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. 2020 Jun;111(6):2104-2115.
doi: 10.1111/cas.14393. Epub 2020 Apr 23.

Predicting the presence of breast cancer using circulating small RNAs, including those in the extracellular vesicles

Yumiko Koi  1   2 , Yasuhiro Tsutani  2 , Yukie Nishiyama  3 , Daisuke Ueda  1   2 , Yuta Ibuki  1   2 , Shinsuke Sasada  2 , Tomoyuki Akita  4 , Norio Masumoto  2 , Takayuki Kadoya  2 , Yuki Yamamoto  3 , Ryou-U Takahashi  3 , Junko Tanaka  4 , Morihito Okada  2 , Hidetoshi Tahara  3   5
Affiliations

Predicting the presence of breast cancer using circulating small RNAs, including those in the extracellular vesicles

Yumiko Koi et al. Cancer Sci. 2020 Jun.

Abstract

Emerging evidence indicates that small RNAs, including microRNAs (miRNAs) and their isoforms (isomiRs), and transfer RNA fragments (tRFs), are differently expressed in breast cancer (BC) and can be detected in blood circulation. Circulating small RNAs and small RNAs in extracellular vesicles (EVs) have emerged as ideal markers in small RNA-based applications for cancer detection. In this study, we first undertook small RNA sequencing to assess the expression of circulating small RNAs in the serum of BC patients and cancer-free individuals (controls). Expression of 3 small RNAs, namely isomiR of miR-21-5p (3' addition C), miR-23a-3p and tRF-Lys (TTT), was significantly higher in BC samples and was validated by small RNA sequencing in an independent cohort. Our constructed model using 3 small RNAs showed high diagnostic accuracy with an area under the receiver operating characteristic curve of 0.92 and discriminated early-stage BCs at stage 0 from control. To test the possibility that these small RNAs are released from cancer cells, we next examined EVs from the serum of BC patients and controls. Two of the 3 candidate small RNAs were identified, and shown to be abundant in EVs of BC patients. Interestingly, these 2 small RNAs are also more abundantly detected in culture media of breast cancer cell lines (MCF-7 and MDA-MB-231). The same tendency in selective elevation seen in total serum, serum EV, and EV derived from cell culture media could indicate the efficiency of this model using total serum of patients. These findings indicate that small RNAs serve as significant biomarkers for BC detection.

Keywords: biomarker; breast cancer; extracellular vesicle; serum; small RNA.

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Conflict of interest statement

Professor Hidetoshi Tahara is representative director of a university‐originated venture, MiRTeL Co. A family member, Kanoko Tahara, is an employee of MiRTeL Co. The remaining authors declare no potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Schematic overview of this study into the diagnostic utility of circulating small RNAs for detection of breast cancer (BC). ncRNA, noncoding RNA
FIGURE 2
FIGURE 2
Comparison of normalized reads of 3 significantly upregulated small RNAs in the validation cohort (*P < .01) of breast cancer patients (BC, n = 39) and cancer‐free individuals (N, n = 36). isomiR, microRNA isoform; miR, microRNA; tRF, transfer RNA fragment
FIGURE 3
FIGURE 3
Comparison of microRNA isoform (isomiR) abundance in serum from breast cancer patients (BC, n = 78) and cancer‐free individuals (N, n = 72). The 5 most abundant forms of microRNA (miR) are presented in the y‐axis. A, miR‐21‐5p. B, miR‐23a‐3p. RPM, reads per million; seq, sequence
FIGURE 4
FIGURE 4
Diagnostic utility of 3 small RNAs for breast cancer (BC) detection. A, Analysis of the area under the receiver operating characteristic curve (AUC). B, Relative levels using small RNAs in BC patients and cancer‐free individuals (N) by BC stage. *P < .01. C, Relative levels using small RNAs in BC patients and N by BC subtype. *P < .01. D, Relative levels using small RNAs in BC patients by immunohistochemical (IHC) analysis in patients with invasive BC. E, Comparison of diagnostic values among serum markers carcinoembryonic antigen (CEA) and carbohydrate antigen (CA)15‐3, and our constructed model (C). DCIS, ductal carcinoma in situ; ER, estrogen receptor; HER2, human epidermal growth factor receptor 2; miRNA, microRNA; PR, progesterone receptor.
FIGURE 5
FIGURE 5
Characterization of extracellular vesicles (EVs) isolated with an exosome isolation kit. A, Transmission electron microscopy analysis of EVs. B, Western blot analysis of exosomal markers CD9 and TSG‐101 in samples from breast cancer patients (BC) and cancer‐free individuals (N). C, D, Contribution of nanoparticles of EVs (C), and total concentration of each EV (D). E, ApoB as a marker of lipoproteins by western blot analysis
FIGURE 6
FIGURE 6
Expression of identified small RNAs in extracellular vesicles (EVs) derived from serum of 32 breast cancer patients (BC) and 20 cancer‐free individuals (N), and from cell culture media of 184‐h TERT, MCF7, and MDA‐MB‐231 cell lines. *P < .01
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