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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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Some MicroRNAs are Released Disproportionately.Duplicate microRNA microarrays were hybridized with 1 µg of total cellular or 1 µg of extracellular miRNA from MCF7 cells. Results are plotted as A relative fluorescent intensities of extracellular (x, upper panel) and cellular (c, lower panel) miRNAs, or B ratio of extracellular to cellular miRNAs. The horizontal lines in B indicate the threshold of 2 fold-changes, whereas the red and the green marked populations indicate a greater than 4-fold enrichment in the released extracellular (A, upper panel, x), or in the cells (A, lower panel, c) respectively. C Scatter plot of average reads of the miRNAs quantitated by array. Only miRNAs with a fluorescent value of greater than 500 in the cellular or extracellular population are shown (see Materials and Methods). The numbers next to dots indicate the miRNA the dot represents. D MCF7 cells were cultured for 5 days, and the total amount of specific cellular and extracellular miRNAs were measured by quantitative linker-ligation mediated RT-PCR, and the miRNA ratios were plotted. The average of 3 independent experiments is shown. E Native PAGE of products at end-point of quantitative RT-PCR. The major PCR-products between 32–48 ntds correspond to the mature miRNA (miRNA) as expected by size and determined by sequencing (Table S5). The bands with a migration of less then 25 ntds are the PCR primers (primers) used in the reaction. Bands that retained in the well are amplification-independent reaction components (reaction components). Hsa-miR-923 has since been reclassified as a specific rRNA fragment.
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pone-0013515-g002: Some MicroRNAs are Released Disproportionately.Duplicate microRNA microarrays were hybridized with 1 µg of total cellular or 1 µg of extracellular miRNA from MCF7 cells. Results are plotted as A relative fluorescent intensities of extracellular (x, upper panel) and cellular (c, lower panel) miRNAs, or B ratio of extracellular to cellular miRNAs. The horizontal lines in B indicate the threshold of 2 fold-changes, whereas the red and the green marked populations indicate a greater than 4-fold enrichment in the released extracellular (A, upper panel, x), or in the cells (A, lower panel, c) respectively. C Scatter plot of average reads of the miRNAs quantitated by array. Only miRNAs with a fluorescent value of greater than 500 in the cellular or extracellular population are shown (see Materials and Methods). The numbers next to dots indicate the miRNA the dot represents. D MCF7 cells were cultured for 5 days, and the total amount of specific cellular and extracellular miRNAs were measured by quantitative linker-ligation mediated RT-PCR, and the miRNA ratios were plotted. The average of 3 independent experiments is shown. E Native PAGE of products at end-point of quantitative RT-PCR. The major PCR-products between 32–48 ntds correspond to the mature miRNA (miRNA) as expected by size and determined by sequencing (Table S5). The bands with a migration of less then 25 ntds are the PCR primers (primers) used in the reaction. Bands that retained in the well are amplification-independent reaction components (reaction components). Hsa-miR-923 has since been reclassified as a specific rRNA fragment.

Mentions: Because of the suggested roles of extracellular miRNAs in signaling and diagnosis [27], we investigated whether the intracellular and extracellular miRNA composition are the same. To answer this question, we performed microRNA microarray analyses of MCF7 cellular (c) and extracellular (x) RNAs (Figure 2A), and found that about 66% of the released miRNAs are at an abundance that closely reflects the cellular miRNA abundance (Figures 2B and 2C). This finding is in agreement with a model wherein most, but not all miRNAs are released passively by mass action.


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)

Some MicroRNAs are Released Disproportionately.Duplicate microRNA microarrays were hybridized with 1 µg of total cellular or 1 µg of extracellular miRNA from MCF7 cells. Results are plotted as A relative fluorescent intensities of extracellular (x, upper panel) and cellular (c, lower panel) miRNAs, or B ratio of extracellular to cellular miRNAs. The horizontal lines in B indicate the threshold of 2 fold-changes, whereas the red and the green marked populations indicate a greater than 4-fold enrichment in the released extracellular (A, upper panel, x), or in the cells (A, lower panel, c) respectively. C Scatter plot of average reads of the miRNAs quantitated by array. Only miRNAs with a fluorescent value of greater than 500 in the cellular or extracellular population are shown (see Materials and Methods). The numbers next to dots indicate the miRNA the dot represents. D MCF7 cells were cultured for 5 days, and the total amount of specific cellular and extracellular miRNAs were measured by quantitative linker-ligation mediated RT-PCR, and the miRNA ratios were plotted. The average of 3 independent experiments is shown. E Native PAGE of products at end-point of quantitative RT-PCR. The major PCR-products between 32–48 ntds correspond to the mature miRNA (miRNA) as expected by size and determined by sequencing (Table S5). The bands with a migration of less then 25 ntds are the PCR primers (primers) used in the reaction. Bands that retained in the well are amplification-independent reaction components (reaction components). Hsa-miR-923 has since been reclassified as a specific rRNA fragment.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2958125&req=5

pone-0013515-g002: Some MicroRNAs are Released Disproportionately.Duplicate microRNA microarrays were hybridized with 1 µg of total cellular or 1 µg of extracellular miRNA from MCF7 cells. Results are plotted as A relative fluorescent intensities of extracellular (x, upper panel) and cellular (c, lower panel) miRNAs, or B ratio of extracellular to cellular miRNAs. The horizontal lines in B indicate the threshold of 2 fold-changes, whereas the red and the green marked populations indicate a greater than 4-fold enrichment in the released extracellular (A, upper panel, x), or in the cells (A, lower panel, c) respectively. C Scatter plot of average reads of the miRNAs quantitated by array. Only miRNAs with a fluorescent value of greater than 500 in the cellular or extracellular population are shown (see Materials and Methods). The numbers next to dots indicate the miRNA the dot represents. D MCF7 cells were cultured for 5 days, and the total amount of specific cellular and extracellular miRNAs were measured by quantitative linker-ligation mediated RT-PCR, and the miRNA ratios were plotted. The average of 3 independent experiments is shown. E Native PAGE of products at end-point of quantitative RT-PCR. The major PCR-products between 32–48 ntds correspond to the mature miRNA (miRNA) as expected by size and determined by sequencing (Table S5). The bands with a migration of less then 25 ntds are the PCR primers (primers) used in the reaction. Bands that retained in the well are amplification-independent reaction components (reaction components). Hsa-miR-923 has since been reclassified as a specific rRNA fragment.
Mentions: Because of the suggested roles of extracellular miRNAs in signaling and diagnosis [27], we investigated whether the intracellular and extracellular miRNA composition are the same. To answer this question, we performed microRNA microarray analyses of MCF7 cellular (c) and extracellular (x) RNAs (Figure 2A), and found that about 66% of the released miRNAs are at an abundance that closely reflects the cellular miRNA abundance (Figures 2B and 2C). This finding is in agreement with a model wherein most, but not all miRNAs are released passively by mass action.

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