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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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Extracellular miR-16 Levels Correlate with Nanovesicle Abundance.A Plot of extracellular to intracellular levels of miRNAs quantitated by qRT-PCR using stem-loop primers of cells lines BT-20, MCF 10A, MCF7, MDA-MB-231 and SK-BR-3. MiRNA levels were calculated using standard curves, and corrected for recovery by normalization to a spiked synthetic RNA (INT-RNA) introduced at the time of RNA-extraction (Materials and Methods and Table S4). Error bars indicate the measure of one standard deviation of 3 independent experiments. B Relative levels of miR-16 and absolute levels of CD81 were measured in P70 fractions of seven independent experiments and plotted. The line indicates a best-fit power curve (r2 = 0.95). C The P70 was collected daily from media conditioned by MCF7 cells (solid symbols), or from MCF 10A cells (open symbol) cultures and miRNAs were quantified in triplicate. The release rates were linear; r2 = 0.79 for miR-1246 (inset), r2 = 0.98 for miR-16, r2 = 0.85 for miR-451; and r2 = 0.87 for miR-720 of MCF7 cells.
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pone-0013515-g003: Extracellular miR-16 Levels Correlate with Nanovesicle Abundance.A Plot of extracellular to intracellular levels of miRNAs quantitated by qRT-PCR using stem-loop primers of cells lines BT-20, MCF 10A, MCF7, MDA-MB-231 and SK-BR-3. MiRNA levels were calculated using standard curves, and corrected for recovery by normalization to a spiked synthetic RNA (INT-RNA) introduced at the time of RNA-extraction (Materials and Methods and Table S4). Error bars indicate the measure of one standard deviation of 3 independent experiments. B Relative levels of miR-16 and absolute levels of CD81 were measured in P70 fractions of seven independent experiments and plotted. The line indicates a best-fit power curve (r2 = 0.95). C The P70 was collected daily from media conditioned by MCF7 cells (solid symbols), or from MCF 10A cells (open symbol) cultures and miRNAs were quantified in triplicate. The release rates were linear; r2 = 0.79 for miR-1246 (inset), r2 = 0.98 for miR-16, r2 = 0.85 for miR-451; and r2 = 0.87 for miR-720 of MCF7 cells.

Mentions: Because the transformation status of a cell regulates exosome secretion [47], and thus possibly exosomal miRNA release, we sought to identify a miRNA that faithfully reflects exosomal abundance in order to quantify selective miRNA release into vesicles. For example, miR-103 and its paralog miR-107 have high and consistent expression in both cancerous and normal mammary tissues, and thus have been used for normalizing miRNA studies comparing mammary cells [42]. However, these miRNAs were grossly underrepresented in the extracellular fraction (Figures 2 A and B), highlighting the observation that at least some miRNAs with diagnostic value in cells are not represented in the released population. We therefore considered other miRNAs for normalization. Using the more sensitive stem-loop primer PCR approach [39], we focused on several miRNAs, including the most abundantly released miRNA of MCF7 cells, miR-720; miR-21; miR-16; and microRNAs that were enriched extracellularly, including miR-451, miR-1275 (Figure 3A). We measured the cellular to extracellular ratio of these miRNAs in a set of HMECs including cell lines BT-20, MCF 10A, MCF7, MDA-MB-231, and SK-BR-3. We found that HMECs irrespective of malignancy, released a constant amount of miR-21 reflective of cellular abundance, a miRNA that is upregulated in many breast cancers [6], [7], [8], and miR-16 (Figure 3A). The most consistently released miRNA was miR-16 of which for each released molecule, 160–400 molecules were retained.


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)

Extracellular miR-16 Levels Correlate with Nanovesicle Abundance.A Plot of extracellular to intracellular levels of miRNAs quantitated by qRT-PCR using stem-loop primers of cells lines BT-20, MCF 10A, MCF7, MDA-MB-231 and SK-BR-3. MiRNA levels were calculated using standard curves, and corrected for recovery by normalization to a spiked synthetic RNA (INT-RNA) introduced at the time of RNA-extraction (Materials and Methods and Table S4). Error bars indicate the measure of one standard deviation of 3 independent experiments. B Relative levels of miR-16 and absolute levels of CD81 were measured in P70 fractions of seven independent experiments and plotted. The line indicates a best-fit power curve (r2 = 0.95). C The P70 was collected daily from media conditioned by MCF7 cells (solid symbols), or from MCF 10A cells (open symbol) cultures and miRNAs were quantified in triplicate. The release rates were linear; r2 = 0.79 for miR-1246 (inset), r2 = 0.98 for miR-16, r2 = 0.85 for miR-451; and r2 = 0.87 for miR-720 of MCF7 cells.
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Related In: Results  -  Collection

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pone-0013515-g003: Extracellular miR-16 Levels Correlate with Nanovesicle Abundance.A Plot of extracellular to intracellular levels of miRNAs quantitated by qRT-PCR using stem-loop primers of cells lines BT-20, MCF 10A, MCF7, MDA-MB-231 and SK-BR-3. MiRNA levels were calculated using standard curves, and corrected for recovery by normalization to a spiked synthetic RNA (INT-RNA) introduced at the time of RNA-extraction (Materials and Methods and Table S4). Error bars indicate the measure of one standard deviation of 3 independent experiments. B Relative levels of miR-16 and absolute levels of CD81 were measured in P70 fractions of seven independent experiments and plotted. The line indicates a best-fit power curve (r2 = 0.95). C The P70 was collected daily from media conditioned by MCF7 cells (solid symbols), or from MCF 10A cells (open symbol) cultures and miRNAs were quantified in triplicate. The release rates were linear; r2 = 0.79 for miR-1246 (inset), r2 = 0.98 for miR-16, r2 = 0.85 for miR-451; and r2 = 0.87 for miR-720 of MCF7 cells.
Mentions: Because the transformation status of a cell regulates exosome secretion [47], and thus possibly exosomal miRNA release, we sought to identify a miRNA that faithfully reflects exosomal abundance in order to quantify selective miRNA release into vesicles. For example, miR-103 and its paralog miR-107 have high and consistent expression in both cancerous and normal mammary tissues, and thus have been used for normalizing miRNA studies comparing mammary cells [42]. However, these miRNAs were grossly underrepresented in the extracellular fraction (Figures 2 A and B), highlighting the observation that at least some miRNAs with diagnostic value in cells are not represented in the released population. We therefore considered other miRNAs for normalization. Using the more sensitive stem-loop primer PCR approach [39], we focused on several miRNAs, including the most abundantly released miRNA of MCF7 cells, miR-720; miR-21; miR-16; and microRNAs that were enriched extracellularly, including miR-451, miR-1275 (Figure 3A). We measured the cellular to extracellular ratio of these miRNAs in a set of HMECs including cell lines BT-20, MCF 10A, MCF7, MDA-MB-231, and SK-BR-3. We found that HMECs irrespective of malignancy, released a constant amount of miR-21 reflective of cellular abundance, a miRNA that is upregulated in many breast cancers [6], [7], [8], and miR-16 (Figure 3A). The most consistently released miRNA was miR-16 of which for each released molecule, 160–400 molecules were retained.

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