Étude de l'impact des microARNs sur la carcinogenèse des cancers colorectaux instables sur les séquences répétées microsatellites du génome

La progression tumorale MSI (Microsatellite Instable) est un processus multi-étapes résultant de mutations générées par un processus d'instabilité génétique qui affecte en majorité les motifs répétés en tandem de l'ADN (microsatellites). Ces mutations contribuent à l'oncogenèse lorsqu'elles perturbent la fonction d'oncogènes ou de gènes suppresseurs de tumeurs. Le trait phénotypique MSI est consécutif à l'inactivation du système de réparation des mésappariements de l'ADN (système MMR). Dans ce travail, je me suis intéressé au rôle des microARNs dans l'oncogenèse MSI. Les microARNs régulent l'expression de nombreux gènes pouvant avoir un rôle clé dans le cancer. J'ai donc fait l'hypothèse d'un rôle de ces microARNs lors des différentes étapes du processus tumorigénique MSI. Tout d'abord nous avons mis en évidence une surexpression du miR-155 (ciblant les principales protéines MMR) au niveau de la muqueuse colique non transformée des malades atteints d'une Maladie Inflammatoire Chronique Intestinale, qui pourrait constituer un évènement pré-tumoral favorisant l'émergence de clones MMR-déficients (notion d'effet de champs). Dans une deuxième partie, nous avons pu identifier la première mutation somatique touchant un microARN. Il s'agit du miR-3613 dont la répétition microsatellite est entièrement localisée dans le miR mature. L'instabilité au niveau de ce miR conduit à des changements de séquence à l'extrémité 3' du miR (notion d'IsomiRs). Les isomiRs produits ont un répertoire de cibles qui pour certaines sont communes à la forme sauvage et pour d'autres spécifiques à chacun des variants.MSI tumor progression (Microsatellite Instability) is depicted as a multistage process that results from mutations generated by a process of genetic instability affecting mostly DNA tandem repeats (known as microsatellites). These mutations contribute to tumorigenesis when they disrupt the function of oncogenes or tumor suppressor genes. As a phenotypic trait, MSI is the consequence of DNA mismatch repair inactivation (MMR). This work focused on the role microRNAs might play in MSI tumorigenesis. MicroRNAs regulate the expression of numerous genes and are deregulated in cancer. I have hypothesized a role of theses microRNAs during the various stages of the MSI tumorigenic process, choosing colorectal cancers (CRC) as a working model. First we demonstrated that overexpression of miR-155 (targeting core MMR proteins) in the non-transformed colonic mucosa of patients with Inflammatory Bowel Disease, might constitute a pre-tumoral event promoting the emergence of MMR-deficient clones (a concept known as ?field effect?). In a second part, we were able to identify the first somatic mutation affecting a mature microRNA sequence. A DNA microsatellite repeat is indeed fully embedded within the mature sequence of miR-3613. Instability at this DNA repeat leads to sequence modifications at the 3?end of miR-3613-5p (IsomiRs). IsomiRs display a signature among which some mRNA targets are common to the wild form, while others are specific to each variant.PARIS-JUSSIEU-Bib.électronique (751059901) / SudocSudocFranceF

MiRNA Genes Constitute New Targets for Microsatellite Instability in Colorectal Cancer

Mismatch repair-deficient colorectal cancers (CRC) display widespread instability at DNA microsatellite sequences (MSI). Although MSI has been reported to commonly occur at coding repeats, leading to alterations in the function of a number of genes encoding cancer-related proteins, nothing is known about the putative impact of this process on non-coding microRNAs. In miRbase V15, we identified very few human microRNA genes with mono- or di-nucleotide repeats (n = 27). A mutational analysis of these sequences in a large series of MSI CRC cell lines and primary tumors underscored instability in 15 of the 24 microRNA genes successfully studied at variable frequencies ranging from 2.5% to 100%. Following a maximum likelihood statistical method, microRNA genes were separated into two groups that differed significantly in their mutation frequencies and in their tendency to represent mutations that may or may not be under selective pressures during MSI tumoral progression. The first group included 21 genes that displayed no or few mutations in CRC. The second group contained three genes, i.e., hsa-mir-1273c, hsa-mir-1303 and hsa-mir-567, with frequent (≥80%) and sometimes bi-allelic mutations in MSI tumors. For the only one expressed in colonic tissues, hsa-mir-1303, no direct link was found between the presence or not of mono- or bi-allelic alterations and the levels of mature miR expression in MSI cell lines, as determined by sequencing and quantitative PCR respectively. Overall, our results provide evidence that DNA repeats contained in human miRNA genes are relatively rare and preserved from mutations due to MSI in MMR-deficient cancer cells. Functional studies are now required to conclude whether mutated miRNAs, and especially the miR-1303, might have a role in MSI tumorigenesis

NRAS Mutation Is the Sole Recurrent Somatic Mutation in Large Congenital Melanocytic Nevi

Congenital melanocytic nevus (CMN) is a particular melanocytic in utero proliferation characterized by an increased risk of melanoma transformation during infancy or adulthood. NRAS and BRAF mutations have consistently been reported in CMN samples, but until recently results have been contradictory. We therefore studied a series of large and giant CMNs and compared them with small and medium CMNs using Sanger sequencing, pyrosequencing, high-resolution melting analysis, and mutation enrichment by an enhanced version of ice-COLD-PCR. Large–giant CMNs displayed NRAS mutations in 94.7% of cases (18/19). At that point, the role of additional mutations in CMN pathogenesis had to be investigated. We therefore performed exome sequencing on five specimens of large–giant nevi. The results showed that NRAS mutation was the sole recurrent somatic event found in such melanocytic proliferations. The genetic profile of small–medium CMNs was significantly different, with 70% of cases bearing NRAS mutations and 30% showing BRAF mutations. These findings strongly suggest that NRAS mutations are sufficient to drive melanocytic benign proliferations in utero

Étude de l'impact des microARNs sur la carcinogenèse des cancers colorectaux instables sur les séquences répétées microsatellites du génome

MSI tumor progression (Microsatellite Instability) is depicted as a multistage process that results from mutations generated by a process of genetic instability affecting mostly DNA tandem repeats (known as microsatellites). These mutations contribute to tumorigenesis when they disrupt the function of oncogenes or tumor suppressor genes. As a phenotypic trait, MSI is the consequence of DNA mismatch repair inactivation (MMR). This work focused on the role microRNAs might play in MSI tumorigenesis. MicroRNAs regulate the expression of numerous genes and are deregulated in cancer. I have hypothesized a role of theses microRNAs during the various stages of the MSI tumorigenic process, choosing colorectal cancers (CRC) as a working model. First we demonstrated that overexpression of miR-155 (targeting core MMR proteins) in the non-transformed colonic mucosa of patients with Inflammatory Bowel Disease, might constitute a pre-tumoral event promoting the emergence of MMR-deficient clones (a concept known as ?field effect?). In a second part, we were able to identify the first somatic mutation affecting a mature microRNA sequence. A DNA microsatellite repeat is indeed fully embedded within the mature sequence of miR-3613. Instability at this DNA repeat leads to sequence modifications at the 3?end of miR-3613-5p (IsomiRs). IsomiRs display a signature among which some mRNA targets are common to the wild form, while others are specific to each variant.La progression tumorale MSI (Microsatellite Instable) est un processus multi-étapes résultant de mutations générées par un processus d'instabilité génétique qui affecte en majorité les motifs répétés en tandem de l'ADN (microsatellites). Ces mutations contribuent à l'oncogenèse lorsqu'elles perturbent la fonction d'oncogènes ou de gènes suppresseurs de tumeurs. Le trait phénotypique MSI est consécutif à l'inactivation du système de réparation des mésappariements de l'ADN (système MMR). Dans ce travail, je me suis intéressé au rôle des microARNs dans l'oncogenèse MSI. Les microARNs régulent l'expression de nombreux gènes pouvant avoir un rôle clé dans le cancer. J'ai donc fait l'hypothèse d'un rôle de ces microARNs lors des différentes étapes du processus tumorigénique MSI. Tout d'abord nous avons mis en évidence une surexpression du miR-155 (ciblant les principales protéines MMR) au niveau de la muqueuse colique non transformée des malades atteints d'une Maladie Inflammatoire Chronique Intestinale, qui pourrait constituer un évènement pré-tumoral favorisant l'émergence de clones MMR-déficients (notion d'effet de champs). Dans une deuxième partie, nous avons pu identifier la première mutation somatique touchant un microARN. Il s'agit du miR-3613 dont la répétition microsatellite est entièrement localisée dans le miR mature. L'instabilité au niveau de ce miR conduit à des changements de séquence à l'extrémité 3' du miR (notion d'IsomiRs). Les isomiRs produits ont un répertoire de cibles qui pour certaines sont communes à la forme sauvage et pour d'autres spécifiques à chacun des variants

Study of the impact of microARNs on the carcinogenèse of cancers unstable colorectaux on repeated sequences microsatellites of the genome

La progression tumorale MSI (Microsatellite Instable) est un processus multi-étapes résultant de mutations générées par un processus d'instabilité génétique qui affecte en majorité les motifs répétés en tandem de l'ADN (microsatellites). Ces mutations contribuent à l'oncogenèse lorsqu'elles perturbent la fonction d'oncogènes ou de gènes suppresseurs de tumeurs. Le trait phénotypique MSI est consécutif à l'inactivation du système de réparation des mésappariements de l'ADN (système MMR). Dans ce travail, je me suis intéressé au rôle des microARNs dans l'oncogenèse MSI. Les microARNs régulent l'expression de nombreux gènes pouvant avoir un rôle clé dans le cancer. J'ai donc fait l'hypothèse d'un rôle de ces microARNs lors des différentes étapes du processus tumorigénique MSI. Tout d'abord nous avons mis en évidence une surexpression du miR-155 (ciblant les principales protéines MMR) au niveau de la muqueuse colique non transformée des malades atteints d'une Maladie Inflammatoire Chronique Intestinale, qui pourrait constituer un évènement pré-tumoral favorisant l'émergence de clones MMR-déficients (notion d'effet de champs). Dans une deuxième partie, nous avons pu identifier la première mutation somatique touchant un microARN. Il s'agit du miR-3613 dont la répétition microsatellite est entièrement localisée dans le miR mature. L'instabilité au niveau de ce miR conduit à des changements de séquence à l'extrémité 3' du miR (notion d'IsomiRs). Les isomiRs produits ont un répertoire de cibles qui pour certaines sont communes à la forme sauvage et pour d'autres spécifiques à chacun des variants.MSI tumor progression (Microsatellite Instability) is depicted as a multistage process that results from mutations generated by a process of genetic instability affecting mostly DNA tandem repeats (known as microsatellites). These mutations contribute to tumorigenesis when they disrupt the function of oncogenes or tumor suppressor genes. As a phenotypic trait, MSI is the consequence of DNA mismatch repair inactivation (MMR). This work focused on the role microRNAs might play in MSI tumorigenesis. MicroRNAs regulate the expression of numerous genes and are deregulated in cancer. I have hypothesized a role of theses microRNAs during the various stages of the MSI tumorigenic process, choosing colorectal cancers (CRC) as a working model. First we demonstrated that overexpression of miR-155 (targeting core MMR proteins) in the non-transformed colonic mucosa of patients with Inflammatory Bowel Disease, might constitute a pre-tumoral event promoting the emergence of MMR-deficient clones (a concept known as ?field effect?). In a second part, we were able to identify the first somatic mutation affecting a mature microRNA sequence. A DNA microsatellite repeat is indeed fully embedded within the mature sequence of miR-3613. Instability at this DNA repeat leads to sequence modifications at the 3?end of miR-3613-5p (IsomiRs). IsomiRs display a signature among which some mRNA targets are common to the wild form, while others are specific to each variant

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

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

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

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

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