Circulating microRNAs as biomarkers in cancer diagnosis

Journal Pre-proof Circulating microRNAs as biomarkers in cancer diagnosis

Md Mahmodul Hasan Sohel PII:

S0024-3205(20)30221-6

DOI:

https://doi.org/10.1016/j.lfs.2020.117473

Reference:

LFS 117473

To appear in:

Life Sciences

Received date:

9 January 2020

Revised date:

24 February 2020

Accepted date:

24 February 2020

Please cite this article as: M.M.H. Sohel, Circulating microRNAs as biomarkers in cancer diagnosis, Life Sciences(2020), https://doi.org/10.1016/j.lfs.2020.117473

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

© 2020 Published by Elsevier.

Journal Pre-proof Circulating microRNAs as biomarkers in cancer diagnosis Md Mahmodul Hasan Sohela, b, * a

= Genome and Stem Cell Centre, Erciyes University, Kayseri 38039, Turkey.

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= Department of Genetics, Faculty of Veterinary Medicine, Erciyes University, Kayseri 38039,

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Turkey.

*

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= Corresponding author

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Md Mahmodul Hasan Sohel

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Department of Genetics, Faculty of Veterinary Medicine, Erciyes University, Kayseri 38039,

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Turkey.

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e-mail address: [email protected]

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Journal Pre-proof Abstract MicroRNAs (miRNAs) are a group of tiny molecules of 18-22 nucleotide long noncoding RNA that regulate the post-transcriptional gene expression through translational inhibition and/or mRNA destabilization. Because of their involvement in important developmental processes, it is highly likely that the altered expression of miRNAs could be associated with abnormal conditions like suboptimal growth or diseases. Thus, the expression of miRNAs can be used as

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biomarkers in pathophysiological conditions. Recently, a handful of miRNAs are detected in

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cell-free conditions including biofluids and cell culture media and they exhibit specific

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expression patterns that are associated with altered physiological conditions. Extracellular

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miRNAs are not only extremely stable outside cells in a variety of biofluids but also they are

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easy to acquire. These characteristics led to the idea of using extracellular miRNAs as a potential biomarker for the onset and prognosis of cancer. Although miRNAs have been proposed as a

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potential diagnostic tool for cancer detection, their application in the routine clinical

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investigation is yet to come. First, this review will provide an insight into the extracellular miRNAs, particularly, their release mechanisms and characteristics, and the potential of

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extracellular miRNAs as a biomarker in cancer detection. Finally, it will discuss the potential of using extracellular miRNAs in different cancer diagnoses and challenges associated with the clinical application of extracellular miRNAs as noninvasive biomarkers.

Keywords: Extracellular microRNA; Circulating microRNA; Biomarker; Cancer detection; Exosome

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Journal Pre-proof Introduction Despite the tremendous advancement in the field of medical science in terms of advanced diagnosis and pharmacological treatment modalities, we still have to go a long way to reach a point where there will be an accurate diagnosis and complete cure for complex diseases like cancer. The treatment/health care cost, disability, premature death due to cancer create a huge socioeconomic burden worth 2.5 trillion a year [1]. It has been identified that cancer is the single

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most important barrier to increase the average life expectancy in every country in the 21st

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century. The rapid growth of cancer incidence and mortality worldwide could be related to

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several factors including aging and growth of population, as well as the distribution and

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prevalence of cancer risk factors which are often associated with socioeconomic development.

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Approximately 70% of all death from cancer occur in low- and middle-income countries. Despite considerable advancement has been made in the prevention and treatment of many types

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of cancers that lead to prolonged survival or even sometimes cure, early detection remains a

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significant challenge in many cancer subtypes due to the lack of suitable and reliable markers as

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a diagnostic tool. Approximately two decades ago, scientists realized that additional components of the human genome, commonly known as microRNAs, are essential to cancer biology. Expression studies showed that a number of miRNAs are differentially expressed in various types of cancer. Very recently, Hunter and colleagues demonstrated that a handful of miRNAs are present in circulation which is enwrapped by lipid-membraned microvesicles [2]. Since their discovery, the extracellular miRNA expressions have been shown to be associated with cancer and many other diseases that have ignited considerable interest in biomarker development [3]. A particular area of intense focus has been the development of noninvasive biomarkers for cancer diagnosis, early detection, and therapeutics. However, many studies focused on noninvasive 3

Journal Pre-proof biomarker development have limited acceptability due to lack of reproducibility and an unclear understanding of the biological relevance of these extracellular miRNAs in the circulation. This review will particularly focus on the extracellular miRNA biogenesis, the growing field of exosomal extracellular miRNAs in biomarker development and the challenges in clinical settings.

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A glimpse of cellular microRNAs

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A variety of short non-coding RNAs including PIWI-interacting RNA (piRNA), small interfering RNA (siRNA), and microRNA (miRNA) have been discovered in the eukaryotic genome.

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Initially, these non-coding RNAs were considered as ‘RNA junk’ because of the lack of their

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known functions. These so-called junk RNA molecules were thought to be the evolutionary

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debris lack of true functions. Although the definition of RNA junk is still elusive, many of these small non-coding RNAs are now recognized because of their ability to control gene expression in

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developmental processes and modulate cell signaling regulations. In addition, genome-wide

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transcriptional studies revealed that most of the transcribed mammalian genomic sequences are

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non-coding RNAs. Notable characteristics of small RNA are their association with Argonaut proteins, particularly AGO2, and their length (approximately 18-30 nucleotide). Among all the short RNAs, miRNAs are the dominating category that is involved in the regulation of a wide range of cellular processes including growth, apoptosis, proliferation, and differentiation [4]. Because of their vital role in different biological processes, differential expression of miRNAs creates an alteration of normal cellular and biological function, hence participating in pathological conditions including cancer [5]. Off the short RNAs, miRNAs are studied extensively and have been shown to be associated with every step of cancer starting from initiation of the tumor to metastasis. miRNAs are a 4

Journal Pre-proof considerable family of small-sized endogenous, noncoding RNA molecules of typically 18-22 nucleotide long that have the ability to alter the gene expression at the post-transcriptional level [6]. In the majority of the cases, miRNAs either destabilize their target messenger RNA (mRNA) sequence or inhibit the translation of protein by an improper binding at 3´-untranslated region (3´-UTR). In total, approximately 38,589 mature miRNAs have been identified in all organisms investigated so far (miRBase, Release 22). miRNAs are estimated to comprise more than 5% of

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the mammalian genome. While, only about 2,654 mature human miRNAs have been identified

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so far (according to miRBase, Release 22), computational analysis of genome predicts that as

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many as 50,000 miRNAs could be existed in the human genome [7] where each miRNA could

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target several genes based on the sequence similarity at the 3´-UTR of mRNAs. Indeed, bioinformatics analysis has predicted that approximately 60% of mammalian protein-coding

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genes can be targeted by at least one miRNA.

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The first miRNA was reported in the early 1990s in Caenorhabditis elegans [8]. Following this

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discovery, miRNAs have been detected in several organisms starting from single-cell algae to multicellular mammals and plants indicating that miRNA-mediated regulation of biological

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process is a very complex and ancient process. Intense conservation of both individual miRNAs and their processing machinery further reinforces the importance of miRNA function. The source of the majority of miRNAs is an ‘independent’ gene which is also known as the intergenic miRNA gene. However, more than 25% of miRNAs are generated from introns placed within the canonical gene sequence. Indeed, miRNA genes could be located in the intergenic area as well as in the exons and introns of protein-coding mRNAs. In addition, the total number of miRNAs may represent the complexity of the developmental process of an organism, as mammals having the largest set of functional miRNAs.

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Journal Pre-proof miRNA and their biogenesis miRNA biogenesis is multi-step processing and involves both nuclear and cytoplasmic processing (Figure 1). miRNAs are transcribed from both intragenic and intergenic chromosomal DNA region. About half of the known miRNAs have an intragenic origin (mostly from introns and few from exons) while remaining other miRNAs are from the intergenic origin [9]. At the beginning of the biogenesis of canonical miRNAs, polymerase II enzyme is activated to produce

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primary miRNA transcripts (pri-miRNAs) of various lengths (generally 1 – 3-kilo bites) from the

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non-coding fraction of the genome. A microprocessor composed of a ribonuclease III (Drosha)

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and an RNA binding protein called DiGeorge syndrome critical region gene 8 (DGCR8/Pasha)

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further processes these pri-miRNAs [6]. Within the pri-miRNA, DGCR8 binds at the N6-

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methyladenylated GGAC and other motifs and Drosha cleaves the pri-miRNA duplex to produce a ~70 nucleotides long hair-pin steam-loop structure called precursor miRNA (pre-miRNA).

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Once pre-miRNAs are generated, a transporter complex consists of Exportin-5 and Ran-GTPase

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carries them to the cytoplasm. In the cytoplasm, a micro-scissor called Dicer (RNase III enzyme) coupled with the TRBP and PACT proteins removes the terminal loop to produce mature

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miRNA duplex. The mature miRNA name is determined by the directionality of the miRNA strand. For instance, the 5p form of a mature miRNA originates from the 5´end while the 3p strand arises from 3´ end of the pre-miRNA. Both the 5p and 3p strands are loaded in the Argonaute (Ago) protein family, particularly Ago 1-4 in mammals, based on the thermodynamic stability at the 5´end of the miRNA duplex. Among the four members of the Ago family, Argonaute2 (Ago2) contains an RNaseH-like PIWI domain that mediates miRNA cleavage and becomes the most important component of RNA-induced silencing complex (RISC). The proportion of 5p or 3p strand loaded into Ago-protein varies greatly on the cellular type and

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Journal Pre-proof environment and the thermodynamic stability at the 5´end of the miRNA duplex [10]. It could be distributed nearly in equal proportions or predominantly one or another. Generally, on a preferential basis, the strand with 5´ uracil or lower 5´ stability is loaded into the Ago2 protein complex and considered as a guide strand. On the other hand, the unloaded strand is deemed as a passenger strand. Through a variety of mechanisms, the passenger strand will be unwound from the guide strand and cleaved by Ago2 depending on the mismatches followed by cellular

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machinery that led to the degradation [9].

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It is important to note that many of the protein components of the canonical pathway including

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Exportin 5, Drosha, Dicer, and Ago2 have also been found to be important in the non-canonical

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pathway-mediated miRNA processing. Although several non-canonical pathways have been

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identified, they are generally grouped into two major groups such as Dicer-independent and DGCR8/Drosha-independent pathway.

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miRNA-mediated gene regulatory mechanisms

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Genome-wide identification and computational prediction of miRNA targets revealed that a

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single miRNA may regulate several genes, suggesting a significant proportion of genome (more than 60% in mammals) could potentially be regulated by miRNAs. Because of this complex relationship between miRNA and mRNA, it becomes the biggest challenge for the researchers to deal with miRNA functional studies in different biological processes. Scientists from around the world are trying to understand the miRNA mediated gene regulatory mechanism. To date, the majority of studies showed the miRNA mediated gene regulation through sequencing specific binding at the 3´ UTR of their target genes. A sequence-specific binding could induce decapping, mRNA deadenylation, and translational repression [11]. Although the detailed mechanism of

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Journal Pre-proof miRNA mediated gene expression regulation will not be discussed in this review, a glimpse of known mechanisms will be provided to understand the underlying molecular mechanisms. Although the miRNA mediated gene regulation is yet to discovers in detail, several models have been proposed to explain the mechanism. The current knowledge sheds light that it can regulate the expression of genes primarily in three ways- post-transcriptional gene silencing in the cytoplasm, translational activation, and transcriptional and post-transcriptional regulation within

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the nucleus. In the miRNA mediated gene silencing model, a multiprotein complex called

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miRISC (miRNA-induced gene silencing complex) is involved which consists of several proteins

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including Dicer, TRBP, and Ago2 and a guide strand [12]. Depending on the matching of

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complementary sequence on the target mRNA, it can be silenced in two ways. In a complete

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matching (a fully complementary miRNA:mRNA responsive element), an Ago2 dependent silencing takes place where Ago2 endonuclease activity initiates mRNA cleavage, reviewed in

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[9]. On the other hand, in case of incomplete matching (happens mostly in animal cells),

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miRISC-mediated translational inhibition takes place and subsequently degrades the mRNA [9]. Although it is not entirely clear, some studies have reported miRNA mediated upregulation of

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their target genes [13,14]. In many of the cases, miRNA mediated translational activation takes place under specific environmental conditions such as amino acid starvation, serum starvation, and cell cycle arrest. In the last mechanism, it has been suggested miRISC may interact with mRNAs within the nucleus and promote the degradation of nuclear mRNA. In this regard Importin-8 or Exportin-1 play a significant role by shuttling Ago2 from the nucleus and cytoplasm. Although the mechanism is yet to fully elucidate, it has been demonstrated that the miRISC with comparatively low molecular weight can target mRNAs within the nucleus and subsequently degrade them [9].

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Journal Pre-proof Extracellular miRNAs (ECmiRNAs) and their characteristics The discovery of miRNAs in 1993 by Ambros and colleagues opened a new era of research related to genome regulation. Previously, miRNA, like other RNAs, has been considered as solely cellular property and thought to be degraded quickly in the extracellular environment. However, in 2007 Valadi and colleagues showed that miRNAs can be released in the extracellular environment in cell culture medium and the showed specific expression pattern

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[15]. Two subsequent studies in 2008 showed that miRNAs are present in the circulation and

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their expression pattern is associated with cancer [2,16]. These findings ignited the idea of the

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existence of miRNAs outside the cellular environment and subsequently termed as

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extracellular/circulating miRNAs depending on their origin. miRNAs found in circulation are

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known as circulating miRNAs and those found in other biofluids and cell culture media are termed as extracellular miRNAs. All miRNAs found outside cells are extracellular miRNAs

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(ECmiRNAs) but only miRNAs found in circulation are circulating miRNAs [17].

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Since their discovery in 2007, ECmiRNAs are found in almost all biological fluids including

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plasma and serum [18–20], follicular fluid [21–23], seminal fluid [24], synovial fluid [25], breast milk [26], saliva [27], cerebrospinal fluid [28], amniotic fluid, tears, peritoneal fluid [29]. Many of the studies reported specific expression patterns of ECmiRNAs in biological fluids in association with a variety of pathophysiological events. Therefore, it appears that the release of miRNAs from cells is not random rather they could release from cells in a selective manner. Cellular miRNAs or other RNAs undergo a quick degradation in the extracellular environment because of the presence of the RNase enzyme. A quick degradation of synthetic miRNAs was observed when they were spiked into plasma. However, inactivating the RNase activity by denaturing solution rescue the exogenous miRNAs from quick degradation [16]. In contrast to 9

Journal Pre-proof cellular miRNAs, ECmiRNAs show remarkable stability in biofluids. Some reports have demonstrated that ECmiRNAs are stable even after they are exposed to harsh environments including low to high pH, boiling, long storage, and several freeze-thaw cycles [30–32]. Surprisingly, these ECmiRNAs are detectable and gave a specific expression pattern even after incubation for four days at room temperature [16]. The extraordinary stability of ECmiRNAs suggesting that they might skip the high RNase activity through some protective mechanisms.

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Because of their accessibility, remarkable stability in different bio-fluids and specific expression

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patterns in association with diseases, they are frequently used as non-invasive biomarkers for

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early detection of several complex diseases.

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Loading and release mechanisms of ECmiRNAs

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The mechanisms underlying the outstanding stability of ECmiRNAs are not clearly understood. To explain this extraordinary stability of ECmiRNAs, several hypotheses have been proposed.

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Despite their resistance against high RNase activity in the extracellular matrix, ECmiRNAs are

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highly susceptible to proteinase K [33] and detergent activity [34] suggesting lipoprotein

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membrane encapsulated or protein associated existence and release of miRNAs from cells to the extracellular matrix. Based on these observations, two hypotheses have been proposed to explain the release and existence of ECmiRNAs. The first one is incorporation with lipoprotein membrane-derived vesicles such as high-density lipoproteins, exosomes, microvesicles, and apoptotic bodies. Although the entire mechanisms of loading of miRNAs into these vesicles are not known [35,36], the suggested mechanisms are divalent cation bridging between miRNAs and HDL [37], a special motif GGAG in 3´ end region of miRNAs controlling the sorting and loading of miRNAs into exosomes [38], or neural sphingomyelinase 2 (nSMase2) mediated miRNA loading into exosomes [39]. However, the sorting and loading mechanisms of miRNAs 10

Journal Pre-proof into shedding vesicles and apoptotic bodies are yet to discover. The loading and unloading of exosomal miRNAs are discussed elaborately by Beuzelin and Kaeffer [40]. The second hypothesis suggests that the ECmiRNAs are associated with protein complex, particularly the Argonaut 2 (a miRNA processing gene) [41]. Valadi and colleagues first reported the release and existence of miRNAs in the cell culture media. In addition, they also demonstrated that these exosome bound miRNAs are transferrable

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and when they are transferred to the new cell they could modulate the protein expression [15]. In

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the following year, Hunter and colleagues demonstrated the presence of miRNAs in

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microvesicles derived from peripheral blood [2] which further establish the idea of vesicle

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encapsulated existence of ECmiRNAs. Extracellular vesicles are a heterogeneous population that

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contains both exosomes and microvesicles. Exosomes are a more homogenous population of vesicles whose diameter ranged from 30-100 nm [42], while the microvesicles are heterogenous

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ranged from 100 – 1000 nm in diameter [43]. Exosomes are producing through an endocytic recycling pathway (inward budding). During the biogenesis of exosomes, endosomal sorting

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complexes required for transport (ESCRT) proteins such as HSC70, HSP90β, TSG101, and Alix

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play a central role. Therefore, these proteins are expected to be found in the exosomes regardless of the origin of cells [44,45]. In addition, plasma membrane proteins like transmembrane proteins (CD9, CD63, and CD81) are also found in exosomes [44,46]. Apart from the exosomesspecific proteins, the protein content of exosome can differ depending on the cell types. For instance, using proteomic analysis, it has been shown that the exosome from U87 glioblastoma cells and Huh7 hepatocellular carcinoma cells share similar protein patterns, while bone-merrow derived mesenchymal stem cells (MSCs)-derived exosomes showed different protein pattern [47]. Although minor quantities of cytosolic lipids can present in exosomes, membrane lipids are

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Journal Pre-proof the major constituents of exosomal lipids. Therefore, exosomal lipid composition resembles the composition of the lipid bilayer of the cell of origin [48]. It is important to note that the phosphatidylcholine and sphingolipids are located in the outer leaflet and other lipid classes are present in the inner leaflet of exosomal lipid bilayer [48]. The lipidomic profile of exosomes can also be similar or different based on their origin. It is important to note that the whole proteomic structure of exosomes from different cells may have a similar pattern, but they could have

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different lipidomic patterns [47]. Microvesicles are produced via outward budding and fission.

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Because of the fact that microvesicles are formed from the plasma membrane, it is highly likely

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that it contains primarily plasma membrane-associated and cytosolic proteins [44]. The

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proteomic profiles of exosomes and microvesicles are different. While microvesicles contain a higher level of mitochondrial, proteasome, and endoplasmic reticulum proteins, exosomes

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contain proteins related to adhesion function, immune response, and extracellular matrix [47].

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For extensive discussion about extracellular vesicle biogenesis and composition please see reviews by Doyle and Wang [44] and Skotland et al. [48]. Soon after the first report, several

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biofluids.

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studies have demonstrated the exosome and/or microvesicles-associated miRNAs in a variety of

In 2011, vesicle-associated miRNA theory faced a huge challenge when two independent studies reported that the majority of the ECmiRNAs in cell culture media [33] and plasma [41] are not associated with vesicles. The authors claimed that potentially 90% ECmiRNAs are associated with protein complex, particularly Ago2, and only 10% ECmiRNAs are released in the extracellular matrix through vesicles [41]. Ago2 is a miRNA machinery gene which is extremely stable in the extracellular matrix. These statements explain the loading, release, and existence of Ago2 incorporated ECmiRNAs in the extracellular environment.

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Journal Pre-proof To date, many studies have demonstrated the existence of miRNAs through high-density lipoprotein [49], apoptotic bodies [50], Ago2 protein [41], microvesicles [2], and exosomes [36,42]. The size and characteristics of different extracellular vesicles and HDL hosting miRNAs are presented in Table 1. A schematic diagram of the release of miRNAs is presented in Figure 2. Dying cells release the apoptotic bodies in the extracellular space. Both the processing mechanism and content of

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apoptotic bodies may differ from exosomes and microvesicles. When the cell shrinks in size,

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increase hydrostatic pressure mediates the separation of plasma membrane from the cytoskeleton

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to facilitate the generation of apoptotic bodies. In contrast to microvesicles and exosomes,

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apoptotic bodies may contain glycosylated proteins, chromatin, and intact organelles [44]. It is

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currently unknown whether the miRNAs are loaded into apoptotic bodies in a selective manner or randomly from the cytosol. The existence of miRNAs in association with apoptotic bodies is

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questionable because of the size of the vesicles (similar to the size of platelet and cellular debris).

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Furthermore, during the sample processing step at the time of the initial centrifugation, apoptotic bodies are removed along with cellular debris. Therefore, this fraction of ECmiRNAs is

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overlooked and understudied.

Differential regulation of circulating miRNAs in different cancers with biomarker potential Despite the initial belief that ECmiRNAs were simply byproducts of cellular damage and apoptosis or death with no real biological functions, increasing evidence has suggested that the release of these miRNAs in the extracellular environment is a regulated process and may represent signaling molecules with a definite role in cell-cell communication. Subsequent findings of ECmiRNAs in microvesicles or exosomes or other carriers in addition to their specific expression patterns in various biofluids in association with pathophysiological 13

Journal Pre-proof conditions provoked us to postulate that they are the potential molecule to develop a noninvasive biomarker for early identification of diseases like cancer. In agreement with this concept, several studies have shown the utility of ECmiRNAs as a cancer biomarker. Since cellular miRNAs are found to be involved in regulating a variety of physiological, cellular, and developmental processes, their aberrant expression potentially indicates the scenario of dysregulation and/or disease including cancer. In fact, a number of miRNAs have been shown to

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be located in the vulnerable fragment of DNA which is associated with cancer [7]. ECmiRNAs

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in the circulation is a subpopulation of cellular miRNAs, therefore, it is logical to think that they

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could also represent a cellular condition in the circulation. Indeed, many studies have

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demonstrated the association of cancer and dysregulated ECmiRNA expressions in the

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circulation.

We have observed in the last two decades, the breast cancer-associated mortality has been

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decreased due to early detection and improved treatment. Indeed, cancer patients have a

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significantly higher survival rate if it is detected in the primary stage during disease progression

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before the formation of tumors and metastasis. However, the symptoms and signs may not appear early stage of the disease. Therefore, the major challenge in cancer treatment is the early detection or prediction of disease which can be done by analyzing the miRNA abundance in circulation. Differential expression of ECmiRNAs with biomarker potential in common cancers is listed in the following subsections. Lung cancer The most common form of cancer is lung cancer (LC) which is currently the leading cause of cancer-related death. Despite significant improvements that have been achieved in the diagnosis

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Journal Pre-proof and treatments of LC, the 5-year survival rate of LC is still under 20%. The primary reason is that the disease diagnosed at the later stages. Indeed, the 5-year survival rate significantly improves up to 70% if the LC is diagnosed at an early stage (stage IA). Early detection can drastically be improved by using circulating miRNA as biomarkers. The correlation between LC and differential expression of miRNAs has been shown in many studies. It is important to note that cellular miR-21 is considered as one of the most important onco-miRs and enhanced

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expression of miR-21 has been observed in many cancer types. In addition to the cellular level,

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extracellular circulating miR-21 has also been found to be differentially regulated in several

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cancer types including LC and has the potential to be used as a biomarker. A recent study

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showed that the expression of circulating miR-21 along with miR-210 was elevated in the LC patient and was able to discriminate early-stage lung cancer patients from non-smoking healthy

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individuals [53]. Furthermore, the authors demonstrated that circulating miR-486-5p expression

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was significantly reduced in the blood plasma of malignant solitary pulmonary nodules (SPNs) patients. These three circulating miRNAs were consistently differentiating LC patients from

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healthy individuals with 75% specificity and 84.95% accuracy [53]. Validation of these miRNAs

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in testing sets further confirms the potential of these miRNAs as a noninvasive biomarker for LC detection and prognosis. A significant increase in the expression of circulating miR-31 in LC patients compared to healthy controls was reported by Yan and colleagues. Furthermore, they showed that the average survival of the patients with high circulating miR-31 expression group was shorter (25.23 months) than that of the low expression group (38.44 months) [54]. A study reported that the miR-34 family can effectively be used in LC diagnosis where the overexpression of miR-34a and miR-34c were associated with a longer disease-free as well as overall survival [55]. The expression of miR-944 and miR-3662 was analyzed in the plasma of

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Journal Pre-proof 90 LC patients and 85 healthy individuals. Subsequently, they found that the higher expression of these miRNAs in the LC group compared to control and surprisingly the expression of these miRNAs is higher in the advanced stage [56]. In a later study, the authors reported another two circulating miRNAs, namely miR-448 and miR-4478, with the potential of noninvasive biomarkers for the early detection of LC patients from healthy individuals [57]. The expression of both miRNAs was significantly upregulated in the LC-patient’s plasma compared to those of

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healthy individuals. Furthermore, the authors reported that combine analysis of both miRNAs

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provides test sensitivity of 89.4% and specificity of 79% with an area under the curve (AUC) of

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0.895 [57] which is significantly higher form the previous studies where the authors used an

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individual miRNA. The most promising LC-specific circulating miRNAs are presented in Table

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2. Gastric cancer

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The second leading cause of cancer-related death is Gastric cancer (GC). Although with the

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tremendous advancement of treatments including surgery, chemotherapy, and radiotherapy, there

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is a significant 5-year increase in the survival rate at an early stage of GC, the prognosis for GC at an advanced stage still remains poor. Therefore, a reliable method to detect the GC at the early stage is extremely important and highly desirable. One of the earliest studies in ECmiRNA research reported the usability of circulating miRNAs as a biomarker for GC detection at an early stage where the authors demonstrated that the plasma concentration of miR-17-5p, miR-21, miR-106a, and miR-106b were significantly lower in GC patients than that of the healthy individuals [69]. Subsequently, several other studies showed the usability of plasma/serum ECmiRNAs as a biomarker to identify GC cancer at an early stage. For instance, the expression level of miR-221-3p, -376c-3p, and -744-5p changed significantly in the serum of GC patients 5 16

Journal Pre-proof years before the appearance of clinical symptoms [70]. In addition, it has been shown that the serum circulating miR-203 and miR-218 expression level is closely associated with GC metastasis [71,72]. In another study, using plasma samples, Tsai and colleagues showed that circulating miR-196a can be served as a potential biomarker for discriminating GC as its expression reduced significantly after the surgical resection and it showed higher specificity and accuracy than carbohydrate-antigen method [73]. Using quantitative droplet digital PCR

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(ddPCR), Zhao and colleagues demonstrated that the circulating miR-21, miR-93, miR-106a, and

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miR-106b expression levels at the training phase were significantly higher in the serum of GC

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patient and passed the validation phase with 82.2% accuracy [74]. It is worth to note that the

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crucial prognosis factor in GC is the lymph node metastasis (LNM) and its removal has been shown to be significantly increased the GC survival and the expression level of serum circulating

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miR-143-3p, miR-146a, miR-451a, and miR-501-3p seems promising in detecting the GC

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patients that develop LNM and provides prognostic information for LNM [75]. Although circulating miR-21 has been proposed as GC biomarker in several studies, it is considered one of

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the most studied oncomiRs and found to be associated with many other cancer types. Therefore,

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circulating miR-21 can be considered as a global biomarker for various cancer types, but not for specific cancer like GC. Table 3 presents the circulating miRNAs in GC. Breast cancer Breast cancer (BC) is responsible for the second-largest cancer-related death among women worldwide. Several studies showed that the potential of serum/plasma ECmiRNAs as a noninvasive biomarker for the discrimination of BC at an early stage. For instance, using global miRNA microarray expression profiling and subsequent validation, Hamam and colleagues reported that expression of nine miRNAs including miR-188-5p, miR-1202, miR-1225-5p, and 17

Journal Pre-proof miR-4270 is consistently higher in the early stages (stage I – III) compared to stage IV in BC patients [82]. The authors suggested that this panel of miRNA can be used for the early diagnosis and stratification of BC patients. However, the authors did not study the specificity and sensitivity of these miRNAs in BC samples. Therefore, the use of these miRNAs to detect BC at an early stage is still questionable. Based on large-scale data from the TCGA database, Yu and colleagues developed an effective and novel approach for the early detection of BC. The authors

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reported that a combination of three miRNAs (miR-21-3p, miR-21-5p, and miR-99a 5p) has

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produced highly accurate results in detecting early BC which achieved an AUC of 0.97 with a

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specificity of 73.5% and a sensitivity of 97.3% [83]. The circulating miRNA-34 family has been

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shown to be important in tumor regulation. Particularly, circulating miR-34c expression was significantly lower in triple-negative BC patients which were correlated with tumor grade and

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distant metastasis [84]. The reduced expression of circulating miR-34c is highly associated with

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tumor progression and poor prognosis. It is important to note that approximately 20 – 30% of cases of early-stage BC eventually develop distant metastasis and experience recurrence. A study

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showed that the expression of circulating miR-376c-3p, miR-382-5p, and miR-411-5p is

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consistently significantly lower in patients with recurrences compared to those without recurrences [85]. The authors suggested that these miRNAs can be used as a noninvasive biomarker for continuous monitoring of breast cancer recurrence. Among the other treatment options, neoadjuvant chemotherapy (NACT) recently became a reasonable option for the treatment of BC due to the fact that it can reduce the need for a complete axillary lymph node dissection and improves the breast-conserving surgery rates. It has been shown that the circulating miR-451 expression was significantly lower in BC patients with higher NACTresistance [86]. However, whether circulating miR-451 plays a role in the NACT-resistance is

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Journal Pre-proof not known. Due to the presence of a huge number of articles, it is extremely difficult to discuss all the studies in this article. The most promising findings regarding extracellular circulating miRNA-based biomarkers in BC are presented in Table 4. Colorectal Carcinoma It is important to mention that one of the major reasons for cancer-related death is the distance

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metastasis in cancer patients including those suffering from colorectal cancer (CRC) which is

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considered to be the most common malignant tumor worldwide. Similar to other cancer types, CRC has a poor prognosis due to the detection in the later stages. Circulating miRNA signature

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has been shown to be useful in the early detection of colorectal cancer in many studies. In one of

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the first studies, Chang and colleagues showed that circulating miR-141 was significantly

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associated with stage IV colon cancer [92]. The sensitivity and specificity of the candidate circulating miRNA were determined using Receiver Operating Characteristics (ROC) analysis.

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Furthermore, the authors observed that a combined examination of circulating miR-141 and

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carcinoembryonic antigen (CEA), a marker for CRC, improved the accuracy of CRC detection

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[92]. Although it is significantly associated with other cancer types and subtypes, one of the widely studied and consistently reported circulating miRNAs in CRC is miR-21. Maria and colleagues investigated a panel of circulating miRNAs in the plasma of CRC patients to find a reliable biomarker for early detection. Subsequently, they found that only miR-21 showed a distinct temporal increase during the 3-year period before diagnosis [93]. However, the authors suggested that further studies on larger patient groups are required to find the optimal panel of miRNA for the early screening of CRC. In another study conducted by Tsukamoto and colleagues found that the expression of miR-21 was significantly higher in metastasis tissues, primary tumor tissues, and plasma-derived exosomes [94]. In addition, the authors reported that 19

Journal Pre-proof the temporal expression changes of circulating miR-21 across different stages of CRC were associated with disease-free survival (DFS) and overall survival (OS). The patients with overexpression of plasma-derived exosomal miR-21 had significantly lower OS and DFS compared to the patients with lower circulating miR-21 [94]. The stage-specific investigation revealed that exosomal miR-21 was an independent prognostic factor for DFS and OS in patients with TNM stage II or III, and for OS in patients with TNM stage IV [94]. It could be deduced

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from the results that circulating miR-21 is a potential biomarker for the early detection,

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prediction of recurrence, and poor prognosis of CRC. Plasma or serum-derived miRNAs that are

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associated with colorectal cancer with a diagnostic potential are listed in Table 5.

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Hepatocellular Carcinoma

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Liver cancer is considered the sixth deadliest cancer across the world and hepatocellular carcinoma (HCC) is the major subtype that accounts for more than 85% of all liver cancer cases.

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The prognosis of HCC remains poor mainly because of liver cirrhosis. Commonly used

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techniques such as imaging, histological analysis of tissue biopsies, and plasmatic alpha-

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fetoprotein detection have several disadvantages including less sensitivity and false-positive detection. Although histological analysis of liver biopsy is considered as the gold standard for HCC diagnosis, it is an invasive procedure and only effective when the nodules reached considerable dimensions. Therefore, scientists and practitioners are looking for a non-invasive and highly accurate detection tool for the detection of HCC at an early stage. In this regard circulating miRNAs could be a trustable option. To date, several studies have demonstrated the potential of circulating miRNAs as a biomarker for HCC. For instance, circulating miR-200 family members are significantly deregulated in patients with liver cirrhosis and HCC [101]. Among the members of the miR-200 family, miR-141 and miR-200a were significantly 20

Journal Pre-proof downregulated in the serum of HCC patients compared to healthy controls (p < 0.002) and noncancerous liver cirrhosis (p < 0.007). While both miRNAs were able to discriminate patients with cirrhosis-associate HCC from healthy controls with high accuracy, the ability to differentiate between non-cancerous liver cirrhosis and HCC was fair [101]. Nevertheless, the applicability of these circulating miRNAs should be further investigated to confirm their diagnostic value. Cellular miR-122 has been shown to be exclusively associated with the liver and accounts for

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70% of the total miRNAs in the liver that plays an important role in liver homeostasis and

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hepatocarcinogenesis [102]. Therefore, it is logical to think that circulating miR-122 could also

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be associated with HCC and maybe the potential to be a potential biomarker for the detection of

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HCC. It has been shown that the higher expression of circulating miR-122 represents poor OS with a confidence interval of 95% in patients with HBV-related carcinoma who underwent

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radiofrequency ablation [103]. Other circulating miRNAs such as miR-224, miR-26a, and miR-

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29a were shown to be associated with HBV-related HCC patients and are independent prognostic markers for poor disease-free survival [104,105]. In order to asses, the risk of developing HCC in

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chronic liver cirrhosis patients Huang and colleagues proposed a circulating miRNA-based

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model where they showed that differential expression of circulating miRNAs in the serum of HBV and HCV patients and miRNAs can be deregulated long before the development of HCC [106]. A combination of traditional biomarkers and other RNA molecules with circulating miRNA signature has been shown to be highly accurate in discriminating the HCC patients from a healthy individual. For instance, low AUC, sensitivity, and specificity values were obtained when individual miRNA or alpha‐ fetoprotein were applied. The values were remarkably increased when a combination of miRNA and alpha‐ fetoprotein was used [107]. A similar study reported that a combination of serum level miR-548-a-3p, lncRNA-TSIX, and SOGA1 showed

21

Journal Pre-proof better diagnostic potential to differentiate HCC patients [108]. Table 6 listed the name of circulating miRNAs with biomarker potential in hepatocellular carcinoma discrimination. Pancreatic carcinoma Pancreatic cancer is an aggressive malignancy characterized by an abundant stroma, remarkable resistance to radiotherapy and chemotherapy, and the majority of the cases are detected at the

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advanced stage. As a result, the 5-year survival rate with pancreatic cancer is less the 5% which

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warrant improved medical intervention and early detection of the disease. The usefulness of circulating miRNAs as a diagnostic biomarker for the early detection of pancreatic cancer has

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been shown in many studies. Chronic pancreatitis (CP) often misdiagnosed as pancreatic cancer

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leading to unwanted pancreases resection. Significantly higher expression of circulating

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exosomal miR-10b, miR-181a, miR-21, and miR-30c and lower expression of miR-let7a effectively discriminate pancreatic cancer patients from normal control and CP patients [112].

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The authors further reported the circulating exosomal miRNA signature was superior to

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exosomal glypican-1 in differentiating pancreatic cancer patients from normal controls. In

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addition, the higher level of circulating exosomal miRNA reached a normal level within 24 h after pancreatic cancer resection [112]. Circulating miR-182 is another miRNA that might be considered as a potential biomarker for the discrimination of pancreatic cancer patients from the healthy controls as its expression was found to be remarkably higher in the circulation of pancreatic cancer patients [113]. Members of the miR-99 family including miR-99a, miR-99b, and miR-100 were found to be associated with pancreatic cancer progression. Circulating miR100 could well discriminate pancreatic cancer from normal healthy controls with high sensitivity and accuracy [113]. All of the six miRNAs (miR-17-5p, miR126-5p, miR-155, miR-1290, miR21-5p, and miR-375) tested in 26 healthy individuals and 25 pancreatic cancer patients showed 22

Journal Pre-proof significant upregulation of these miRNAs in the blood plasma of pancreatic cancer patients compared to healthy individuals where miR-21-5p exhibited the highest potential to be a noninvasive biomarker [114]. Increased expression of plasma circulating miR-375 and miR-21-5p was significantly associated with the overall survival of pancreatic cancer patients [114]. Circulating miRNAs that are found to be associated with pancreatic cancer are listed in Table 7.

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Challenges of using extracellular miRNAs as cancer biomarkers

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Although circulating miRNA based noninvasive biomarker discovery in cancer detection holds significant and exciting promises, widespread concerns emerged as the results of circulating

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miRNA have shown considerable inconsistency between different studies. Majority of the

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reported ECmiRNAs related to cancer biomarkers have been failed to enter from the

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experimental phase to clinical trials due to the lack of reproducible assay, variations among the results, use of the different statistical method, variability within a healthy population, and finally

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the inadequate performance of detected ECmiRNAs. The major reasons behind these variations

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in results perhaps are the sample acquiring and processing methods, starting materials, detection

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platform and last but not least data analysis. Therefore, in order to have consistency among the cancer biomarker studies, a standardize procedure/strategy should be followed from sample collection-data analysis to clinical trials (for more details please see [3]). In order to develop a circulating miRNA based noninvasive biomarker for early detection of cancer may confront many challenges. Firstly, the lack of each cancer-specific miRNA. It means that one miRNA may highly be expressed in the sera of different cancer types. The golden example is circulating miR-21 which is found to be associated with many cancer types such as lung cancer, gastric cancer, breast cancer, pancreatic cancer, etc. (see above subsections for more details). Secondly, the lack of standardization of circulating miRNAs. It includes standardization 23

Journal Pre-proof of the starting materials whether plasma or serum should be used, preparation and handling of plasma/serum samples, whole serum/plasma or exosomal fraction, etc. In recent years, several studies reported that the exosomal miRNAs are more accurate compared to whole serum or plasma miRNAs. However, inconsistency remains a problem here too. Lai and colleagues reported that the sensitivity and specificity of plasma-derived miRNAs are higher than that of exosomal miRNAs [112]. In all cases, complete removal of the cellular fraction is mandatory as

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it may have a significant contribution to the miRNA expression. Because of variation in sample,

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collection and handling techniques, significant differences were observed in similar studies.

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Therefore, in order to establish circulating miRNA-based biomarkers for the early detection of

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cancer, a global standard procedure must be followed by all the researchers in every corner of the world. Thirdly, and perhaps most importantly, the quantification of miRNAs. It includes

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extraction methods: phenol/chloroform-based or column or bead-based, detection platform: next-

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generation sequencing, microarray or qPCR, data analysis: in case of q-PCR which one will be the housekeeping gene to normalize the data, etc. For detail information regarding these

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Conclusion

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challenges and their possible solutions please see [3,17].

The discovery of miRNAs in general and particularly of circulating miRNAs is one of the major scientific breakthroughs in the modern era and has revolutionized our understanding of current cell biology and medical science. miRNA-based therapeutic strategies hold great promises given miRNAs are able to significantly alter the biological processes as well as cellular behavior. Currently, we have enough evidence to understand the biology of circulating miRNAs and confidently conclude that circulating miRNAs are a potential regulator of developmental processes and could be a promising biomarker for several diseases including cancer. However, 24

Journal Pre-proof inconsistent results across different studies due to variation in the experimental setup and technical differences create much controversy about whether ECmiRNAs is a reliable biomarker for cancer detection. Despite it holds a great promise, this particular field of circulating miRNA or ECmiRNA is still in infancy and has to travel a long way to achieve its goal. It requires a significant technological advancement along with the standardization of the protocol which may lead us to develop a noninvasive biomarker for the early detection of cancers in the near future.

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Within a few years, we will know whether circulating miRNAs alone or in combination with

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other modalities will have significant impacts on disease detection or treatment of various

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diseases including cancer. However, this concept should be rapidly translating from bench to

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bedside and should validate this novel approach through carefully planned studies. The era of circulating miRNA-based biomarkers for rapid detection of disease is forthcoming if we can

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maintain the momentum of current translational research.

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Conflict of interest

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No conflict of interest exists. Acknowledgement None Funding

This work did not receive any grants from government or privet organizations

25

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Journal Pre-proof Figure 1 Nuclear and cytoplasmic processing of miRNAs and mechanisms of gene expression regulation. Canonical miRNA biogenesis divided into nuclear processing and cytoplasmic processing. At the beginning of nuclear processing, pri-miRNA transcripts are generated from miRNA genes with the help of RNA polymerase II. Drosha and DGCR8 microprocessor complex cleaved the primiRNAs to produce precursor miRNAs (pre-miRNAs). Exportin5 transports these pre-miRNAs

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from the nucleus to the cytoplasm. In the cytoplasm, pre-miRNAs are further processed by Dicer

ro

to produce miRNA duplex (miRNA/miRNA*). Finally, either miRNA or miRNA* can be loaded

-p

into the AGO family to produce the miRISC complex. miRISC complex binds with the target

re

mRNA to induce translational repression or mRNA destabilization. DiGeorge syndrome critical

lP

region gene 8 (DGCR8), transactivation response element RNA-binding protein (TRBP),

Figure 2

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Argonaut proteins 1 – 4 (AGO 1- 4), miRNA-induced silencing complex (miRISC).

ur

Release mechanisms of circulating miRNAs. 1) Through exosomes: at the beginning, membrane

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invaginates to form endosomes. miRNAs are sorted and loaded into the exosome precursors (intraluminal vesicles, ILVs) at the late endosome phase. Mature multivesicular bodies (MVBs) either fuse with the lysosome to degrade their contents or fuse with the plasma membrane and release the ILVs as exosomes. 2) Through AGO proteins: AGO2 is a miRNA processing protein. Mature miRNAs are interacting with AGO2 to form the miRISC complex. AGO2 bound miRNAs can be released into the extracellular space or can be loaded into microvesicles. 3) Through microvesicles: microvesicles are spontaneously generated from healthy cells. During the biogenesis of microvesicles, mature miRNAs or AGO2-bound miRNAs can be loaded along with other molecules. 4) Through HDL: mature miRNAs can be attached to HDL through 46

Journal Pre-proof divalent cation bridging to form the miRNA-HDL complex. This complex can be released from

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na

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-p

ro

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cells through exocytosis.

47

Journal Pre-proof

Table 1: Overview of size and characteristics of extracellular vesicles carrying miRNAs Vesicle

Size (nm)

Properties

Reference

Apoptotic bodies

800 - 5000 - Largest of all vesicles

[44,51]

- Forms through plasma membrane blebbing during programmed cells death

Microvesicles

100 - 1000 - Larger than exosomes

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- Unlike exosomes and microvesicles, it may contain intact organelles, chromatin, and glycosylated proteins. [43,44,47]

ro

- Forms through outword budding and fisson of cell membran

30 - 100

- Widely studied vesicles

re

Exosomes

-p

- Contains bioactive molecules like proteins, mRNA and miRNA [15,42]

lP

- Forms through an endocytic recycling pathway (inward budding)

na

- Contains bioactive molecules including miRNAs - May mediate cell-cell communication - Smallest in size, not a vesicle

ur

8 12

- Endogenous nanoparticles

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High density lipoprotein

- Participate in the cholesterol, phospholipids, and triglycerides metabolization and transportation - May contain miRNAs

48

[37,52]

Journal Pre-proof Table 2: Differential expression regulation of circulating miRNAs in lung cancer with biomarker potential Name of the miRNA

Expression Area Sensitivity Biomarker Reference regulation under the & specificity potential curve

Serum

↑

0.954

Plasma

↑

0.855

miR-486-5p

Plasma

↓

0.855

miR-31

Serum

↑

0.785

Plasma

↑

Serum

↑

0.898 0.914 0.842

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miR-106a-5p, miR-20a-5p & miR-93-5p miRs-21 & miR-210

Sample type

Serum

↑

0.96

Serum

↓

Serum

↑

miR-339-5p

Plasma

↓

miR-21

Plasma

Jo

miR-21-5p, miR-223-3p, Serum & miR-9-5p miR-16, miR205 & miRPlasma 486 miR-28-5p, miR-362-5p, Plasma & miR-660-5p miR-422a miR-19b-3p, miR-21-5p, miR-221-3p, miR-409-3p, miR-425-5p & miR-584-5p

Plasma

0.900 0.880

Yes

[53]

Yes

[53] [54]

86% & 79%

Yes

[59]

95.31% & 82.98%

Yes

[60]

Yes

[61]

Yes

[62]

Yes

[63]

Yes

[63]

-p

0.856

[58]

Yes

re

lP 0.893

Yes

of

75.0% & 84.8% 75.0% & 84.8% 76.9% &74.5%

na ↑

ur

miR-944 & miR-3662 miR-210-3p miR-146b, miR-205, miR-29c, & miR-30b miR-145 & miR-223 miR-141

-

85.7% & 80.0% 82.1% & 92.9% 82.1% & 96.4%

↑

0.886

82.69% & 88.00%

Yes

[64]

↑

0.898

80% & 95%

Yes

[65]

↓

0.876

63.41% & 96.88%

Yes

[66]

↑

0.880

86.22% & 96.55%

Yes

[67]

↑

0.73

68% & 70%

Yes

[68]

49

Journal Pre-proof

Jo

ur

na

lP

re

-p

ro

of

↑= Upregulated; ↓= downregulated.

50

Journal Pre-proof Table 3: Differential expression regulation of circulating miRNAs in gastric cancer with biomarker potential

↑

0.70 – 0.74

miR-203

Serum

↓

0.707

miR-196a

Plasma

↑

0.864

Plasma

↑

0.887

Serum

↓

0.891

miR-21

Plasma

↑

0.893

miRNA-22-3p

Plasma

↓

miR-101

Plasma

↓

Jo

miR-217

Not calculated 69.5% & 97.6% 84.8% & 79.2%

lP

0.8494 0.740

na ↑

Plasma

Yes

[70]

-

[72]

Yes

[73]

63.33% & 87.78% 86.7% & 72.2% 91.70% & 65.40% 56.3% & 82.5%

Yes

[74]

Yes

[75]

Yes

[76]

Yes

[77]

Yes

[78]

0.826

95% & 90%

Yes

[79]

↑

0.95

85.7% & 95.8%

Yes

[80]

↓

0.893

81.3% & 83.2%

Yes

[81]

ur

miR-19b-3p & Serum miR-106a-5p exosome miR-106a, miR-18a, miR-20b, Plasma miR-486-5p, & miR-584

82.4% & 58.8%

of

Serum

ro

miR-221, miR-744, & miR-376c

miR-106a, miR-106b, miR-21, & miR-93 miR-143-3p, miR-146a, miR-451a, & miR-501-3p

Expression Area Sensitivity Biomarker Reference regulation under the & specificity potential curve

-p

Sample type

re

Name of the miRNA

↑= Upregulated; ↓= downregulated.

51

Journal Pre-proof Table 4: Differential expression regulation of circulating miRNAs in breast cancer with biomarker potential

Serum

↑

Plasma exosome

↑

Serum

↓

Plasma

↓

Serum exosome

↑

Serum

-

Sensitivity & specificity

Biomarker Reference potential

-

???

[82]

97.9% & 73.5%

Yes

[83]

ro

of

↑

-

Yes

[87]

97.9% & 73.5%

Yes

[83]

-

Yes

[84]

Not studied

Yes

[88]

92.9% & 77.4%

Yes

[85]

-p

0.843

re

miR-34a & miR-34c miR-188-3p, miR-500a-5p & miR-501-5p miR-376c-3p, miR-382-5p, & miR-411-5p miR-451 miR-24-2-5p & miR-375

Plasma & serum

lP

miR-99a 5p

Expression Area regulation under the curve

0.811

na

miR-188-5p, miR-1202, miR-1225-5p, & miR-4270 miR-21-3p & miR-21-5p miR‐ 122‐ 5p, miR‐ 146b‐ 5p, miR‐ 210‐ 3p, miR‐ 215‐ 5p & let‐ 7b‐ 5p

Sample type

↓

ur

Name of the miRNA

↓

-

-

No

[86]

↑

0.808

-

Yes

[89]

↓

0.808

-

Yes

[89]

↓

0.79

69% & 76%

Yes

[90]

↑

0.79

69% & 76%

Yes

[90]

miR-17–5p

↑

0.848

100% & 75.4%

Yes

[91]

Jo

Serum Plasma exosome Plasma miR‐ 548b‐ 5p exosome miR-296-3p, miR-575, miR3160-5p miRSerum 4483, miR4710, & miR4755-3p miR-5698 & Serum miR-8089 Serum

↑= Upregulated; ↓= downregulated.

52

Jo

ur

na

lP

re

-p

ro

of

Journal Pre-proof

53

Journal Pre-proof Table 5: Differential regulation and the potential of circulating miRNAs in colorectal cancer Sample type

miR-141

Plasma

miR-21

Plasma

Expression Area regulation under the curve ↑ 0.861

Sensitivity Biomarker Reference & specificity potential

0.93

miR-139-3p & Plasma miR-145-3p & exosome miR-150-3p & Plasma let-7b-3p & exosome miR-144-3p, Plasma miR-425-5p, & miR-1260b miR-17-5p, , Plasma miR-18a-5p, exosome miR-181a-5p & miR-18b-5p miR-497 Serum

↓

0.927

↑

0.927

-

↓

0.954

↑

0.895

miR-1290 & miR-320d miR‐ 338‐ 5p

↓

[92]

Yes

[93]

Yes

[95]

[95]

93.8% & 91.3%

Yes

[96]

0.769 & 0.867

Yes

[97]

80.9% & 81.4% 90.9% & 93.3% 85% & 88.8%

Yes

[98]

Yes

[99]

Yes

[100]

-p

Yes

re lP 0.883 0.98

↑

0.923

ur

Serum

↓

na

Plasma

Yes

of

↓

77.1% & 89.7% 90% and 81% -

ro

Name of the miRNA

Jo

↑= Upregulated; ↓= downregulated.

54

Journal Pre-proof Table 6: Differential regulation and the potential of circulating miRNAs in hepatocellular cancer

Plasma

↑

0.908

miR-122-5p, miR-486-5p, & miR-142-3p miR‐ 122, miR‐ 148a, and miR‐ 1246 miR-122a miR-548-a-3p

Serum

↑

0.94

Serum exosome

↑

0.947

Serum Serum

↓ ↓

0.897 0.883

miR-1246

Serum

↑

0.762

Yes

[101]

-

Yes -

[103] [104]

93.1% & 80.0% 80% & 95%

Yes

[105]

Yes

[109]

87.0% & 90.0%

lP

Jo

ur

na

↑= Upregulated; ↓= downregulated.

79% & 72%

of

Serum

Sensitivity Biomarker Reference & specificity potential

ro

miR-141 & miR-200a miR-122 miR-26a & miR-29a miR-224

Plasma Plasma

Expression Area regulation under the curve ↓ 0.82 0.85 ↑ 0.787 ↓ -

Yes

[107]

92% & 69.2% 54.1% & 77.4%

Yes Yes

[110] [108]

Yes

[111]

-p

Sample type

re

Name of the miRNA

55

Journal Pre-proof Table 7: Differential regulation and the potential of circulating miRNAs in pancreatic cancer Expression Area regulation under the curve ↑ 0.97 - 1

Sensitivity Biomarker Reference & specificity potential

miR-10b, miR-21, miR30c, & miR181a miR-182

Plasma & exosome

100% & 100%

Yes

[112]

Plasma

↑

0.775

Yes

[113]

miR-21-5p

Plasma

↑

0.99

Yes

[114]

let‐ 7b‐ 5p, miR‐ 19a‐ 3p, miR‐ 19b‐ 3p, miR‐ 25‐ 3p miR‐ 192‐ 5p, & miR‐ 223‐ 3p miR-100 miR-221-3p

Serum

↑

0.910

64.1% & 82.6% 85% & 100% 95.3% & 76.7%

Serum Plasma

↓ ↑

0.81 0.687

miR-21-5p MiR-196 & miR-200

Serum Serum

↑ ↑

of

Sample type

Yes

[115]

Yes Yes

[116] [117]

Yes Yes

[118] [119]

lP

re

-p

ro

Name of the miRNA

na

0.78 0.74 0.79

Jo

ur

↑= Upregulated; ↓= downregulated.

56

76.3% & 63.6% 77% & 80% 94% & 82%

Figure 1

Figure 2