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Selective release of microRNA species from normal and malignant mammary epithelial cells.

Pigati L, Yaddanapudi SC, Iyengar R, Kim DJ, Hearn SA, Danforth D, Hastings ML, Duelli DM - PLoS ONE (2010)

Bottom Line: Here we report that released miRNAs do not necessarily reflect the abundance of miRNA in the cell of origin.Our findings suggest the existence of a cellular selection mechanism for miRNA release and indicate that the extracellular and cellular miRNA profiles differ.This selective release of miRNAs is an important consideration for the identification of circulating miRNAs as biomarkers of disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, The Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, Illinois, United States of America.

ABSTRACT
MicroRNAs (miRNAs) in body fluids are candidate diagnostics for a variety of conditions and diseases, including breast cancer. One premise for using extracellular miRNAs to diagnose disease is the notion that the abundance of the miRNAs in body fluids reflects their abundance in the abnormal cells causing the disease. As a result, the search for such diagnostics in body fluids has focused on miRNAs that are abundant in the cells of origin. Here we report that released miRNAs do not necessarily reflect the abundance of miRNA in the cell of origin. We find that release of miRNAs from cells into blood, milk and ductal fluids is selective and that the selection of released miRNAs may correlate with malignancy. In particular, the bulk of miR-451 and miR-1246 produced by malignant mammary epithelial cells was released, but the majority of these miRNAs produced by non-malignant mammary epithelial cells was retained. Our findings suggest the existence of a cellular selection mechanism for miRNA release and indicate that the extracellular and cellular miRNA profiles differ. This selective release of miRNAs is an important consideration for the identification of circulating miRNAs as biomarkers of disease.

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Differential Cellular Release and Retention of Small RNAs.Medium conditioned by MCF7 cells for 5 days was enriched for exosomes by a filtration and ultracentrifugation protocol producing a P70 preparation. A The P70 was subjected to negative-staining EM. B The abundance of tetraspanin CD81, an exosome-marker was assessed in the filtered conditioned medium, the P70 pellet obtained by ultracentrifugation, and the supernatant (S70) using slot-blot (inset, n = 2). C The surface antigens CD81, CD63 and Mucin-1 were detected in the P70 fraction of the mammary epithelial cells using slot-blot. The absolute amount of bound antibody was quantified using standard-curves of antibody dilutions, and expressed as a percent of total antigenicity for the P70 of each cell line. The data of two replicate experiments for the indicated cell lines are shown. D Radiolabeled small RNAs isolated from MCF7 cells (c) and the extracellular preparation P70 (x) were separated by PAGE on a 12% denaturing gel. Star: Extracellular enriched RNA; Circle: Some extracellular RNAs identified by sequencing (see text and Tables S1 and S2). E Quantitation of labeled RNA species of D. The thin line indicates abundance of cellular small RNAs, whereas the thick line indicates the abundance of the extracellular miRNAs.
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pone-0013515-g001: Differential Cellular Release and Retention of Small RNAs.Medium conditioned by MCF7 cells for 5 days was enriched for exosomes by a filtration and ultracentrifugation protocol producing a P70 preparation. A The P70 was subjected to negative-staining EM. B The abundance of tetraspanin CD81, an exosome-marker was assessed in the filtered conditioned medium, the P70 pellet obtained by ultracentrifugation, and the supernatant (S70) using slot-blot (inset, n = 2). C The surface antigens CD81, CD63 and Mucin-1 were detected in the P70 fraction of the mammary epithelial cells using slot-blot. The absolute amount of bound antibody was quantified using standard-curves of antibody dilutions, and expressed as a percent of total antigenicity for the P70 of each cell line. The data of two replicate experiments for the indicated cell lines are shown. D Radiolabeled small RNAs isolated from MCF7 cells (c) and the extracellular preparation P70 (x) were separated by PAGE on a 12% denaturing gel. Star: Extracellular enriched RNA; Circle: Some extracellular RNAs identified by sequencing (see text and Tables S1 and S2). E Quantitation of labeled RNA species of D. The thin line indicates abundance of cellular small RNAs, whereas the thick line indicates the abundance of the extracellular miRNAs.

Mentions: To compare intracellular and extracellular miRNA populations, we analyzed the breast cancer cell line MCF7, which releases exosomes [36]. We focused on RNA contained in vesicles, because such vesicles are released from cells in vitro, as well as into blood, urine, saliva and other body fluids [14], [15], [19], [20], [23], [24], [25], [27], [30], [37]. We enriched for vesicles by centrifugation at 70,000 g from media conditioned by MCF7 cells (Figure 1). This preparation (P70) included cup-shaped vesicles of about 100 nm (Figure 1A), which is consistent with exosomes; and was enriched in CD81, a marker protein of exosomes (Figure 1B).


Selective release of microRNA species from normal and malignant mammary epithelial cells.

Pigati L, Yaddanapudi SC, Iyengar R, Kim DJ, Hearn SA, Danforth D, Hastings ML, Duelli DM - PLoS ONE (2010)

Differential Cellular Release and Retention of Small RNAs.Medium conditioned by MCF7 cells for 5 days was enriched for exosomes by a filtration and ultracentrifugation protocol producing a P70 preparation. A The P70 was subjected to negative-staining EM. B The abundance of tetraspanin CD81, an exosome-marker was assessed in the filtered conditioned medium, the P70 pellet obtained by ultracentrifugation, and the supernatant (S70) using slot-blot (inset, n = 2). C The surface antigens CD81, CD63 and Mucin-1 were detected in the P70 fraction of the mammary epithelial cells using slot-blot. The absolute amount of bound antibody was quantified using standard-curves of antibody dilutions, and expressed as a percent of total antigenicity for the P70 of each cell line. The data of two replicate experiments for the indicated cell lines are shown. D Radiolabeled small RNAs isolated from MCF7 cells (c) and the extracellular preparation P70 (x) were separated by PAGE on a 12% denaturing gel. Star: Extracellular enriched RNA; Circle: Some extracellular RNAs identified by sequencing (see text and Tables S1 and S2). E Quantitation of labeled RNA species of D. The thin line indicates abundance of cellular small RNAs, whereas the thick line indicates the abundance of the extracellular miRNAs.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2958125&req=5

pone-0013515-g001: Differential Cellular Release and Retention of Small RNAs.Medium conditioned by MCF7 cells for 5 days was enriched for exosomes by a filtration and ultracentrifugation protocol producing a P70 preparation. A The P70 was subjected to negative-staining EM. B The abundance of tetraspanin CD81, an exosome-marker was assessed in the filtered conditioned medium, the P70 pellet obtained by ultracentrifugation, and the supernatant (S70) using slot-blot (inset, n = 2). C The surface antigens CD81, CD63 and Mucin-1 were detected in the P70 fraction of the mammary epithelial cells using slot-blot. The absolute amount of bound antibody was quantified using standard-curves of antibody dilutions, and expressed as a percent of total antigenicity for the P70 of each cell line. The data of two replicate experiments for the indicated cell lines are shown. D Radiolabeled small RNAs isolated from MCF7 cells (c) and the extracellular preparation P70 (x) were separated by PAGE on a 12% denaturing gel. Star: Extracellular enriched RNA; Circle: Some extracellular RNAs identified by sequencing (see text and Tables S1 and S2). E Quantitation of labeled RNA species of D. The thin line indicates abundance of cellular small RNAs, whereas the thick line indicates the abundance of the extracellular miRNAs.
Mentions: To compare intracellular and extracellular miRNA populations, we analyzed the breast cancer cell line MCF7, which releases exosomes [36]. We focused on RNA contained in vesicles, because such vesicles are released from cells in vitro, as well as into blood, urine, saliva and other body fluids [14], [15], [19], [20], [23], [24], [25], [27], [30], [37]. We enriched for vesicles by centrifugation at 70,000 g from media conditioned by MCF7 cells (Figure 1). This preparation (P70) included cup-shaped vesicles of about 100 nm (Figure 1A), which is consistent with exosomes; and was enriched in CD81, a marker protein of exosomes (Figure 1B).

Bottom Line: Here we report that released miRNAs do not necessarily reflect the abundance of miRNA in the cell of origin.Our findings suggest the existence of a cellular selection mechanism for miRNA release and indicate that the extracellular and cellular miRNA profiles differ.This selective release of miRNAs is an important consideration for the identification of circulating miRNAs as biomarkers of disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, The Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, Illinois, United States of America.

ABSTRACT
MicroRNAs (miRNAs) in body fluids are candidate diagnostics for a variety of conditions and diseases, including breast cancer. One premise for using extracellular miRNAs to diagnose disease is the notion that the abundance of the miRNAs in body fluids reflects their abundance in the abnormal cells causing the disease. As a result, the search for such diagnostics in body fluids has focused on miRNAs that are abundant in the cells of origin. Here we report that released miRNAs do not necessarily reflect the abundance of miRNA in the cell of origin. We find that release of miRNAs from cells into blood, milk and ductal fluids is selective and that the selection of released miRNAs may correlate with malignancy. In particular, the bulk of miR-451 and miR-1246 produced by malignant mammary epithelial cells was released, but the majority of these miRNAs produced by non-malignant mammary epithelial cells was retained. Our findings suggest the existence of a cellular selection mechanism for miRNA release and indicate that the extracellular and cellular miRNA profiles differ. This selective release of miRNAs is an important consideration for the identification of circulating miRNAs as biomarkers of disease.

Show MeSH
Related in: MedlinePlus