MicroRNAs (miRNAs) represent a promising source of biomarkers for GvHD because they play critical roles in the development and function of the immune system (Banerjee
et al. 2010; Rodriguez et al. 2007). MicroRNAs are a family of 19-24 nucleotide
noncoding RNAs, which affect the regulation of gene expression in eukaryotic cells by binding to the 3´-untranslated region of target messenger RNAs (mRNAs). They play an important role in many cellular processes such as development, stem cell division, apoptosis and cancer (Ajit, 2012).
MiRNAs regulate gene expression by binding to the mRNA and the seed sequence is essential for this binding. The seed sequence (or seed region) is a conserved
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heptametrical sequence which is mostly situated at positions 2-7 from the miRNA 5´- end. Even though perfect base pairing of miRNA and its target mRNA is required, the “seed sequence” has to be perfectly complementary (Felekkis et al., 2010). Human miRNA biogenesis is a two-step process including both nuclear and cytoplasmic cleavage events, performed by two ribonuclease III endonucleases, Drosha and Dicer (Figure 1.11) (Denli et al., 2004; Gregory et al., 2004; Han et al., 2004). The miRNA- processing pathway includes the production of the primary miRNA transcript (pri- miRNA) by RNA polymerase II or III and cleavage of the pri-miRNA by the microprocessor complex Drosha–DGCR8 (Pasha) in the nucleus. The resulting precursor hairpin, the pre-miRNA, is exported from the nucleus by Exportin-5–Ran- GTP (Du and Zamore, 2005). In the cytoplasm, the RNase Dicer in complex with the double-stranded RNA-binding protein TRBP cleaves the pre-miRNA hairpin to its mature form (Lee et al., 2007b). The functional strand of the mature miRNA is loaded together with Argonaute (Ago2) proteins into the RNA-induced silencing complex (RISC), where it guides RISC to silence target mRNAs. This may be via cleavage, translational repression or deadenylation, and the passenger strand is usually degraded (Winter et al., 2009) (Figure 1.7).
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According to Bentwich et al, around 50% of all genes are regulated by miRNAs, which makes the investigation of their roles in different diseases very important (Bentwich et
al., 2005). In this context, Atarod et al, have reviewed the possible interaction between
some miRNA pathways and GvHD using in silico approaches and eight microRNAs (miR-146a, miR-155, miR-515, miR-346, miR-143, miR-373, miR-31a and miR-29)
Figure 1.7 MicroRNA biogenesis (Esquela-Kerscher and Slack, 2006). Pri-
miRNA is transcribed from the miRNA gene and to pre-miRNA by Drosha in the nucleus. This pre-miRNA is then exported into the cytoplasm by Exportin 5 and cleaved into mature miRNA by Dicer. Mature miRNA is loaded onto RISC/miRISC and delivered to the mRNA where it represses translation and/or results in mRNA cleavage. Pol II: Polymerase II; Pri-miRNA: Primary MicroRNA; Pre-miRNA: Precursor MicroRNA; miRISC: MicroRNA Induced Silencing Complex; ORF: Open Reading Frame; UTR: Untranslated Region; mRNA: Messenger RNA.
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were predicted to impact on different molecules in the GvHD signalling pathway (Atarod and Dickinson, 2013). MiR-155 was one of the first miRNAs to be associated with the regulation of aGvHD. This miRNA is required for the normal function of B and T lymphocytes (Rodriguez et al., 2007). Ranganathan et al showed that miR-155 was up-regulated in patients with intestinal aGvHD, making this miRNA a novel target for therapeutic intervention (Ranganathan et al., 2012).
1.7.3.1 MicroRNA-146a involvement in GvHD
At present, there is extensive knowledge on the cellular mechanisms of GvHD but less is known about the molecular biology of the disease. Molecular studies carried out to date have focused on identifying SNPs (Dickinson and Holler, 2008) and specific genes involved in the development of GvHD (Baron et al., 2007). However, there have been fewer studies focusing on the molecular regulation of GvHD. Recently, the potential role of microRNAs as biomarkers for GvHD has been highlighted (Paczesny
et al., 2013).
MicroRNA-146 is increasingly being recognized as a ‘fine-tuner’ of cell function and differentiation in both innate and adaptive immunity. MiR-146a controls innate immune cell and T-cell responses, and its deficiency was shown to be responsible of autoimmunity (Boldin et al., 2011). MiR-146a is expressed within a family that shares the same seed sequence, but is coded by different loci in the human genome. The miR-146a gene is located on human chromosome 5, corresponding to chromosome 11 in mouse (Garcia et al, 2011) (Garcia et al., 2011).
Mechanistically, miR-146a has been shown to directly target two adapter proteins in the nuclear factor (NF) κB activation pathway, tumour necrosis factor (TNF) receptor- associated factor 6 (TRAF6) and IL-1 receptor-associated kinase 1 (IRAK1), both in innate immune cells and T cells (Taganov et al., 2006b; Boldin et al., 2011; Yang et al., 2012). In addition, the survival and maturation of human plasmacytoid dendritic cells that are involved in GvHD were shown to be regulated by miR-146a (Koyama et al., 2009; Karrich et al., 2013).
MiR-146a gene expression analysis has demonstrated induction in response to microbial components such as LPS which triggers GvHD pathology (Cooke et al., 2001; Taganov et al., 2006b). Upon stimulation with LPS or monocyte activation via
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cell surface receptors such as TLR4, miR-146a has been shown, both in vivo and in
vitro, to target IRAK1 and TRAF6 that become associated with the IL-1 receptor upon
stimulation and are partially responsible for IL-1-induced upregulation of NF-kB (Figure 1.8) (Boldin et al., 2011).
Such binding results in the suppression of the expression of NF-κB target genes such as the interleukins IL-6, IL-8, IL-1β, and TNF-α (Pauley et al., 2008; Tang et al., 2009; Boldin et al., 2011). Taganov et al, established that IRAK1 is regulated by miR-146a (Taganov et al., 2006). IRAK1 is considered as a linker of the TLR with the TRAF6 intracytoplasmic activator of transcription factor of NF-κB and is subject to a negative feedback by miR-146a (Figure 1.12) (Chatzikyriakidou et al., 2010).
Figure 1.8 MicroRNA-146a and IRAK1 interaction and their association with NF-κB signalling (adapted from Rusca and Monticelli, 2011). MiR-
146a negatively regulates signal transduction pathways leading to NF-κB activation. Upon activation of cell surface receptor such as TLR4, a molecular cascade including TRAF6 and IRAK1 leads to IκBα phosphorylation and degradation and to activation and nuclear translocation. NF-κB activation induces transcription of many genes, including pri-miR- 146a. Once translocated to the cytoplasm and loaded onto the RISC complex, mature miR-146a contributes to attenuate receptor signalling through the down-modulation of IRAK1 and TRAF6 (Taganov et al., 2006b; Taganov et al., 2007; Rusca and Monticelli, 2011b).
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In addition to being studied as potential biomarkers for GvHD, microRNAs also have promising potential to be used in the therapy of different diseases including GvHD (Paczesny, 2013a). As miRNAs are being increasingly studied as key regulators of gene expression, several SNPs in miRNA genes (miRNA-related SNPs) have also been shown to be associated with human diseases by affecting the miRNA mediated regulatory function (Gong et al., 2012).
1.8
Non-invasive biomarkers for HSCT outcome
Unlike the genome, the proteome varies with time and is defined as the proteins present in a single sample at a certain time point. Ideal clinical tests are based on non- invasive collection, which allows for repetitive sample collection from the same patient in short amount of time. Thus, proteins represent ideal biomarkers in the post- transplantation setting and have been widely studied, as detailed in the following sections.
GvHD biomarkers may be produced by several sources such as donor cells, the local or systemic cytokine milieu, or recipient target tissues during disease development. These biomarkers may then be released into a variety of body fluids. For non-invasive tests used in diagnostics, bio-fluids such as plasma, sera (Paczesny et al., 2009; McDonald et al., 2015), or urine, are the preferred samples. Enormous effort has been placed into developing standardized methods for clinical sample collection (Rai et al., 2005; Court et al., 2011). Plasma and sera are the most frequently analysed bio-fluids. The levels of individual blood proteins represent a summation of multiple, disparate events that occur in every organ system. Plasma and sera contain proteins shed by the affected tissue as well as proteins that reflect secondary systemic changes. Urine samples represent an alternative to plasma/sera samples for biomarker discovery. Urine has 3 main advantages compared with plasma/sera: (1) it can be obtained in large quantities; (2) the protein mixture is far less complex and the variation in protein abundance is low (Thongboonkerd, 2007) ; and (3) it is more stable (Schaub
et al., 2004). However, urine yields better information about diseases in organs that
are directly involved in its production and excretion, such as the kidneys (Paczesny, 2013a).
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Although blood biomarkers are ideal for use in a clinical setting, one goal of research into the fundamental biology of GvHD is to identify markers that are target-tissue specific. Thus, the ideal sample for discovery of biologically relevant GvHD proteins may be the target tissue itself. However, finding tissue-specific markers has thus far proven difficult because of the cellular heterogeneity of tissues, and the limited material available in biopsies for tissue proteomics. To date, there is no method capable of amplifying the amount of proteins requiring, at best, pooling of several biopsies (Tangrea et al., 2004; Hwang et al., 2007).