Presence of Circulating miR-145, miR-155, and miR-382 in Exosomes Isolated from Serum of Breast Cancer Patients and Healthy Donors.

Vianey Gonzalez-Villasana,Diana Resendez-Perez,Cristina Rodriguez-Padilla,Jose Manuel Vazquez-Guillen,Yessica Gonzalez-Cantú,Recep Bayraktar,Jorge Luis Menchaca-Arredondo,Mohammed H Rashed,Gabriel Lopez-Berestein

doi:10.1155/2019/6852917

Abstract

miR-145, miR-155, and miR-382 have been proposed as noninvasive biomarkers to distinguish breast cancer patients from healthy individuals. However, it is unknown if these three miRNAs are secreted by exosomes. Thus, we hypothesized that miR-145, miR-155, and miR-382 in breast cancer patients are present in exosomes. We isolated exosomes from serum of breast cancer patients and healthy donors, then we characterized them according to their shape, size, and exosome markers by scanning electron microscopy, atomic force microscopy, nanoparticle tracking analysis (NTA), and Western blot and determined the exosome concentration in all samples by NTA. Later, exosomal small RNA extraction was done to determine the expression levels of miR-145, miR-155, and miR-382 by qRT-PCR. We observed a round shape of exosomes with a mean size of 119.84 nm in breast cancer patients and 115.4 nm in healthy donors. All exosomes present the proteins CD63, Alix, Tsg, CD9, and CD81 commonly used as markers. Moreover, we found a significantly high concentration of exosomes in breast cancer patients with stages I, III, and IV compared to healthy donors. We detected miR-145, miR-155, and miR-382 in the exosomes isolated from serum of breast cancer patients and healthy donors. Our results show that the exosomes isolated from the serum of breast cancer patients and healthy donors contains miR-145, miR-155, and miR-382 but not in a selective manner in breast cancer patients. Moreover, our data support the association between exosome concentration and the presence of breast cancer, opening the possibility to study how miRNAs packaged into exosomes play a role in BC progression.

Highlights

Breast cancer (BC) is the most common cancer among women and the second cause of cancer deaths in women after lung cancer [1]
To determine whether miR-145, miR-155, and miR-382 are secreted by exosomes in breast cancer patients, we first isolated exosomes from serum of BC patients and healthy donors using ExoQuick reagent
We performed the characterization of exosomes through their shape and size using Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM) which showed a round shape of exosomal vesicles (Figures 1(a)–1(b)) and a mean size of 134.34 nm in exosomes isolated from BC patients (Figure 1(c))

Summary

Introduction

Breast cancer (BC) is the most common cancer among women and the second cause of cancer deaths in women after lung cancer [1]. The overall five-year relative survival rate for women diagnosed with localized BC is 99%, whereas that for patients with distant-stage disease is 27% [2]. For these reasons, many efforts have been made to identify Disease Markers. MiRNAs are present and highly stable in biofluids like urine, plasma, and serum [7,8,9]. These characteristics have allowed miRNAs to emerge as biomarkers in many types of cancer including BC [10, 11]. The miRNAs are carried out and protected from endogenous RNase activity in blood by their association with exosomes, argonaute-2 (Ago-2), and high-density lipoproteins [8, 12]

Methods

Results

Conclusion