Author details

Serena Bertozzi<sup>1</sup> \*† , Ambrogio P Londero2† , Luca Seriau<sup>1</sup> , Roberta Di Vora1 , Carla Cedolini<sup>1</sup> and Laura Mariuzzi<sup>3</sup>

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\*Address all correspondence to: ambrogio.londero@gmail.com

1 Breast Unit, Clinic of Surgery, DAME, University of Udine, University Hospital of Udine, Udine, Italy

2 Clinic of Obstetrics and Gynecology, University Hospital of Udine, Udine, Italy

3 Institute of Pathologic Anatomy, DAME, University of Udine, University Hospital of Udine, Udine, Italy

† These authors contributed equally.

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Author details

Serena Bertozzi<sup>1</sup>

Udine, Italy

Udine, Italy

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**Chapter 2**

**Provisional chapter**

**Circulating MicroRNA Profiling in Cancer Biomarker**

**Circulating MicroRNA Profiling in Cancer Biomarker** 

MicroRNAs (miRNAs) are a class of small non-coding RNA molecules of approximately 22 nucleotides that regulate gene expression at the post-transcriptional level. Alterations in miRNA expression patterns correlate with a wide spectrum of pathological conditions, including cancer. miRNA profiling was mostly performed, in solid tissues, obtained by invasive diagnostic procedures. However, miRNAs in biofluids, such as serum and plasma, show high stability resulting from the formation of complexes with specific protein or incorporation within circulating exosomes or other microvesicles. Circulating miRNAs could be reliable biomarkers for early-stage cancer diagnosis, prognosis and response to therapy. In this chapter, we analyze the major pre-analytical and analytical challenges in experimental design for circulating miRNA detection, focusing on exosome

MicroRNAs (miRNAs) are small evolutionary conserved non-coding RNAs of 19–25 nucleotides that bind to the 3′-untranslated region (3'-UTR) of target mRNAs, resulting in a negative regulation of gene expression by suppressing translation or causing mRNA degradation [1]. The complex miRNA network plays an important role in the regulation of cellular processes such as development, proliferation, differentiation and apoptosis. Significant changes of tissue miRNA "signatures" occur in various diseases, including cancer [2–4]. More than 50% of human miRNAs are mapped to chromosomal region of genomic instability due to extensive

**Keywords:** miRNAs, exosomes, miRNA profiling, biomarkers discovery

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

DOI: 10.5772/intechopen.75981

**Discovery**

**Abstract**

**1. Introduction**

**Discovery**

Francesca Scionti, Pierosandro Tagliaferri,

Francesca Scionti, Pierosandro Tagliaferri,

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

fraction and microarray-based approach.

http://dx.doi.org/10.5772/intechopen.75981

Pierfrancesco Tassone and Maria Teresa Di Martino

Pierfrancesco Tassone and Maria Teresa Di Martino

#### **Circulating MicroRNA Profiling in Cancer Biomarker Discovery Circulating MicroRNA Profiling in Cancer Biomarker Discovery**

DOI: 10.5772/intechopen.75981

Francesca Scionti, Pierosandro Tagliaferri, Pierfrancesco Tassone and Maria Teresa Di Martino Francesca Scionti, Pierosandro Tagliaferri, Pierfrancesco Tassone and Maria Teresa Di Martino

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.75981

#### **Abstract**

MicroRNAs (miRNAs) are a class of small non-coding RNA molecules of approximately 22 nucleotides that regulate gene expression at the post-transcriptional level. Alterations in miRNA expression patterns correlate with a wide spectrum of pathological conditions, including cancer. miRNA profiling was mostly performed, in solid tissues, obtained by invasive diagnostic procedures. However, miRNAs in biofluids, such as serum and plasma, show high stability resulting from the formation of complexes with specific protein or incorporation within circulating exosomes or other microvesicles. Circulating miRNAs could be reliable biomarkers for early-stage cancer diagnosis, prognosis and response to therapy. In this chapter, we analyze the major pre-analytical and analytical challenges in experimental design for circulating miRNA detection, focusing on exosome fraction and microarray-based approach.

**Keywords:** miRNAs, exosomes, miRNA profiling, biomarkers discovery

#### **1. Introduction**

MicroRNAs (miRNAs) are small evolutionary conserved non-coding RNAs of 19–25 nucleotides that bind to the 3′-untranslated region (3'-UTR) of target mRNAs, resulting in a negative regulation of gene expression by suppressing translation or causing mRNA degradation [1]. The complex miRNA network plays an important role in the regulation of cellular processes such as development, proliferation, differentiation and apoptosis. Significant changes of tissue miRNA "signatures" occur in various diseases, including cancer [2–4]. More than 50% of human miRNAs are mapped to chromosomal region of genomic instability due to extensive

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

repetitive sequences resulting in structural mutations (deletion, duplication and translocation) during tumor development [5]. Regulation of miRNA expression is an important mechanism by which tumor-suppressor proteins and oncogenic proteins exert some of their effects. A decrease or mutation in tumor-suppressor miRNAs can lead to overexpression of oncogenic proteins, in contrast to an overexpression of oncogenic miRNAs which can reduce expression of tumor suppressors. For example, tumor suppressor of the let-7 family targets RAS oncogene, which is involved in cell growth, differentiation and survival; reduced expression of let-7 miRNAs correlates with poor survival in many cancers [6]. In contrast, oncogenic miR-NAs in the cluster miR-17-92, comprising six miRNAs (miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1 and miR-92a-1), inhibits PTEN to activate AKT signaling and promote cancer cell survival [7].

**2. Methodological challenges in miRNA profiling design**

**Figure 1.** Summary of workflow in designing miRNA profiling from blood serum.

Blood sample processing has a substantial impact on the results of miRNA profiling. During blood collection, it is important to avoid cellular contamination and hemolysis that can occur during phlebotomy as miRNAs derived from red and white blood cells risk to mask the intensities of truly circulating miRNA species. Residual platelets and microparticles can also affect the miRNA profile so an additional centrifugation is recommended prior to freezing samples. Moreover, biofluidics contain inhibitors of the reverse transcriptase and polymerase enzymes that can inhibit the enzymatic reactions in RT-qPCR so it is important to minimize the car-

Circulating MicroRNA Profiling in Cancer Biomarker Discovery

http://dx.doi.org/10.5772/intechopen.75981

33

In our protocol, blood samples were collected and processed according to the national cancer institute (NCI's) Early Detection Research Network (EDRN) standard operating procedures

Whole blood samples were collected in red-top vacutainer tubes. Blood samples were incubated at room temperature for 30 min to allow complete coagulation. Coagulated samples were then centrifuged at 1500 × *g* for 20 min at room temperature to separate serum. The serum was transferred to new cryotubes with care so as to not to disturb the red blood layer and then centrifuged for 5 min at 3000 × *g* to remove cells. Aliquots of 1.5 ml of supernatant containing the cell-free serum were stored in cryotubes at −80°C until RNA extraction.

The established standard for exosome isolation is ultracentrifugation [16]. However, this method cannot discriminate between exosomes and other microvesicles because different vesi-

Recently, methods claiming fast and simple exosome-purification procedures without ultracentrifugation are commercially available by various firms that use polymer-based precipita-

However, according to the International Society for Extracellular Vesicles (ISEV) the separation of non-vesicular entities, such as protein complexes, from EV is not fully achievable by

cles of similar size as well as protein aggregates can co-sediment at 100,000 × *g*.

**2.1. Sample collection**

ryover of inhibitors into the RNA.

for the collection and preparation of serum [15].

**2.2. Exosome isolation and characterization**

tion or immune capture by antibody-coated beads.

Hemolyzed samples were excluded from further analysis.

miRNAs have been considered promising candidates as diagnostic and prognostic biomarkers for the strong correlation between expression patterns of miRNAs and disease status and for the differences between normal and cancer tissues. This reflects the current evidence that some miRNAs are overexpressed or downregulated exclusively or preferentially in certain cancer types.

Although miRNA profiling of tumors has been reported in solid tissues, obtained by invasive procedures, as an excellent prognostic test [8], routine biopsies from any organ for miRNA profiling are not practical options. Different studies suggest that circulating miRNAs are reliable indicators of pathological change because of their stability and protection against RNase digestion resulting from the formation of complexes with specific protein or incorporation within circulating exosomes or other extracellular vesicles (EV) [9]. In this context, accumulating evidence suggests that tumor cells are able to alter the function of both local and distant normal cells, thereby promoting tumor growth and metastasis, through the transfer of EV cargo [10]. miR-21, which targets the tumor-suppressor gene PTEN and programmed cell death 4 (PDCD4), is one of the first discovered and most investigated circulating miRNAs. Its upregulation seems to be of diagnostic and prognostic value in a variety of solid and hematological malignancies. High serum levels of miR-21 were strongly associated with lymph node metastasis, advanced-stage clinical disease and poor survival [11]. In patients with ovarian cancer, high serum levels of miR-34a were associated with lymph node disease and distant metastases [12].

Also, in patients with prostate cancer, plasma levels of miR-21, miR-141 and miR-221 were significantly higher in patients with metastases as compared to patients with localized or locally advanced-stage disease [13].

Currently, a variety of miRNA detection methods, including northern blotting, in situ hybridization, quantitative reverse transcription PCR (qRT-PCR), microarray and deep sequencing, are commonly used [14]. However, miRNA profiling in biofluid samples is affected by a range of pre-analytical and analytical challenges in experimental design, from sample collection to profiling and data analysis (**Figure 1**). In this chapter, we will propose a workflow for exosomal miRNA detection from sera samples using the Affymetrix GeneChip microarray platform as a powerful molecular approach for biomarker discovery to translate into clinical practice.

Circulating MicroRNA Profiling in Cancer Biomarker Discovery http://dx.doi.org/10.5772/intechopen.75981 33

**Figure 1.** Summary of workflow in designing miRNA profiling from blood serum.
