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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]


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[5] Ballman, K. V. (2015). Biomarker: predictive or prognostic? . Retrieved from Journal of Clinical Oncology :

[6] Carolina N. Correia, Nicolas C. Nalpas, Kirsten E. McLoughlin, John A. Browne, Stephen V. Gordon, David E. MacHugh, and Ronan G. Shaughnessy. (2017 , February 16). Circulating microRNAs as Potential Biomarkers of infectious Disease. Retrieved from frontiers in Immunology :

[7] Lee RC, Feinbaum RL, Ambros V. . (1993, December 03). The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Retrieved from ScienceDirect :

[8] Wightman B, Ha I, Ruvkun G. . (1993, December 03). Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans. . Retrieved from ScienceDirect :

[9] Kozomara A, Griffiths-Jones S. (2014, January 01 ). miRBase: annotating high confidence microRNAs using deep sequencing data. Retrieved from Oxford Academic :

[10] Wheeler BM, Heimberg AM, Moy VN, Sperling EA, Holstein TW, Heber S, et al. (2009 , January 19). The deep evolution of metazoan microRNAs. . Retrieved from Wiley Online Library :

[11] E., B. (2011, November 18 ). Evolutio of microRNA diversity and regulation in animals. Retrieved from nature reviews genetics:

[12] O’Connell RM, Rao DS, Chaudhuri AA, Baltimore D. . (2010 , February ). Physiological and pathological roles for microRNAs in the immune system. Retrieved from nature reviews immunology :

[13] O’Neill LA, Sheedy FJ, McCoy CE. (2011, February 18). microRNAs: the fine-tuners of toll-like receptor signalling. . Retrieved from nature reviews immunology :

[14] Shenoy A, Blelloch RH. (2014, August 13). Regulation of microRNA function in somatic stem cell proliferation and differentiation . Retrieved from nature reviews molecular cell biology:

[15] Lee Y, Kim M, Han J, Yeom KH, Lee S, Baek SH, et al. (2004 ). microRNA genes are transcribed by RNA polymerase II. . Retrieved from The EMBO Journal :

[16] Lee Y, Ahn C, Han J, Choi H, Kim J, Yim J, et al. (2003). The nuclear RNase III Drosha initiates microRNA processing. . Retrieved from nature :

[17] Lund E, Guttinger S, Calado A, Dahlberg JE, Kutay U. (2004, January 02). Nuclear export of microRNA precursors. Retrieved from Science:

[18] Zhang HD, Kolb FA, Brondani V, Billy E, Filipowicz W. (2002, November 01). Human Dicer preferentially cleaves dsRNAs at their termini without a requirement for ATP. . Retrieved from The EMBO Journal:

[19] Chendrimada TP, Gregory RI, Kumaraswamy E, Norman J, Cooch N, Nishikura K, et al. (2005, June 22). TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing. . Retrieved from nature:

[20] Lewis BP, Shih IH, Jones-Rhoades MW, Bartel DP, Burge CB. (2003, December 26). Prediction of mammalian microRNA targets. . Retrieved from ScienceDirect :

[21] Schwarz DS, Hutvagner G, Du T, Xu Z, Aronin N, Zamore PD. (2003, October 17). Asymmetry in the assembly of the RNAi enzyme complex. Retrieved from ScienceDirect :

[22] Kim Y, Yeo J, Lee JH, Cho J, Seo D, Kim JS, et al. . (2014, November 06). Deletion of human tarbp2 reveals cellular microRNA targets and cell-cycle function of TRBP. Retrieved from ScienceDirect :

[23] Morin RD, O’Connor MD, Griffith M, Kuchenbauer F, Delaney A, Prabhu AL, et al. (2008). Application of massively parallel sequencing to microRNA profiling and discovery in human embryonic stem cells. . Retrieved from Genome Research :

[24] Guo L, Chen F. (2014, July 01). A challenge for miRNA: multiple isomiRs in miRNAomics. Retrieved from ScienceDirect :

[25] Ruby JG, Jan CH, Bartel DP. . (2007, June 24). Intronic microRNA precursors that bypass Drosha processing. Retrieved from nature :

[26] Robbins PD, Morelli AE. (2014 , February 25). Regulation of immune responses by extracellular vesicles . Retrieved from nature reviews immunology :

[27] Weber JA, Baxter DH, Zhang S, Huang DY, Huang KH, Lee MJ, et al. (2010 , November 01). The microRNA spectrum in 12 body fluids. Retrieved from Oxford Academic :

[28] Turchinovich A, Weiz L, Langheinz A, Burwinkel B. . (2011, September 01). Characterization of extracellular circulating microRNA. . Retrieved from Nucleic Acid Research :

[29] Zhang Y, Liu D, Chen X, Li J, Li L, Bian Z, et al. (2010 , July 09 ). Secreted monocytic miR-150 enhances targeted endothelial cell migration. Retrieved from ScienceDirect :

[30]Wang K, Zhang S, Weber J, Baxter D, Galas DJ. (2010 , November 01 ). Export of microRNAs and microRNA-protective protein by mammalian cells. Retrieved from Nucleic Acids Research :

[31] Cortez MA, Bueso-Ramos C, Ferdin J, Lopez-Berestein G, Sood AK, Calin GA. . (2011, June 07). microRNAs in body fluids – the mix of hormones and biomarkers. Retrieved from nature reviews clinical oncology :


This article was prepared by Emeralda Erna Nordin


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