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Plasma exosome microRNAs are indicative of breast cancer

View Article: PubMed Central - PubMed

ABSTRACT

Background: microRNAs are promising candidate breast cancer biomarkers due to their cancer-specific expression profiles. However, efforts to develop circulating breast cancer biomarkers are challenged by the heterogeneity of microRNAs in the blood. To overcome this challenge, we aimed to develop a molecular profile of microRNAs specifically secreted from breast cancer cells. Our first step towards this direction relates to capturing and analyzing the contents of exosomes, which are small secretory vesicles that selectively encapsulate microRNAs indicative of their cell of origin. To our knowledge, circulating exosome microRNAs have not been well-evaluated as biomarkers for breast cancer diagnosis or monitoring.

Methods: Exosomes were collected from the conditioned media of human breast cancer cell lines, mouse plasma of patient-derived orthotopic xenograft models (PDX), and human plasma samples. Exosomes were verified by electron microscopy, nanoparticle tracking analysis, and western blot. Cellular and exosome microRNAs from breast cancer cell lines were profiled by next-generation small RNA sequencing. Plasma exosome microRNA expression was analyzed by qRT-PCR analysis.

Results: Small RNA sequencing and qRT-PCR analysis showed that several microRNAs are selectively encapsulated or highly enriched in breast cancer exosomes. Importantly, the selectively enriched exosome microRNA, human miR-1246, was detected at significantly higher levels in exosomes isolated from PDX mouse plasma, indicating that tumor exosome microRNAs are released into the circulation and can serve as plasma biomarkers for breast cancer. This observation was extended to human plasma samples where miR-1246 and miR-21 were detected at significantly higher levels in the plasma exosomes of 16 patients with breast cancer as compared to the plasma exosomes of healthy control subjects. Receiver operating characteristic curve analysis indicated that the combination of plasma exosome miR-1246 and miR-21 is a better indicator of breast cancer than their individual levels.

Conclusions: Our results demonstrate that certain microRNA species, such as miR-21 and miR-1246, are selectively enriched in human breast cancer exosomes and significantly elevated in the plasma of patients with breast cancer. These findings indicate a potential new strategy to selectively analyze plasma breast cancer microRNAs indicative of the presence of breast cancer.

Electronic supplementary material: The online version of this article (doi:10.1186/s13058-016-0753-x) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus

Small RNA next generation sequencing analysis of microRNAs in mammary epithelial cells and breast cancer cell lines versus exosomes. Cellular and exosome RNA was isolated from normal mammary cells (MCF10A) and breast cancer cell lines (MCF7 and MDA-MB-231). microRNAs were differentially expressed in MCF10A (a), MCF7 (c) and MDA-MB-231 (e) exosomes with a fold-change ≥6.0 (exosome vs. cell, p < 0.05) with mapped reads ≥ 200 are plotted. The abundance of the microRNAs highly expressed in exosomes vs. the cellular RNA in MCF10A (b), MCF7 (d) and MDA-MB-231 (f) are plotted by their mean number of reads per million reads
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Fig2: Small RNA next generation sequencing analysis of microRNAs in mammary epithelial cells and breast cancer cell lines versus exosomes. Cellular and exosome RNA was isolated from normal mammary cells (MCF10A) and breast cancer cell lines (MCF7 and MDA-MB-231). microRNAs were differentially expressed in MCF10A (a), MCF7 (c) and MDA-MB-231 (e) exosomes with a fold-change ≥6.0 (exosome vs. cell, p < 0.05) with mapped reads ≥ 200 are plotted. The abundance of the microRNAs highly expressed in exosomes vs. the cellular RNA in MCF10A (b), MCF7 (d) and MDA-MB-231 (f) are plotted by their mean number of reads per million reads

Mentions: To determine whether the microRNA profiles of mammary epithelial cells and breast cancer cells may differ, total RNA was isolated from the cells and exosomes and a small RNA library was prepared using equal quantities of RNA. The resulting cDNA library was sequenced and the reads were mapped onto the human genome (complete RNA sequencing data is available in Additional file 1). We first compared the cellular microRNA content to the matched exosome microRNAs for each cell line (Fig. 2). In MCF10A, 168 microRNAs were differentially expressed in the exosomes compared to the cells, 33 of these microRNAs were higher in the exosomes than the cells (selectively encapsulated by exosomes), 9 of these were at least six-fold greater in the exosomes versus the cell (Fig. 2a), and 11 of the selectively secreted microRNAs were detected at a level of 1000 reads or more (Fig. 2b). In MCF7, 196 microRNAs were differentially expressed in the exosomes compared to the cells, 6 of them were at least 20-fold higher in the exosomes versus the cell (Fig. 2c), and 4 of the 6 were detected at a level of 200 reads or more (Fig. 2d). In MDA-MB-231, 101 microRNAs were differentially expressed in the exosomes compared to the cells, with 63 higher in the exosome versus the cell; 7 of these were at least six-fold greater in the exosome versus the cell (Fig. 2e) and 10 were detected at a level of 1000 reads or more (Fig. 2f). Interestingly, the top ranked microRNAs that are selectively encapsulated by exosomes differed among MCF7, MDA-MB-231, and MCF10A cells (Fig. 2 and Table 1), indicating different profiles of cancer exosome microRNAs compared to normal epithelial exosome microRNAs.Fig. 2


Plasma exosome microRNAs are indicative of breast cancer
Small RNA next generation sequencing analysis of microRNAs in mammary epithelial cells and breast cancer cell lines versus exosomes. Cellular and exosome RNA was isolated from normal mammary cells (MCF10A) and breast cancer cell lines (MCF7 and MDA-MB-231). microRNAs were differentially expressed in MCF10A (a), MCF7 (c) and MDA-MB-231 (e) exosomes with a fold-change ≥6.0 (exosome vs. cell, p < 0.05) with mapped reads ≥ 200 are plotted. The abundance of the microRNAs highly expressed in exosomes vs. the cellular RNA in MCF10A (b), MCF7 (d) and MDA-MB-231 (f) are plotted by their mean number of reads per million reads
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getmorefigures.php?uid=PMC5016889&req=5

Fig2: Small RNA next generation sequencing analysis of microRNAs in mammary epithelial cells and breast cancer cell lines versus exosomes. Cellular and exosome RNA was isolated from normal mammary cells (MCF10A) and breast cancer cell lines (MCF7 and MDA-MB-231). microRNAs were differentially expressed in MCF10A (a), MCF7 (c) and MDA-MB-231 (e) exosomes with a fold-change ≥6.0 (exosome vs. cell, p < 0.05) with mapped reads ≥ 200 are plotted. The abundance of the microRNAs highly expressed in exosomes vs. the cellular RNA in MCF10A (b), MCF7 (d) and MDA-MB-231 (f) are plotted by their mean number of reads per million reads
Mentions: To determine whether the microRNA profiles of mammary epithelial cells and breast cancer cells may differ, total RNA was isolated from the cells and exosomes and a small RNA library was prepared using equal quantities of RNA. The resulting cDNA library was sequenced and the reads were mapped onto the human genome (complete RNA sequencing data is available in Additional file 1). We first compared the cellular microRNA content to the matched exosome microRNAs for each cell line (Fig. 2). In MCF10A, 168 microRNAs were differentially expressed in the exosomes compared to the cells, 33 of these microRNAs were higher in the exosomes than the cells (selectively encapsulated by exosomes), 9 of these were at least six-fold greater in the exosomes versus the cell (Fig. 2a), and 11 of the selectively secreted microRNAs were detected at a level of 1000 reads or more (Fig. 2b). In MCF7, 196 microRNAs were differentially expressed in the exosomes compared to the cells, 6 of them were at least 20-fold higher in the exosomes versus the cell (Fig. 2c), and 4 of the 6 were detected at a level of 200 reads or more (Fig. 2d). In MDA-MB-231, 101 microRNAs were differentially expressed in the exosomes compared to the cells, with 63 higher in the exosome versus the cell; 7 of these were at least six-fold greater in the exosome versus the cell (Fig. 2e) and 10 were detected at a level of 1000 reads or more (Fig. 2f). Interestingly, the top ranked microRNAs that are selectively encapsulated by exosomes differed among MCF7, MDA-MB-231, and MCF10A cells (Fig. 2 and Table 1), indicating different profiles of cancer exosome microRNAs compared to normal epithelial exosome microRNAs.Fig. 2

View Article: PubMed Central - PubMed

ABSTRACT

Background: microRNAs are promising candidate breast cancer biomarkers due to their cancer-specific expression profiles. However, efforts to develop circulating breast cancer biomarkers are challenged by the heterogeneity of microRNAs in the blood. To overcome this challenge, we aimed to develop a molecular profile of microRNAs specifically secreted from breast cancer cells. Our first step towards this direction relates to capturing and analyzing the contents of exosomes, which are small secretory vesicles that selectively encapsulate microRNAs indicative of their cell of origin. To our knowledge, circulating exosome microRNAs have not been well-evaluated as biomarkers for breast cancer diagnosis or monitoring.

Methods: Exosomes were collected from the conditioned media of human breast cancer cell lines, mouse plasma of patient-derived orthotopic xenograft models (PDX), and human plasma samples. Exosomes were verified by electron microscopy, nanoparticle tracking analysis, and western blot. Cellular and exosome microRNAs from breast cancer cell lines were profiled by next-generation small RNA sequencing. Plasma exosome microRNA expression was analyzed by qRT-PCR analysis.

Results: Small RNA sequencing and qRT-PCR analysis showed that several microRNAs are selectively encapsulated or highly enriched in breast cancer exosomes. Importantly, the selectively enriched exosome microRNA, human miR-1246, was detected at significantly higher levels in exosomes isolated from PDX mouse plasma, indicating that tumor exosome microRNAs are released into the circulation and can serve as plasma biomarkers for breast cancer. This observation was extended to human plasma samples where miR-1246 and miR-21 were detected at significantly higher levels in the plasma exosomes of 16 patients with breast cancer as compared to the plasma exosomes of healthy control subjects. Receiver operating characteristic curve analysis indicated that the combination of plasma exosome miR-1246 and miR-21 is a better indicator of breast cancer than their individual levels.

Conclusions: Our results demonstrate that certain microRNA species, such as miR-21 and miR-1246, are selectively enriched in human breast cancer exosomes and significantly elevated in the plasma of patients with breast cancer. These findings indicate a potential new strategy to selectively analyze plasma breast cancer microRNAs indicative of the presence of breast cancer.

Electronic supplementary material: The online version of this article (doi:10.1186/s13058-016-0753-x) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus