Dataset Information

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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE22235 | GEO | 2010/09/10

SECONDARY ACCESSION(S): PRJNA128849

REPOSITORIES: GEO

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Project description: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.

Project description:Currently, micro RNAs (miRNAs) constitute a promising models for cell-to-cell communication since they are transferred between cells to execute essential roles in many processes. Their structure and size, in addition to various transport mechanisms, allow them to remain stable within various biological fluids, surviving in extremely adverse conditions, including low pH, boiling, and freezing. The presence of miRNAs in reproductive fluids, such as follicular, uterine and seminal fluid, indicate potential roles in the reproductive system. These extracellular miRNAs can be transported by lipoproteins (both HDL and LDL) or other proteins, including Argonaute2 (AGO2) and nucleophosmin1 (NPM1). Another transport system is mediated by extracellular vesicles (EVs), such as apoptotic bodies, microvesicles (MVs) and/or exosome-like vesicles. EVs protect miRNAs from degradation and contribute to their stability within biological fluids. Furthermore, EVs can transport a wide range of components packaged in a selective way. For instance, Squadrito et al. (2014), used macrophages and endothelial cells to demonstrate that the sorting of miRNAs into EVs for heterotrophic cell communication is altered by both, the presence of target transcripts and the self-presence of the respectively miRNA. In addition, the signature of miRNAs found in the exosomes significantly differed for those detected in the parent cells. To date, mechanisms controlling the specific loading of miRNAs into exosomes remain unclear. Indeed, several mechanisms may govern exosome sorting of specific subsets of miRNAs. MiRNA sorting appears to be influenced by different pathways and molecules in different cell types and tissues, and miRNAs contain well defined motifs (i.e., EXOmotifs), that direct the miRNA allocation into exosomes before delivery into recipient cells. A recent study showed that this RNA sequence can be recognized by the sumoylated form of the heterogeneous ribonucleoprotein A2B1 (hnRNPA2B1). Moreover, a terminal 30 nucleotide addition in miRNAs affects their selective sorting in B cells. Another hypothesis suggests that RNAs are selectively sorted depending on the differential affinity of RNA motifs towards the raft-like region of the cytoplasmic surface of microvesicular body (MVB) limiting membranes. Current data indicate that cells can communicate with each other through the transfer of miRNA-loaded exosomes. For example, monocyte-derived exosomes deliver miR-150 to endothelial cells and enhance endothelial cell migration by reducing c-myb expression. The miRNA content of exosomes plays a critical role in such cell-to-cell communication and determines the fate of recipient cells. Thus, exosomes derived from the bone marrow mesenchymal stromal cells of myeloma patients promote tumor growth depending on the content of miR-15a in exosomes. Our group has described a novel cell-to-cell communication mechanism involving the delivery of endometrial miRNAs from the maternal endometrium to the trophectoderm cells of preimplantation embryos. Specifically, in B6C3 derived mouse embryos, we found EV-associated and free miR-30d to cause overexpression of genes involved in embryonic adhesion processes, including Itb3, Itga7 and Cdh5. Furthermore, supplementing murine embryos with miR-30d significantly improved embryo adhesion, suggesting that external miRNAs may have a functional role as transcriptomic modifiers of preimplantation embryos. Based on profiling of miRNAs in endometrial fluid, maternally-derived miRNAs are present within EVs in the uterine microenvironment. The internalization of maternally-derived exosomes has been visualized, but the mechanism by which miR-30d becomes incorporated into exosomes remains unknown. The present study aimed to elucidate the underlying mechanism of hsa-miR-30d transfer from human endometrial epithelial cells (hEECs) to the interior of exosomes and eventually to early-stage blastocysts, using a mir-30d knockout murine mode.

Project description:Most cancer-related deaths are caused by distant metastases, which are tumour cells that have escaped from a primary tumour and passed into the bloodstream to colonize a new organ. In this context, communication between tumour and stromal cells is essential. Indeed, tumor cells interact with cells in the tumor microenvironment and are able to modify them to their advantage. Both extracellular vesicles (EVs) and exosomes are heterogeneous populations of small vesicles present in the tumor microenvironment and in body fluids that have recently emerged as powerful mediators involved in this communication and their transport in fluids. Tumor cells release large quantities of exosomes containing tumor markers, which can then spread to distant locations. The exosomes are of endosomal origin. They are composed of proteins, lipids, RNA and DNA, and they circulate in the bloodstream. They can be internalized by specific distant cells and thus deliver a functional message. It has recently been shown that tumor exosomes containing pro-metastatic factors form pre-metastatic niches, before the tumor cells actually arrive, while determining the metastatic organotropism of tumors. These properties are now opening up new avenues of research in tumor biomarkers. In recent years, several studies have highlighted different markers contained specifically in exosomes derived from cancer cells. Consequently, exosomes are considered as potential reservoirs of tumor biomarkers that could be clinically useful for the non-invasive diagnosis of cancer, with the advantage of being performed by liquid biopsy. The study of microRNA (miRNA) is of particular interest. Indeed, miRNAs are small non-coding RNAs (between 21 and 25 nucleotides) involved in the regulation of gene expression and which are frequently deregulated in cancer. Several studies underline that the variation of free miRNAs in the blood is correlated with the progression of the disease, particularly in colon cancer. However, the stability of free miRNAs is controversial. Therefore, exosomes represent a very advantageous means of transporting miRNAs in the blood, as they are able to protect miRNAs from degradation by RNAase. The hypothesis of the project is that circulating exosomes derived from tumours contain markers including specific miRNAs that could be used as biomarkers of early prognosis (survival and progression), easily measured in blood samples from patients with colon cancer. But other molecules contained in exosomes could also be of interest.