age and gender In this section, we analyzed the relative expression of the miRNAs tested (miR801, miR-369-3p, miR-16-5p and miR-24-1-5p) in the peripheral blood fractions (serum, plasma and WBCs) according to the age and gender. Since our study population had a median age of 48.5, we divided the results in two different groups (< 48 years and ≥ 48 years). As shown in Figure 7, the majority of miRNAs showed no statistically significant differences according to the age in the three samples tested (serum, plasma and WBCs). The only case that exhibited significant differences was miR-369-3p in serum and plasma. According to the results, the expression levels of miR-369-3p were significantly higher in young individuals (age < 48 years) than in the older group (age ≥ 48 years) in serum (P=0.027) and in plasma samples (P=0.023). Figure 7 – miRNA relative quantification of miR-801, miR-369-3p, miR-16-5p and miR-24-1-5p in serum, plasma and white blood cells (WBCs) according to age (mean ± standard error of the mean; * P < 0.050). In the analysis according to gender, the situation is similar to what it was observed for age, since predominantly weren’t found any statistically significant differences according to this characteristic, with the exception of miR-16-5p in serum (P=0.027) and miR-801 in WBCs (P=0.025), which were the only cases that exhibited significant differences, i.e., the expression levels of miR-801 in WBCs and of miR-16-5p in serum were significant higher in males, than in females (Figure 8). -18 -13 -8 -3 2 7 12 miRNA relative quantification (-ΔCt) < 48 years ≥ 48 years Serum Plasma WBCs * * 4. Results & Discussion microRNA biomarkers for peripheral blood fractions identification: possible forensic applications 56 Figure 8 – miRNA relative quantification of miR-801, miR-369-3p, miR-16-5p and miR-24-1-5p in serum, plasma and white blood cells (WBCs) according to gender. (mean ± standard error of the mean; * P < 0.050). 4.4. Specificity and sensitivity analysis of miR-24-1-5p as a WBCs biomarker In order to clarify and evaluate the effective potential of miR-24-1-5p as a WBCs biomarker, a ROC curve was constructed and odd ratio (OR), specificity (or true negative rate, TNR), sensitivity (or true positive rate, TPR), positive predictive value or precision (PPV), negative predictive value (NPV) and accuracy (ACC) values were determined (Table 3, Figure 9). These statistics were performed since they are able to reflect the ability of the correct identification of a peripheral blood fraction as WBCs or non-WBCs (serum and plasma) based on the detection or non-detection of this miRNA in the sample in question [79, 80]. The odd ratio (OR) represents the odds of the sample being WBCs, given that miR-24-1-5p is present, compared to the odds of the sample being plasma or serum in the presence of miR-24-1-5p [81]. The specificity reveals the proportion of the non-detection of miR-24-1-5p in a sample that isn’t WBCs (serum and plasma) [80]. On the other hand, the sensitivity reflects the proportion of the detection of miR-24-1-5p in WBCs samples [80]. The positive predictive value (PPV) is the proportion of WBCs samples identified in which miR-24-1-5p was detected, whereas the negative predictive value (NPV) indicates the proportion of serum and plasma in the samples in which miR-24-1-5p wasn’t detected [80]. Lastly, the accuracy (ACC) is the proportion of correctly identified fractions [80]. -15 -10 -5 0 5 10 15 20 miRNA relative quantification (-ΔCt) Male Female Serum Plasma WBCs * * 4. Results & Discussion microRNA biomarkers for peripheral blood fractions identification: possible forensic applications 57 According to our results, we observed that the presence of miR-24-1-5p in biological samples presents a likelihood of 45 times higher to be WBCs than serum or plasma (OR=45) (Table 3, Figure 9). Moreover, the specificity of miR-24-1-5p is 81.82%, and the sensitivity is 90.91%. Additionally, the PPV value is 71.43%, and the NPV value is 94.74%. The ACC is 84.85%, in other words, with the screening of miR-24-1-5p in an unknown fraction, 84.85% of the samples tested are correctly identified as WBCs or nonWBCs (serum and plasma). These results suggest that miR-24-1-5p is a potential biomarker for WBCs identification. Table 3 – Odd Ratio (OR), specificity, sensitivity, positive predictive value or precision (PPV), negative predictive value (NPV) and accuracy (ACC) values for the evaluation of miR-24-1-5p as a WBCs biomarker. OR Specificity (%) Sensitivity (%) PPV (%) NPV (%) ACC (%) All samples 45 81.82 90.91 71.43 94.74 84.85 Age < 48 years 10 94.44 100.00 90.00 100.00 96.30 ≥ 48 years 14 73.08 84.62 61.11 90.48 76.92 Gender Female 92 85.29 94.12 76.19 96.97 88.00 Male 92 70.00 80.00 57.14 87.50 73.33 Figure 9 – ROC curve of miR-24-1-5p as a WBCs biomarker. AUC – Area under the curve. AUC = 0.864 5. Conclusion & Future Perspectives microRNA biomarkers for peripheral blood fractions identification: possible forensic applications 64 performed with caution, as it quantification exhibited statistically significant differences according to gender (Table 6). Table 6 – Main conclusions reached concerning the biomarker potential of the analyzed miRNAs. miRNAs Biomarker miR-24-1-5p WBCs  Detected almost exclusively in WBCs  Sensitivity of 90.91% and specificity of 81.82% for WBCs identification miR-16-5p Serum Peripheral blood  Overly expressed in serum, comparing to plasma and WBCs (statistically significant)  Expressed in all peripheral blood fractions miR-801 and miR-369-3p Peripheral blood  Expressed in all peripheral blood fractions Furthermore, the fact that, overall, the miRNA expression pattern of the analyzed miRNAs didn’t reveal any major differences between samples from individuals of different gender or age, in a forensic perspective, represents a solid result, since that, in forensic body fluid identification what is desirable is a miRNA that could allow the correct identification of the fluid/fraction throughout the population, without displaying individual variations. Overall, our results demonstrated that miR-801, miR-369-3p and miR-16-5p could be biomarkers for whole blood identification; miR-16-5p could additionally be a biomarker for serum identification and miR-24-1-5p for WBCs identification. In the future it would be useful to work towards the standardization of the procedures for miRNA analysis, in order to allow a more reliable interpretation of the results between studies. Moreover, as future perspectives it would be also interesting to explore the expression patterns and levels of these and others miRNAs in peripheral blood fractions in a larger and diverse set of samples, to uncover other miRNAs that could be used as biomarkers for these fractions, as well as possible associations with pathologic conditions. Particularly, it would be interesting to pursue this line of investigation for miR24-1-5p. 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