Persistent upregulation of U6:SNORD44 small RNA ratio in the serum of breast cancer patients

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.

Circulating Serum Exosomal miRNAs As Potential Biomarkers for Esophageal Adenocarcinoma

“…The spliceosome complex, which catalyzes pre-mRNA splicing, and in which U6 is a key functional component, is frequently deregulated in a wide variety of cancers [30]. Consistent with this and with our findings, it has been shown that serum RNU6-1 levels are differentially expressed in serum from individuals with breast cancer compared to healthy controls [31]. In addition, a recent miRNA profiling study performed on the serum exosomes in individuals with glioblastoma identified RNU6-1 as a potential diagnostic cancer biomarker [32].…”

The Oncogenic Role of miR-155 in Breast Cancer

“…The relationship of PR status and miR-155 expression is unresolved, with 2 studies reporting contradictory results (27,29). The topic of serum miRNAs is also somewhat controversial, with some studies suggesting that serum miRNA levels are robust (35,36), and others claiming that the miRNAs often used as normalization controls are highly variable in sera samples and thus miRNA quantification in sera is not reproducible (37). This suggests analysis of serum alone is not sufficient to determine whether miR-155 is differentially expressed.…”

The R2TP chaperone complex: its involvement in snoRNP assembly and tumorigenesis

“…Intriguingly, in TIC xenografts of mice, knock down of SNORA42 resulted in a decrease of tumorigenesis (46). In other cancer cell lines, it has been identified that SNORD33, SNORD44, SNORD66, and SNORD76 are upregulated, and a depletion of their expression decreases both cancer cell growth and colony formation (47,48). Regarding snoRNA downregulation that is related to tumorigenesis, human U50 box C/D snoRNA (also called SNORD50) is mutated and downregulated in prostate and breast cancers (49,50).…”