Abstract
IntroductionSerum microRNAs have the potential to be valuable biomarkers of cancer. This investigation addresses two issues that impact their utility: a) appropriate normalization controls and b) whether their altered levels persist in patients who are clinically free of the disease.MethodsSera from 40 age-matched healthy women and 39 breast cancer patients without clinical disease at the time of serum collection were analyzed for microRNAs let-7f, miR-16, miR-21 and miR-155 using quantitative real-time PCR. U6 and 5S, which are transcribed by RNA polymerase III (RNAP-III) and the small nucleolar RNU44 (SNORD44), were also analyzed for normalization. Significant results from the initial study were verified using a second set of sera from 15 healthy patients, 15 breast cancer patients without clinical disease and 15 with metastatic disease, and a third set of 12 healthy and 18 patients with metastatic disease. U6 was further verified in the extended second cohort of 75 healthy and 68 breast cancer patients without clinical disease.ResultsU6:SNORD44 ratio was consistently higher in breast cancer patients with or without active disease (fold change range 1.5-6.6, p value range 0.0003 to 0.05). This increase in U6:SNORD44 ratio was observed in the sera of both estrogen receptor-positive (ER+) and ER-negative breast cancer patients. MiR-16 and 5S, which are often used as normalization controls for microRNAs, showed remarkable experimental variability and thus are not ideal for normalization.ConclusionsElevated serum U6 levels in breast cancer patients irrespective of disease activity at the time of serum collection suggest a new paradigm in cancer; persistent systemic changes during cancer progression, which result in elevated activity of RNAP-III and/or the stability/release pathways of U6 in non-cancer tissues. Additionally, these results highlight the need for developing standards for normalization between samples in microRNA-related studies for healthy versus cancer and for inter-laboratory reproducibility. Our studies rule out the utility of miR-16, U6 and 5S RNAs for this purpose.
Highlights
Serum microRNAs have the potential to be valuable biomarkers of cancer
MiRNA and small RNA expression analyses Earlier studies showing the presence of miR-21 and miR-155 in the serum/plasma of patients with cancer [25,26] prompted us to evaluate their levels in the sera of healthy subjects and patients with breast cancer
U6, 5S, miR-16, RNU66, RNU49, RNU19, and small nucleolar RNA 44 (SNORD44) levels were analyzed in these samples to identify a small RNA expressed at a similar level in equal volume of sera from both healthy subjects and patients with cancer to serve as a normalization control
Summary
Serum microRNAs have the potential to be valuable biomarkers of cancer. This investigation addresses two issues that impact their utility: a) appropriate normalization controls and b) whether their altered levels persist in patients who are clinically free of the disease. Approximately 940 miRNAs have been described [3] Their functions include epigenetic control of gene expression, mRNA degradation, and suppression of mRNA translation [4]. These diverse functions of miRNAs are necessary for normal development, metabolism, cellular differentiation, proliferation, cell cycle control, and cell death. Secreted miRNAs have been shown to be in the Argonaute protein complex, which confers stability [10]. These secreted miRNAs are transported through high-density lipoprotein (HDL) and enter heterotypic cells to alter migration/invasive properties [7,8,11,12,13]. Most of which are RNAbinding proteins, are suggested to be involved in exosomal secretion of miRNAs and their stability in circulation [15]
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