Exosome-encapsulated microRNA-223-3p as a minimally invasive biomarker for the early detection of invasive breast cancer

Abstract

Patients diagnosed preoperatively with ductal carcinoma in situ (DCIS) breast cancer have the potential to develop invasive ductal carcinoma (IDC). The present study investigated the usefulness of exosome-encapsulated microRNA-223-3p (miR-223-3p) as a biomarker for detecting IDC in patients initially diagnosed with DCIS by biopsy. The potential association between miR-223-3p and clinicopathological characteristics was examined in patients with breast cancer. Exosomes of 185 patients with breast cancer were separated from plasma by ultracentrifugation. Initially a microRNA (miRNA) microarray was examined to reveal the invasion specific miRNAs using exosomes collected from 6 patients with breast cancer, including 3 DCIS patients, 3 IDC patients and 3 healthy controls. In the miR microarray analysis the miR-223-3p levels of IDC patients demonstrated the highest fold-change compared with those in the DCIS patients and healthy controls. The potential of miR-223-3p for cell proliferation and cell invasion were examined in vitro using MCF7 cells transfected with the miR-223-3p gene. MCF7 cells transfected with the miR-223-3p gene significantly promoted cell proliferation and cell invasive ability (P<0.05). The plasma exosomal miR-223-3p levels of the other 179 patients with breast cancer and 20 healthy controls were measured using TaqMan miR assays. The exosomal miR-223-3p levels of the patients with breast cancer were significantly increased compared with the healthy controls (P<0.01). A statistically significant association was observed between the exosomal miR-223-3p levels and histological type, pT stage, pN stage, pathological stage, lymphatic invasion and nuclear grade (P<0.05). The exosomal miR-223-3p levels of IDC patients (stage I) and upstaged IDC patients (stage I) were significantly higher compared with the DCIS patients (P<0.05). These results suggest that exosomal miR-223-3p may be a useful preoperative biomarker to identify the invasive lesions of DCIS patients diagnosed by biopsy. In addition, plasma exosome-encapsulated miR-223-3p level was significantly associated with the malignancy of breast cancer.

Keywords: biomarker; breast cancer; detecting invasive ductal carcinoma; ductal carcinoma in situ; exosome; microRNA-223-3p.

Figure 1.

Plasma exosome image by transmission electron microscope.

Figure 2.

Expression of miR-223-3p gene in MCF7 cells transfected with miR-223-3p. (A) Expression of the miR-223-3p gene in miR-223-3p-transfected MCF7 cells and mock-transfected MCF7 cells were examined by reverse transcription-quantitative PCR. (B) Expression of the miR-223-3p gene in MCF miR-223 and MCF7 mock were examined by digital PCR. *P<0.01. DW, distilled water; PCR, polymerase chain reaction; miR, microRNA.

Figure 3.

Potential of cell proliferation and cell invasion of MCF7 cells transfected with miR-223-3p. (A) Cell proliferation assays were examined using the MCF7 miR-223-3p and MCF7 mock. (B) Cell invasion assays were examined using the MCF7 miR-223-3p and MCF7 mock. ^#P<0.05, *P<0.05 and ⁺P<0.05. miR, microRNA.

Figure 4.

Correlation between miR-223-3p levels in plasma exosomes and breast cancer tissues. This study consisted of 40 patients with breast cancer, including DCIS (n=5), stage I (n=15), stage II (n=15) and stage III patients (n=5). The pair samples of plasma and tissue were collected from the same patients. DCIS, ductal carcinoma in situ; miR, microRNA.

Figure 5.

Comparison of exosomal miR-223-3p levels in healthy control and patients with breast cancer at each tumor stage. Exosomal miR-223-3p levels of healthy controls (n=20) and patients with breast cancer at stage 0 (n=25), stage I (n=94), stage II (n=52) and stage III (n=8) were measured by reverse transcription-quantitative polymerase chain reaction. *P<0.05, **P<0.01. miR, microRNA.

Figure 6.

Comparison of exosomal miR-223-3p levels between DCIS patients, upstage IDC patients and IDC patients. Exosomal miR-223-3p levels of DCIS patients (Stage 0), upstaged IDC patients (Stage I) and IDC patients (Stage I) were measured by reverse transcription-quantitative polymerase chain reaction. DCIS patients who were initially diagnosed by a needle biopsy prior to surgery were re-diagnosed following the operation using the completely excised specimen. Patients upstaged to IDC from DCIS on the final pathological report were described as upstage IDC patients. DCIS, ductal carcinoma in situ; IDC, invasive ductal carcinoma; miR, microRNA.

Figure 7.

Correlation between miR-223-3p and EPB41L3 mRNA levels in breast cancer tissues. This study consisted of 40 breast cancer patients, including DCIS patients (n=5) and stage I patients (n=15), stage II patients (n=15) and stage III patients (n=5). DCIS, ductal carcinoma in situ; miR, microRNA.