Monday Article #45: Potential Biomarkers - Circulating microRNAs

A biomarker has to be something that can be measured: accurately and reproducibly. There are several definitions of a biomarker, The World Health of Organization (WHO) definition of a biomarker includes the statement: ‘any measurement reflecting an interaction between a biological system and a potential hazard, which may be chemical, physical, or biological. The measured response may be functional and physiological, biochemical at the cellular level, or a molecular interaction. [1] The ideal biomarker has high specificity and sensitivity, is detectable by minimally invasive sampling procedures, and its concentration should be indicative of a disease state [2-4] Diagnostic biomarkers can be used to evaluate disease status, prognostic biomarkers are informative of disease outcome, and predictive biomarkers help determine treatment efficacy when experimental groups are compared to controls [5]

MicroRNAs (miRNAs) are a class of small non-coding endogenous RNA molecules that regulate a wide range of biological processes by post-transcriptionally regulating gene expression. The fundamental regulatory role of miRNAs in a variety of biological processes suggests that differential expression of these transcripts may be exploited as a novel source of molecular biomarkers for many different disease pathologies or abnormalities. This has been emphasized by the recent discovery of remarkably stable miRNAs in mammalian biofluids, which may originate from intracellular processes elsewhere in the body. [6]

The role of miRNAs in post-transcriptional regulation of gene expression was discovered in 1993 through analyses of the lin-4 locus in the roundworm Caenorhabditis elegans. Two contemporaneous studies showed that an RNA transcript from lin-4 repressed translation of the lin-14 messenger RNA (mRNA), thereby exerting temporal developmental control on a diverse range of cell lineages [7, 8] Since then, it has been demonstrated that eukaryotic organisms contain hundreds to thousands of these small non-coding regulatory RNA molecules [9]. Many miRNAs are evolutionarily conserved across divergent metazoan taxa [10, 11] , highlighting the extensive roles that these small RNAs play in the regulatory networks and pathways governing complex biological processes such as cell fate specification, and innate and adaptive immunity [12-14]

Canonical biogenesis of miRNA in mammalian cells starts with transcription of a long RNA molecule called the primary-miRNA (pri-miRNA) by RNA polymerase II [15]. Within the nucleus, pri-miRNA undergoes cleavage by the microprocessor complex, which consists of a Drosha ribonuclease III and the RNA-binding DGCR8 microprocessor complex subunit protein [16]. The intermediate product is a precursor-miRNA (pre-miRNA) hairpin of ~70 nucleotides in length that is transported to the cytoplasm by the exportin-5 protein [17] . An additional cleavage occurs near the pre-miRNA terminal loop through the action of endoribonuclease Dicer [18]. The final product is an 18–25 nucleotide double-stranded RNA with short 3′ overhangs that binds to argonaute (AGO) proteins and is loaded into the RNA-induced silencing complex (RISC) by the RISC-loading complex (RLC), which is formed by endoribonuclease Dicer, RLC subunit TARBP2, and AGO1–4 proteins [19]. One strand of the RNA duplex, the mature miRNA, remains within the RLC and is used as a guide by the RISC for complementary nucleotide base pairing with a target mRNA [20]. The second strand is known as miRNA* (or passenger strand) and is normally degraded after its release from the RLC. Further details on canonical biogenesis [21] and the processes driving mature miRNA strand selection [22] have been extensively reviewed elsewhere. The development of HTS technologies has facilitated high-resolution miRNA-sequencing (miRNA-seq), revealing the existence of multiple functional mature variants that are termed isomiRs [23, 24]. In addition, non-canonical pathways have been identified as alternative mechanisms of miRNA biogenesis. [25]

However, there is a growing consensus that immune and non-immune cells routinely and actively release miRNAs into extracellular environments [26]. Commonly associated with RNA-binding proteins, high-density lipoprotein particles or enclosed within lipid vesicles (Figure 1), miRNAs have been found to be extremely stable in extracellular fluids of mammals, such as blood plasma, serum, urine, saliva, and semen [27, 28] miRNAs released by a human THP-1 monocyte cell line may be taken up by recipient cells in an alternative means of cell-to-cell communication [29]. Wang and colleagues have shown that nucleophosmin, an RNA-binding protein involved with nuclear export of ribosomes, mediates export and protection of circulating miRNAs against degradation in several human cell lines (HepG2, A549, T98, and BSEA2B) immediately after serum deprivation, which is suggestive of an active response to stress [30]. Active release of extracellular circulating miRNAs supports the hypothesis that they may act as “hormones” in cell-to-cell communication. [30, 31]

Mycobacterial pathogen-associated molecular patterns are recognized by toll-like receptors (TLRs) and other pattern recognition receptors, which result in the upregulation of primary-miRNAs in macrophages. These transcripts are subsequently cleaved in the nucleus and cytoplasm by Drosha and Dicer, respectively, resulting in 21–25 nucleotide mature miRNAs that act to fine-tune intracellular immune processes. Specific pathways and components of the immune response may be regulated by different miRNA subsets. Concurrently, the surrounding T lymphocytes involved in granuloma formation/maintenance upregulate T cell subset-specific miRNAs as a means of modulating the type of adaptive immune response. Mature miRNAs generated in macrophages and T cells may also be released into the extracellular environment within exosomes, heterogeneous microvesicles, or in association with high-density lipoprotein, LDL, or other protein complexes. Subsequently, by means not yet fully understood, these extracellular miRNAs move from local sites of infection to the circulatory system. This process can therefore give rise to infection-specific circulating miRNA expression signatures that can readily be accessed from multiple biological fluids (e.g., serum, plasma, or sputum). [2]

Further work is required to fully understand how the release of extracellular miRNAs and uptake by target cell populations influences biomolecular signalling networks. Regardless of their precise functions, the main utility of miRNAs in the field of diagnostics and prognostics is based on the premise that different miRNA expression signatures are linked to different pathological states. [2]

Reference(s)

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This article was prepared by Emeralda Erna Nordin

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