Circulating MicroRNAs as Cancer Biomarkers: Can They Play a Role in Clinical Practice? Short Review

microRNAs (miRNAs) are a large family of short noncoding RNA sequences which modulate gene expression and regulate a wide range of biological processes. There is evidence that miRNAs may have a role in molecular mechanisms linked to tumorigenesis and a lot of studies have proven that some miRNAs are closely correlated with cancer. miRNAs are not only contained in tissue cells but they are also detectable in extracellular sites, as plasma, urine, cerebrospinal and other body fluids where they are remarkably stable, so that may be identified and measured. Since tumors alter the normal concentrations of circulating miRNAs, these oligo­nucleotides can be used as cancer biomarkers. Circulating miRNAs detection may be used for early diagnosis, staging, follow up, assessment of therapeutic responses and therapy outcomes in several types of human cancer as colorectal cancer, pancreatic adenocarcinoma, lung cancer and malignant pleural mesothelioma, urinary and prostate cancer, breast cancer, hematologic malignancies, glioblastoma and others. Quantitative real-time Polimerase Chain Reaction (qRT-PCR) is one of the most sensitive techniques for quantifying circulating miRNAs. Deep sequencing technology has recently emerged as an attractive approach for miRNA analysis; in some cases this technique showed more specificity and sensitivity compared to qRT-PCR and Microarray, also allowing identification of novel MiRNA isoforms. Given that current serological cancer biomarkers, commonly employed in follow up, have low specificity and sensi­tivity, it is plausible that circulating miRNA detection can be included in future routine clinical examinations for management of cancer patients, although costs and wide availability of quantifying techniques could represent a critic limit. Further comparative studies will be required.

Introduction

microRNAs (miRNAs) are a large family of short noncoding RNA sequences [1] which modulate gene expression and regulate a wide range of biological cell processes [2].

There is evidence that miRNAs may have a role in molecular mechanisms linked to cellular pathways of certain diseases, as viral infections, diabetes and cardiovascular disease; moreover, miRNA have been shown to regulate several processes involved in tumorigenesis [2-5].

miRNAs are not only contained in tissue cells but they are also detectable in extracellular sites, as plasma, urine, cerebrospinal and other body fluids; miRNAs are carried in body fluids within small membrane vesicles (exosomes), in the form of high-density lipoprotein complexes, or complexed to carrier proteins (argonaute-2 proteins). Extracellular microRNAs in exosomes may be transferred to other cells, altering gene expression and changing the functional effects of receivers [6-8].

The presence of endogenous miRNAs in microparticles makes circulating miRNAs remarkably stable in the bloodstream even under conditions as harsh as boiling, low or high pH, long-time storage at room temperature, and multiple freeze-thaw cycles, so that they may be identified and measured in the circulation and can be used as potential disease biomarkers [5].

A lot of studies have proven that expression of some miRNAs is closely correlated with cancer development and altered levels of miRNA have been related to the maintenance of cancer stem cells, neoangiogenesis, metastasis and epithelial-mesenchymal transition, which contribute to the malignancy [9-11]. It has also been shown that microRNAs may be responsible for other biological processes, such as cancer-associated inflammation and tumor drug resistance [12].

Aim of the Research

The purpose of the present review was to examine the latest evidences on circulating miRNAs as cancer biomarkers and evaluate whether the detection of these nucleotides can play a role in daily clinical practice. 

Material and Methods

A review of recent literature has been carried out via Pub Med database, using these search term: microRNA, cancer biomarkers, clinical practice. Search was not limited by language or human subjects. All the found items, published in the last five years were analysed. Additional articles were selected from the bibliographies of the quoted references.

Results

91 items were obtained: 55 reviews (9 systematic reviews), 4 meta-analysis, 2 clinical studies, 1 randomized phase III clinical trial, 1 editorial, 1 comment; 35 items were research support and 14 were laboratory studies; the remaining items were prevalently other journal articles or other publication types. Comparative studies were not found, neither laboratory trials nor guidelines nor consensus. Article types was determined using filters available on Pub Med database. Other data were deduced from retrospective analysis and by careful assessment of the obtained items and their references.

The analysis of obtained data showed that a lot of miRNA is associated with cancer and many types of miRNA have been identified in several types of human cancer, as summarized in Table 1.

There is wide evidence that tumors alter the normal concentrations of miRNAs in biological fluid, thus these oligo­nucleotides may serve as cancer biomarkers. These findings will be discussed in this brief review with related references.

In cancer management, circulating miRNAs detection may be used for early diagnosis, cancer staging, prognosis and patient follow up to individuate early relapses; moreover, there are promising data to extend this assay for predicting specific therapeutic responses and also to assess therapy outcomes (Table 1).

Discussion

microRNAs (miRNAs) are a large family of short noncoding RNA sequences, approximately 20-22 nucleotides, synthesized in the nucleus, through a complex multi-step biosynthetic process, starting from RNA polymerase II; it is estimated that the human genome contains more than 2500 mature miRNAs [1,2].

These nucleotides modulate gene expression, by binding the 3′-untranslated region of target messenger RNA (mRNA), both degrading mRNA and inhibiting translation into protein. miRNAs regulate a wide range of biological processes as cell differentiation, proliferation and development, cell-to-cell communication, cell metabolism and apoptosis [2,3].

In 2002, for the first time, the link between miRNA and cancer was reported in patients with B-cell chronic lymphocytic leukemia; in these patients was found a down-regulation of miR-15a and miR-16-1 [13]. Subsequently, a lot of studies have proven that expression of some miRNAs is closely correlated with cancer development and some miRNAs can function as oncogenes or tumor suppressors. Indeed, there is evidence that some of these oligonucleotides act as oncogenes (oncomiRs) and their overexpression leads to cancer growth; conversely, other miRNAs are tumor suppressors (anti-oncomiRs) in normal cells, so that their underexpression correlates with tumor progression [9-11].

Since miRNAs are also detectable in extracellular sites, as plasma and other body fluids where they may be measured and since tumors alter the normal concentrations of circulating miRNAs, these oligo­nucleotides can be used as biomarkers for cancer detection [14].

The study of miRNA has thus become a rapidly emerging field in oncology and the detection of miRNA expression is a very important first step in miRNA exploration. 

Several techniques are available for quantifying circulating miRNAs, such as quantitative real-time Polimerase Chain Reaction (qRT-PCR) [15], Northern blotting [16], bead-based flow Cytometry [17], Microarray [18] or Deep sequencing [19]. However, of these assay, qRT-PCR seems superior because of its high sensitivity, specificity and reproducibility; moreover qRT-PCR requires less amount of RNA sample, usually more than 1 μg, but the number of miRNAs possible to analyze and RNA quantity may represent limitations for this assay [15]. Conversely, Deep sequencing technology has recently emerged as an attractive approach for miRNA analysis; in some cases this technique showed most specificity and sensitivity compared to qRT-PCR and Microarray, also allowing identification of novel miRNA isoforms [20]. Actually, it is well known that a single gene may, in turn, be regulated by multiple miRNAs, therefore, given the large number of miRNAs annotated in the human genome, 30%–80% of human genes are predicted to be influenced by miRNAs. Moreover, a single miRNA influences the expression of hundreds of unique miRNAs and aberrant miRNA expression may affect a multitude of transcripts and profoundly influence cancer-related signaling pathways. This situation generates a complex net­work, and the analysis of miRNA panels is con­sequently more efficient in cancer studies than the analysis of a single miRNA [21].

Thanks to these analysis methods and techniques that allow measurement of multiple miRNA types, these circulating nucleotides may be used for early diagnosis, staging, follow up, assessment of therapeutic responses and therapy outcomes in several types of human cancer as colorectal cancer, pancreatic adenocarcinoma, bladder cancer, lung cancer and malignant pleural mesothelioma, urinary and prostate cancer, breast cancer, hematologic malignancies, glioblastoma and others [22].

Currently, conventional cancer biomarkers commonly utilized in clinical practice as, carbohydrate antigens, oncofetal antigens, hormones, enzymes, tissue polypeptide antigen and others, are usually employed in follow up of cancer patients; however they have low specificity and sensi­tivity so that monitoring disease is, perhaps, the most common clinical use of these tumor markers [23-26]. Thus miRNA detection could be a new effective tool in the clinical management of cancer.

The current methods used for miRNAs detection usually requires high costs and this aspect could limit their use in daily clinical practice. However, in this way, research should try to overcome this limiting aspect, although spending review in health care and cost-containment measures could represent a critical ethical problem in a delicate field as oncolgy [27].

In our research we have not found comparative studies, in terms of specificity, sensibility and costs, between miRNAs and other conventional cancer biomarkers commonly used in clinical practice. It is desirable that, in future, comparative studies will be undertaken.

Conclusion: circulating miRNAs detection may be used for early diagnosis, staging, follow up, assessment of therapeutic responses and therapy outcomes in several types of human cancer. Given that current serological cancer biomarkers, commonly employed in follow up, have low specificity and sensi­tivity, it is plausible that circulating miRNA detection can be included in future routine clinical examinations for management of cancer patients, although costs and wide availability of quantifying techniques could represent a critic limit. Research should try to overcome this limiting aspect by developing less expensive detection techniques and by initiating comparative studies. Particularly, new low-cost and non-invasive cancer biomarkers need to be developed to improve screening protocols and therapy and to provide information on chemoresistance and the risk of relapses.

1. Ha M, Kim VN (2014) Regulation of microRNA biogenesis. Nat Rev Mol Cell Biol 15: 509-524. Link: https://goo.gl/jMPKs9
2. Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. cell 116: 281–297. Link: https://goo.gl/TAeBG
3. Bushati N, Cohen SM (2007) microRNA functions. Annu Rev Cell Dev Biol 23: 175–205. Link: https://goo.gl/z68E2S
4. Yang W, Lee DY, Ben-David Y (2011) The roles of microRNAs in tumorigenesis and angiogenesis. Int J Physiol Pathophysiol Pharmacol 3: 140–155. Link: https://goo.gl/rVbhCj
5. Creemers EE , Tijsen AJ , Pinto YM (2012) Circulating MicroRNAs: Novel Biomarkers and Extracellular Communicators in Cardiovascular Disease? Circ Res  110: 483-495. Link: https://goo.gl/kOtS9H
6. Valadi H, Ekström K , Bossios A , Sjöstrand M , Lee JJ , et al. (2007) Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol 9: 654–659. Link: https://goo.gl/Ze7JUl
7. Arroyo JD, Chevillet JR , Kroh EM , Ruf IK , Pritchard CC , et al. (2011) Argonaute2 complexes carry a population of circulating microRNAs independent of vesicles in human plasma. Proc Natl Acad Sci U S A 108: 5003–5008. Link: https://goo.gl/7xL8k3
8. Vickers KC, Palmisano BT , Shoucri BM , Shamburek RD , Remaley AT (2011) MicroRNAs are transported in plasma and delivered to recipient cells by high-density lipoproteins. Nat Cell Biol 13: 423–433. Link: https://goo.gl/UcsxDx
9. Ye J , Wu X , Wu D , Wu P , Ni C , et al. (2013) miRNA-27b targets vascular endothelial growth factor C to inhibit tumor progression and angiogenesis in colorectal cancer. PLoS ONE 8: e60687. Link: https://goo.gl/uT7022
10. Hur K , Toiyama Y , Takahashi M , Balaguer F , Nagasaka T , et al. (2013) MicroRNA-200c modulates epithelial-to-mesenchymal transition (EMT) in human colorectal cancer metastasis. Gut 62: 1315–1326. Link: https://goo.gl/kX9LlC
11. Yu Y , Kanwar SS , Patel BB , Oh PS , Nautiyal J , et al. (2012) MicroRNA-21 induces stemness by downregulating transforming growth factor beta receptor 2 (TGF_R2) in colon cancer cells. Carcinogenesis 33: 68–76. Link: https://goo.gl/0LyzgB
12. Liu J , Zheng M , Tang YL , Liang XH , Yang Q (2011) MicroRNAs, an active and versatile group in cancers. Int J Oral Sci 3: 165-175. Link: https://goo.gl/GoKQ38
13. Calin GA, Dumitru CD, Shimizu M, Bichi R, Zupo S, et al. (2002) Frequent deletions and down-regulation of micro- RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia . Proc Natl Acad Sci U S A 99: 15524-15529. Link: https://goo.gl/xHB96X
14. Mitchell PS, Parkin RK, Kroh EM, Fritz BR, Wyman SK, et al. (2008) Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci U S A 105: 10513–10518. Link: https://goo.gl/C1yREQ
15. Chen C, Ridzon DA , Broomer AJ , Zhou Z , Lee DH , et al. (2005) Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res 33: e179–e179. Link: https://goo.gl/0BaFiM
16. Valoczi A, Hornyik C, Varga N, Burgyan J, Kauppinen S, et al. (2004) Sensitive and specific detection of microRNAs by northern blot analysis using LNA-modified oligonucleotide probes. Nucleic Acids Res 32: 175. Link: https://goo.gl/y9qjbK
17. Lu J, Getz G, Miska EA, Alvarez-Saavedra E, Lamb J, et al. (2005) MicroRNA expression profiles classify human cancers. Nature 435: 834–838. Link: https://goo.gl/XZao9S
18. Zhao H, Shen J, Medico L, Wang D, Ambrosone CB, et al. (2010) A pilot study of circulating miRNAs as potential biomarkers of early stage breast cancer. PLoS One 5: 13735–13747. Link: https://goo.gl/H6KiSm
19. Wu Q, Lu Z, Li H, Lu J, Guo L, et al. (2011) Next-generation sequencing of microRNAs for breast cancer detection. J Biomed Biotechnol 2011: 597145–597152. Link: https://goo.gl/pmCMvg
20. Megiorni F, Cialfi S, McDowell HP, Felsani A, Camero S, et al. (2014) Deep Sequencing the microRNA profile in rhabdomyosarcoma reveals down-regulation of miR-378 family members. BMC Cancer 14: 880. Link: https://goo.gl/LvBdTX
21. Lu J, Clark AG (2012) Impact of microRNA regulation on variation in human gene expression. Genome Res 22: 1243–1254. Link: https://goo.gl/s4rX6P
22. Izzotti A, Carozzo S, PullieroA, Zhabayeva D, Ravetti JL, et al. (2016) Extracellular MicroRNA in liquid biopsy: applicability in cancer diagnosis and prevention. Am J Cancer Res 6: 1461-1493. Link: https://goo.gl/dutple
23. Carpelan-Holmström M, Louhimo J, Stenman UH, Alfthan H, Haglund C (2002) CEA, CA 19-9 and CA 72-4 improve the diagnostic accuracy in gas­trointestinal cancers. Anticancer Res 22: 2311-2316. Link: https://goo.gl/EUqA70
24. Buys SS , Partridge E , Black A , Johnson CC , Lamerato L , et al. (2011) Effect of screening on ovarian cancer mortality: the Prostate, Lung, Colorectal and Ovarian (PLCO) Cancer Screening Randomized Controlled Trial. JAMA  305: 2295-2303. Link: https://goo.gl/w6PjSF
25. Schröder FH, Hugosson J, Roobol MJ, Tammela TL, Zappa M, et al. (2014) Screening and prostate cancer mortality: results of the European Ran­domised Study of Screening for Prostate Can­cer (ERSPC) at 13 years of follow-up. Lancet 384: 2027-2035. Link: https://goo.gl/SGQxf5
26. Sturgeon CM, Duffy MJ, Stenman UH, Lilja H, Brunner N, et al. (2008) National academy of clinical biochemistry laboratory medicine practice guidelines for use of tumor markers in testicular, prostate, colorectal, breast, and ovarian cancers. Clin Chem 54: e11–e79. Link: https://goo.gl/KpRGAq
27. Li W , Zhao B , Jin Y , Ruan K (2010) Development of a low-cost detection method for miRNA microarray. Acta Biochim Biophys Sin (Shanghai) 42: 296-301. Link: https://goo.gl/8ONQuv