Circulating U2 small nuclear RNA fragments as a novel diagnostic biomarker for pancreatic and colorectal adenocarcinoma

Abstract

Improved non-invasive strategies for early cancer detection are urgently needed to reduce morbidity and mortality. Non-coding RNAs, such as microRNAs and small nucleolar RNAs, have been proposed as biomarkers for non-invasive cancer diagnosis. Analyzing serum derived from nude mice implanted with primary human pancreatic ductal adenocarcinoma (PDAC), we identified 15 diagnostic microRNA candidates. Of those miR-1246 was selected based on its high abundance in serum of tumor carrying mice. Subsequently, we noted a cross reactivity of the established miR-1246 assays with RNA fragments derived from U2 small nuclear RNA (RNU2-1). Importantly, we found that the assay signal discriminating tumor from controls was derived from U2 small nuclear RNA (snRNA) fragments (RNU2-1f) and not from miR-1246. In addition, we observed a remarkable stability of RNU2-1f in serum and provide experimental evidence that hsa-miR-1246 is likely a pseudo microRNA. In a next step, RNU2-1f was measured by qRT-PCR and normalized to cel-54 in 191 serum/plasma samples from PDAC and colorectal carcinoma (CRC) patients. In comparison to 129 controls, we were able to classify samples as cancerous with a sensitivity and specificity of 97.7% [95% CI 5 (87.7, 99.9)] and 90.6% [95% CI 5 (80.7, 96.5)], respectively [area under the ROC curve 0.972]. Of note, patients with CRC were detected with our assay as early as UICC Stage II with a sensitivity of 81%. In conclusion, this is the first report showing that fragments of U2 snRNA are highly stable in serum and plasma and may serve as novel diagnostic biomarker for PDAC and CRC for future prospective screening studies.

Circulating U2 Small Nuclear RNA Fragments as a Novel Diagnostic Tool for Patients with Epithelial Ovarian Cancer

“…Previous reports have shown that the Qiagen miR-1246 assay, when used for lysates of tumor cell lines, produces an amplicon of approximately 180 bp in size, corresponding to full-length RNU2-1 and not to its fragmented counterpart found in human serum (RNU2-1f) (16 ). We could confirm the formation of a 180-bp amplicon by this assay, when applied to ovarian cancer tissue (data not shown).…”

“…Absence of syn-cel-miR-54 signal in the human circulation was experimentally verified previously (9 ), and a previous investigation showed a linear correlation between the logarithm of the amount of syn-celmiR-54 input and the Cq for syn-cel-miR-54 as well as RNU2-1f (16 ). From these analyses, we verified the adjusted amount of spike-in (25 fmol per sample) to be in the linear amplification range of the assay (16 ). We determined a normalized Cq value for RNU2-1f relative to the syn-cel-miR-54 signal [Cq ϭ Cq(syn-celmiR-54) Ϫ Cq(RNU2-1f)].…”

“…Importantly, given that the putative miR-1246 is very likely a result of false mapping of RNU2-1f, all of the results reported herein concern circulating RNU2-1f rather than miR-1246 (16,18 ). In the present investigation, we observed significantly increased RNU2-1f concentrations in the patients' blood.…”

“…To relatively quantify RNU2-1f in human serum and tissue, we used the hsa-miR-1246 miScript assay (Qiagen), which has previously been shown to be specific for RNU2-1f in serum and for full-length U2-1 snRNA (RNU2-1) in a cellular context (16 ). We carried out all experimental steps according to the manufacturer's instructions.…”

Elevated Plasma miR-29a Levels Are Associated with Increased Carotid Intima-Media Thickness in Atherosclerosis Patients

“…We evaluated the expression of plasma miRs using the S-Poly (T) reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) method as previously described (Kang et al 2012), using miR-54 as a control (Baraniskin et al 2013;Huang et al 2016). The comparative cycle threshold (Ct) (ΔCt) was used to calculate the relative expression level of miRs.…”