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

David Danforth,Dong-Ja Kim,Lucy Pigati,Steven A. Hearn,Sree C. S. Yaddanapudi,Ravi Iyengar,Dominik M. Duelli,Michelle L. Hastings

doi:10.1371/journal.pone.0013515

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.

Highlights

MicroRNAs are small RNA molecules that are defined by structure, regulatory functions, and mode of biogenesis
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]
To assess if other human mammary epithelial cells release exosomes, we quantified the abundance of CD81 and CD63, an endosomal marker protein, in P70 preparations of breast cancer cell lines MDA-MB-231, BT-20, and the nontumorigenic mammary epithelial cell line MCF 10A

Summary

Introduction

MicroRNAs (miRNAs) are small RNA molecules that are defined by structure, regulatory functions, and mode of biogenesis. MiRNAs are produced as primary miRNA transcripts (pri-miRNA), which are processed by the Microprocessor complex that includes Drosha into pre-miRNA molecules. PremiRNAs are exported from the nucleus, and further processed by Dicer to yield mature miRNAs that associate with the RNAInduced Silencing Complex, RISC and target mRNA. Changes in the abundance of miRNAs have been documented in various diseases including malignancies such as breast cancer. The cellular miRNA composition has been explored for diagnosis and prognosis of breast cancer and other diseases [1,2,3,4,5,6,7,8,9,10]. Because upregulated miRNAs of lymphoma, prostate, lung and breast cancers have been detected in blood plasma and serum [11,12,13,14], circulating miRNAs are currently evaluated as surrogate biomarkers for breast cancer [14,15,16,17], other cancers [13,18,19,20], diseases or conditions [21]

Methods

Results

Conclusion