Secondary structures of WT and mutated <i>hsa-mir-1303</i> and expression levels of miR-1303 in CRC cell lines.

<p>A: Alterations in repeat sequences of <i>hsa-mir-1303</i> (A) and its variant (delA) did not seem to affect overall the secondary structure of the hairpin but the dimension of the loop (annoted inside) is slightly reduced as determined by mfold software (<a href="http://mfold.rna.albany.edu/" target="_blank">http://mfold.rna.albany.edu/</a>). Mature miR (bold letters) and MNR (underlined letters) are shown in both hairpin sequences. The arrows indicate the potential positions of an Adenine deletion that leads to an enlargement of the loop. B: Comparison of the relative expressions of mature miR-1303 in MSS (unaltered MNR) and MSI CRC cell lines with none, mono- or bi-allelic mutations of <i>hsa-mir-1303</i>. MiR expression was normalized to the expression of RNU48. Means are shown for each group (black horizontal line). A significant increase in the expression of miR-1303 was observed between MSS cell lines and normal colonic mucosae (<i>p</i> = 0.012). C: Absence of correlation between the size of mir-1303 loop and the levels of mature miR-1303 expression in MSI cell lines with no (HCT-8, TC7) or bi-allelic mutations (LS411, RKO, LIM2405, KM12, LoVo, HCT116) in MNR of <i>hsa-mir-1303</i>. Note cell lines that produce hairpin precursors with the same size of the loop do express mature miR-1303 at various levels.</p

MNR instabilities in <i>hsa-mir-1273c</i> (T11), <i>hsa-mir-567</i> (A13) and <i>hsa-mir-1303</i> (T13).

<p>Allelic profiles for several MSI CRC cell lines and primary tumors are shown. Normal profiles are defined in LBL and MSS cell lines and primary tumors. For monomorphic genes, a dashed vertical line indicates the unique allele. The polymorphic zone for <i>hsa-mir-1303</i> is defined between two dashed vertical lines going along the 2 alleles (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031862#pone.0031862.s001" target="_blank">Figure S1</a>). Sizes (bp) are indicated in a box below each profile. Various allelic deletions ranging from 1 to 4 bp were observed in MSI CRC cell lines and primary tumors and are indicated in bold. The observed deletions were sometimes bi-allelic in MSI CRC cell lines. In MSI primary tumors, the allelic profiles were also highly suggestive of bi-allelic mutations. Due to the inherent polymorphism that can modify the length of the sequence, the hairpin sequence of <i>hsa-mir-1303</i> was determined for a correct and reliable evaluation of the alterations in MSI CRC cell lines (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031862#pone.0031862.s005" target="_blank">Table S2</a>).</p

Representative scheme of miRNA hairpins with repeats spaning different locations.

<p>The basal segment (BS, single-stranded RNA), stem (S, double-stranded RNA) and terminal loop (L) are designated. The duplex (D, containing one or two potential miRs) is considered as a different entity and therefore distinguished from the stem region. Regions of the hairpin covered by MNRs or DNRs are noted for each miRNAs. To the left of the scheme are miRNA genes whose sequence repeats overlap two regions.</p

Classification of miRNAs with MNR according to their mutation frequencies in MSI CRCs.

<p>Two distinct groups of miRNAs with MNR are established based on their mutation frequencies in MSI primary tumors. The cut-off value is calculated by the ratio of likelihood statistical method and is marked by a dashed vertical line. Note that <i>hsa-mir-644</i> is included in the group of miRNAs rarely or not mutated in MSI CRCs (<i>n</i> = 18, frequency of mutation <25%) whereas <i>hsa-mir-1273c</i>, <i>hsa-mir-567</i> and <i>hsa-mir-1303</i> constitute the group of miRNAs frequently altered (<i>n</i> = 3, frequency of mutation >75%).</p

Correlation between the lengths of mononucleotide repeats in miRNAs and their mutation rates in MSI CRCs.

<p>Note the highly significant correlation observed.</p

Polymorphism and somatic mutation frequency of microsatellite repeats in miRNA genes.

<p>NS, not significant;</p>a<p>the polymorphism rate is the percentage of normal samples showing length variations when compared to the major peak (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031862#pone.0031862.s004" target="_blank">Table S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031862#pone.0031862.s001" target="_blank">Figure S1</a>);</p>b<p>mutation rates were estimated by taking into account sizes that diverge from the normal polymorphism (refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031862#pone.0031862.s001" target="_blank">Figure S1</a>).</p