Unknown,Transcriptomics,Genomics,Proteomics

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Selective Release of miRNA Species from Normal and Malignant Mammary Epithelial Cells

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. miRNA microarrays were screened by LC Sciences. One microgram of MCF7 cellular RNA preparation was labeled with Cy3, and one microgram of the extracellular RNA samples was labeled with Cy5. In brief, the RNA was labeled with Cy5 or Cy3 and hybridized to LCSciences standard arrays for mature miRNA of all species available in the Sanger miRBase database (Release 12.0). The data were analyzed including background subtraction, using a LOWESS (locally weighted regression) method on the background-subtracted data. Only transcripts with a signal intensity higher than 3X (background SD) and spot CV<0.5. CV was calculated by (SD)/(signal intensity), and in which repeating probes on the array produced signals from at least 50% of the repeating probes are above detection level.

ORGANISM(S): Homo sapiens

SUBMITTER: Dominik Duelli

PROVIDER: E-GEOD-22235 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:MicroRNAs (miRNAs) are small noncoding RNAs that negatively regulate gene expression post-transcriptionally. They play a critical role in developmental and physiological processes and have been implicated in the pathogenesis of several diseases including cancer. To identify miRNA signatures associated with different stages of neoplastic development, we examined the expression profile of 776 primate miRNAs in the following cells: primary African green monkey kidney (pAGMK) cells; spontaneously immortalized, non-tumorigenic, low-passage VERO cells (10-87 LP); tumorigenic, high-passage VERO cells (10-87 HP); and a cell line (10-87 T) derived from a 10-87 HP cell tumor xenograft in athymic nude mice. When compared with pAGMK cells, the majority of miRNAs were expressed at lower levels in 10-87 LP, 10-87 HP, and 10-87 T cells. We identified 10 up-regulated miRNAs whose level of expression correlated with VERO cell evolution from a non-tumorigenic phenotype to a tumorigenic phenotype. Several miRNAs that were components of the tumorigenic phenotype-specific signatures in our AGMK model are also found in a variety of human tumors. This may prove to be of general relevance to the biology of neoplastic development as it occurs both in vivo as well as in vitro. In addition, one or more of these miRNAs could be potential biomarkers for the expression of the tumorigenic phenotype of VERO cells. The spontaneousely transformed VERO cells, non-tumorigenic, were pasasged at low density in culture up to 250. The high passage (p250) was found to be tumorigenic. The cell line from xenograft of high passage was also established. We then evaluated patterns of miRNA expression in pAGMK cells and in derivatives of the 10-87 VERO cell line (10-87 LP cells, 10-87 HP cells, and 10-87 T cells) in an attempt to identify the miRNAs whose altered expression might correlate with, and perhaps be involved in, the evolution of the neoplastic phenotypes that occurred during passage of these AGMK cells in tissue culture. performed high-throughput miRNA profiling to audit the expression level of miRNAs in pAGMK cells and in VERO cells at non-tumorigenic and tumorigenic stages of neoplastic development. The analysis involved pAGMK cells, non-tumorigenic 10-87 low-passage VERO cells (10-87 LP) tumorigenic, high-passage VERO cells (10-87 HP) and a cell line (10-87 T) derived from a 10-87 HP cell tumor xenograft in athymic nude mice.

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Selective Release of miRNA Species from Normal and Malignant Mammary Epithelial Cells

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