Alternative Processing of the U2 Small Nuclear RNA Produces a 19–22nt Fragment with Relevance for the Detection of Non-Small Cell Lung Cancer in Human Serum

<div><p>RNU2 exists in two functional forms (RNU2-1 and RNU2-2) distinguishable by the presence of a unique 4-bases motif. Detailed investigation of datasets obtained from deep sequencing of five human lung primary tumors revealed that both forms express at a high rate a 19–22nt fragment (miR-U2-1 and -2) from its 3′ region and contains the 4-bases motif. Deep sequencing of independent pools of serum samples from healthy donors and lung cancer patients revealed that miR-U2-1 and -2 are pervasively processed in lung tissue by means of endonucleolytic cleavages and stably exported to the blood. Then, microarrays hybridization experiments of matched normal/tumor samples revealed a significant over-expression of miR-U2-1 in 14 of 18 lung primary tumors. Subsequently, qRT-PCR of miR-U2-1 using serum from 62 lung cancer patients and 96 various controls demonstrated that its expression levels identify lung cancer patients with 79% sensitivity and 80% specificity. miR-U2-1 expression correlated with the presence or absence of lung cancer in patients with chronic obstructive pulmonary disease (COPD), other diseases of the lung – not cancer, and in healthy controls. These data suggest that RNU2-1 is a new bi-functional ncRNA that produces a 19–22nt fragment which may be useful in detecting lung cancer non-invasively in high risk patients.</p> </div

Microarrays profiling of miR-U2-1.

<p>In a) are indicated the three probes used for detecting miR-U2-1. The boxed region corresponds to the miR-U2-1 longest and most expressed isoforms; upper case letters indicate nucleotides belonging to miR-U2-1, while lower case letters are for nucleotides belonging to the RNU2-1 gene. The red nucleotide points on the difference between miR-1290 and miR-U2-1; b) gives the fold changes calculated between primary tumors and matched normal tissue (column “matched”) or between primary tumors and the averaged value of all normal tissues (column “All”).</p

Demographic and histopathologic data for lung cancer patients enrolled in the microarray-based discovery cohort.

<p>ANT stands for Adjacent Normal Tissue. The number between parentheses indicates primary tumors which were also sequenced: (1) short size fragments (16–30nt long); (2) long size fragments (25–50nt long).</p

Anova of miR-U2 in the serum of the cohort.

<p>“0” stands for the “healthy” group of control individuals; “1” for the “lung diseases not-cancer not-COPD” group; “4” for the “COPD not-lung cancer” group; “5” for COPD patients with a lung cancer; “6” for lung cancer patients without COPD.</p

Normalized number of reads mapped along the RNU2-1 gene.

<p>Small reads detected in Primary tumors (a) and serum specimens (c). Long-reads detected in Primary tumors (e). The color code shows the number of normalized reads detected in each library. The red line locates miR-U2. The number of reads according to their size is given in (b) for the primary tumors, in (d) for the serum specimens.</p

Roc (Receiving Operator Curves) of miR-U2 in the serum of the cohort.

<p>“0” stands for control individuals, “1” for lung cancer patients.</p

Overview of demographic and histopathologic data for serum samples included in the cohort.

<p>Detailed information including Ct values is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060134#pone.0060134.s010" target="_blank">Table S3</a>.</p

Most represented isoforms.

<p>(a) Indicates the sequence of each read detected and the normalized number of each read is given for each tumor specimen. (b) The total number of each type of read is shown as an histogram. Histograms (c) and (d) display the number of reads at the diverse 5′ starts and 3′ ends of miR-U2 respectively.</p

Simplified diagram illustrates the genomic location of miR-4772 family on Chromosome q12.1 within intron 5 of IL-18 RAP.

<p>Simplified diagram illustrates the genomic location of miR-4772 family on Chromosome q12.1 within intron 5 of IL-18 RAP.</p

Demographic and clinical information for patients used for sequencing.

<p>Data are presented as numbers (percentages) for categorical variables and as median values (ranges) for continuous variables. Statistics: The distribution of sex was tested using Pearson Chi-Square Tests, the rest variables were tested using Kruskal-Wallis ANOVA test.</p