Abstract
Extracellular circulating microRNAs (miRNAs) have been suggested to be biomarkers for disease monitoring but data are inconsistent, one reason being that blood miRNA is of heterogeneous origin. Here, we sampled extracellular microRNAs locally in situ using microdialysis. Three different cohorts of women were included; postmenopausal women with ongoing breast cancer investigated within the cancer and in normal adjacent breast tissue, postmenopausal women investigated in their normal healthy breast and subcutaneous fat before and after six weeks of tamoxifen therapy, premenopausal women during the menstrual cycle. Samples were initially screened using TaqMan array cards with subsequently absolute quantification. 124 miRNA were expressed in microdialysates. After absolute quantifications extracellular miRNA-21 was found to be significantly increased in breast cancer. In addition, the levels were significantly higher in pre-menopausal breast tissue compared with postmenopausal. In breast tissue of pre-menopausal women miRNA-21 exhibited a cyclic variation during the menstrual cycle and in postmenopausal women six weeks of tamoxifen treatment decreased miRNA-21 suggesting that this miRNA may be important for breast carcinogenesis. None of these changes were found in plasma or microdialysates from subcutaneous fat. Our data revealed tissue specific changes of extracellular circulating miRNAs that would be otherwise unraveled using blood samples.
Highlights
MicroRNAs are small endogenous noncoding molecules of approximately 21-25 nucleotides that regulate transcription of numerous genes by binding to complementary sequences on the 3 ́un-translated regions (3 ́UTR) of the messenger RNA molecule [1]
We show that extracellular miRNAs in microdialysis samples are stable and exhibit very low extraction variability and that the transport proteins pass the microdialysis membrane. 124 miRNA were expressed in microdialysates of human breast cancer
Out of the 377 miRNAs on the card, 124 were expressed in the samples i.e. exhibited a cycle threshold (CT)-value less than 35, Supplementary Table S1. Out of these 124, 9 miRNAs previously reported to be involved in cancer progression and/or were readily detectable in the microdialysis samples during the screening process were chosen for absolute quantification using real-time PCR with serial diluted synthetic miRNAs as standard curves
Summary
MicroRNAs (miRNAs) are small endogenous noncoding molecules of approximately 21-25 nucleotides that regulate transcription of numerous genes by binding to complementary sequences on the 3 ́un-translated regions (3 ́UTR) of the messenger RNA molecule [1]. Since the discovery of circulating miRNAs in different body fluids it has been suggested that miRNAs act within the cells, but are exported into the extracellular space [3, 4]. These extracellular circulating miRNAs has been shown to be transported in membrane bound vesicles or in different protein-complexes including nucleophosmin 1 (NPM1) and argonaute 2 protein (Ago 2) [4,5,6]. As there are no specific endogenous control for extracellular miRNAs a fixed volume rather than a fixed amount of RNA should be used as an input as recently described [9]
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