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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

qRT-PCR analysis of microRNAs in breast cancer cell line exosomes versus mammary epithelial cell exosomes. qRT-PCR analysis of selected microRNAs that are abundantly present in MCF7, ZR-75-1, T47D, MDA-MB-231, BT-20, BT-474, and SK-BR-3 exosomes vs. MCF10A exosomes. Fold-change in expression is shown for the exosome microRNAs relative to their cellular microRNA levels and normalized to the spike-in cel-miR-54 control
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Fig3: qRT-PCR analysis of microRNAs in breast cancer cell line exosomes versus mammary epithelial cell exosomes. qRT-PCR analysis of selected microRNAs that are abundantly present in MCF7, ZR-75-1, T47D, MDA-MB-231, BT-20, BT-474, and SK-BR-3 exosomes vs. MCF10A exosomes. Fold-change in expression is shown for the exosome microRNAs relative to their cellular microRNA levels and normalized to the spike-in cel-miR-54 control

Mentions: We then selected several microRNAs that are selectively secreted or highly abundant in the breast cancer MCF7 exosomes (miR-1246, miR-451, and miR-122) for validation by real-time PCR analysis in seven breast cancer cell lines representing the various breast cancer subtypes (Fig. 3). The data indicated that these microRNA species are selectively secreted and enriched in breast cancer exosomes relative to their cellular content and compared to mammary epithelial exosomes, with a few exceptions (miR-1246 in T47D and miR-451 in BT-474). Based on their fold-change, the enrichment of all three microRNAs seemed to be more pronounced in the estrogen receptor (ER)+, progesterone receptor (PR)+ cell line-derived exosomes (MCF7, ZR-75-1), and the ER-, PR-, HER2- cell-line-derived exosomes (MDA-MB-213 and BT-20). These observations are consistent with the RNA sequencing results.Fig. 3


Plasma exosome microRNAs are indicative of breast cancer
qRT-PCR analysis of microRNAs in breast cancer cell line exosomes versus mammary epithelial cell exosomes. qRT-PCR analysis of selected microRNAs that are abundantly present in MCF7, ZR-75-1, T47D, MDA-MB-231, BT-20, BT-474, and SK-BR-3 exosomes vs. MCF10A exosomes. Fold-change in expression is shown for the exosome microRNAs relative to their cellular microRNA levels and normalized to the spike-in cel-miR-54 control
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Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC5016889&req=5

Fig3: qRT-PCR analysis of microRNAs in breast cancer cell line exosomes versus mammary epithelial cell exosomes. qRT-PCR analysis of selected microRNAs that are abundantly present in MCF7, ZR-75-1, T47D, MDA-MB-231, BT-20, BT-474, and SK-BR-3 exosomes vs. MCF10A exosomes. Fold-change in expression is shown for the exosome microRNAs relative to their cellular microRNA levels and normalized to the spike-in cel-miR-54 control
Mentions: We then selected several microRNAs that are selectively secreted or highly abundant in the breast cancer MCF7 exosomes (miR-1246, miR-451, and miR-122) for validation by real-time PCR analysis in seven breast cancer cell lines representing the various breast cancer subtypes (Fig. 3). The data indicated that these microRNA species are selectively secreted and enriched in breast cancer exosomes relative to their cellular content and compared to mammary epithelial exosomes, with a few exceptions (miR-1246 in T47D and miR-451 in BT-474). Based on their fold-change, the enrichment of all three microRNAs seemed to be more pronounced in the estrogen receptor (ER)+, progesterone receptor (PR)+ cell line-derived exosomes (MCF7, ZR-75-1), and the ER-, PR-, HER2- cell-line-derived exosomes (MDA-MB-213 and BT-20). These observations are consistent with the RNA sequencing results.Fig. 3

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