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.     

Genetic recombination destabilizes (CTG)n.(CAG)n repeats in E. coli

The expansion of trinucleotide repeats has been implicated in 17 neurological diseases to date. Factors leading to the instability of trinucleotide repeat sequences have thus been an area of intense interest. Certain genes involved in mismatch repair, recombination, nucleotide excision repair, and replication influence the instability of trinucleotide repeats in both Escherichia coli and yeast. Using a genetic assay for repeat deletion in E. coli, the effect of mutations in the recA, recB, and lexA genes on the rate of deletion of (CTG)n.(CAG)n repeats of varying lengths were examined. The results indicate that mutations in recA and recB, which decrease the rate of recombination, had a stabilizing effect on (CAG)n.(CTG)n repeats decreasing the high rates of deletion seen in recombination proficient cells. Thus, recombination proficiency correlates with high rates of genetic instability in triplet repeats. Induction of the SOS system, however, did not appear to play a significant role in repeat instability, nor did the presence of triplet repeats in cells turn on the SOS response. A model is suggested where deletion during exponential growth may result from attempts to restart replication when paused at triplet repeats.     

Target genes of microsatellite sequences in head and neck squamous cell carcinoma: Mononucleotide repeats are not detected

Microsatellite instability (MSI) is detected in a wide variety of tumors. It is thought that mismatch repair gene mutation or inactivation is the major cause of MSI. Microsatellite sequences are predominantly distributed in intergenic or intronic DNA. However, MSI is found in the exonic sequences of some genes, causing their inactivation. In this report, we searched GenBank for candidate genes containing potential MSI sequences in exonic regions. Twenty seven target genes were selected for MSI analysis. Instability was found in 70% of these genes (14/20) with head and neck squamous cell carcinoma (HNSCC). Interestingly, no instability was detected in mononucleotide repeats in genes or in intergenic sequences. We conclude that instability of mononucleotide repeats is a rare event in HNSCC. High MSI phenotype in young HNSCC patients is limited to noncoding regions only. MSI percentage in HNSCC tumor is closely related to the repeat type, repeat location and patient's age.     

Neurofibromatosis type 1 gene as a mutational target in a mismatch repair-deficient cell type

DNA mismatch repair (MMR) is the process by which incorrectly paired DNA nucleotides are recognized and repaired. A germline mutation in one of the genes involved in the process may be responsible for a dominantly inherited cancer syndrome, hereditary nonpolyposis colon cancer. Cancer progression in predisposed individuals results from the somatic inactivation of the normal copy of the MMR gene, leading to a mutator phenotype affecting preferentially repeat sequences (microsatellite instability, MSI). Recently, we identified children with a constitutional deficiency of MMR activity attributable to a mutation in the h MLH1 gene. These children exhibited a constitutional genetic instability associated with clinical features of de novo neurofibromatosis type 1 (NF1) and early onset of extracolonic cancer. Based on these observations, we hypothesized that somatic NF1 gene mutation was a frequent and possibly early event in MMR-deficient cells. To test this hypothesis, we screened for NF1 mutations in cancer cells. Genetic alterations were identified in five out of ten tumor cell lines with MSI, whereas five MMR-proficient tumor cell lines expressed a wild-type NF1 gene. Somatic NF1 mutations were also detected in two primary tumors exhibiting an MSI phenotype. Finally, a 35-bp deletion in the murine Nf1 coding region was identified in mlh1-/- mouse embryonic fibroblasts. These observations demonstrate that the NF1 gene is a mutational target of MMR deficiency and suggest that its inactivation is an important step of the malignant progression of MMR-deficient cells.     

用一个简单快速的RT-PCR技术筛选白细胞介素-6作用相关基因

为了研究白细胞介素-6(IL-6)作用相关基因以及一些可能受IL-6调控的基因,利用一个简单快速的以PCR为基础的方案,检测了IL-6处理和未处理的Sko007细胞中基因表达的差异,克隆并鉴定了差异表达基因的cDNA片段.首先用6-mer寡核苷酸引物进行反转录从而最大限度地将mRNA编码区序列生成cDNA;然后用2或3个较长的随机引物进行PCR扩增,并以不同引物组合重复PCR增扩;扩增产物在2%琼脂糖凝胶上电泳分离,回收差异片段并直接用于克隆、测序及进一步分析.在此研究中,获得了3个表达序列标签(EST),其中一个为新的基因片段,反向RNA杂交有力证实了它们与IL-6作用的相关性.进一步的生物信息学分析表明,新基因片段STRF17在多种组织中表达.     

Cloning and sequencing of the cDNA encoding an isoform of microtubule-associated protein tau containing four tandem repeats: differential expression of tau protein mRNAs in human brain.

We have isolated cDNA clones encoding a 383-amino acid isoform of the human microtubule-associated protein tau. It differs from previously determined tau sequences by the presence of an additional repeat of 31 amino acids, giving four, rather than three, tandem repeats in its carboxy-terminal half. The extra repeat is encoded by a separate exon. Probes derived from cDNA clones encoding the three (type I) and four repeat (type II) tau protein isoforms detected mRNAs for both forms in all adult human brain areas examined. However, in foetal brain only type I mRNA was found. Type I and type II mRNAs were present in pyramidal cells in cerebral cortex. In the hippocampal formation, type I mRNA was found in pyramidal and granule cells; type II mRNA was detected in most, though not all, pyramidal cells but not in granule cells. These observations indicate that tau protein mRNAs are expressed in a stage- and cell-specific manner. Tau protein is found in the protease-resistant core of the paired helical filament, the major constituent of the neurofibrillary tangle in Alzheimer's disease. Taken in conjunction with previous findings, the present results indicate that both the three and four repeat-containing tau protein isoforms are present in the core of the paired helical filament.     

Instability of CAG and CTG trinucleotide repeats in Saccharomyces cerevisiae.

A quantitative genetic assay was developed to monitor alterations in tract lengths of trinucleotide repeat sequences in Saccharomyces cerevisiae. Insertion of (CAG)50 or (CTG)50 repeats into a promoter that drives expression of the reporter gene ADE8 results in loss of expression and white colony color. Contractions within the trinucleotide sequences to repeat lengths of 8 to 38 restore functional expression of the reporter, leading to red colony color. Reporter constructs including (CAG)50 or (CTG)50 repeat sequences were integrated into the yeast genome, and the rate of red colony formation was measured. Both orientations yielded high rates of instability (4 x 10(-4) to 18 x 10(-4) per cell generation). Instability depended on repeat sequences, as a control harboring a randomized (C,A,G)50 sequence was at least 100-fold more stable. PCR analysis of the trinucleotide repeat region indicated an excellent correlation between change in color phenotype and reduction in length of the repeat tracts. No preferential product sizes were observed. Strains containing disruptions of the mismatch repair gene MSH2, MSH3, or PMS1 or the recombination gene RAD52 showed little or no difference in rates of instability or distributions of products, suggesting that neither mismatch repair nor recombination plays an important role in large contractions of trinucleotide repeats in yeast.     

桉树EST序列中微卫星含量及相关特征

通过对桉树属（Eucalyptus）的10000条EST序列进行分析,在其中的1499条序列上共发现1775个微卫星重复序列。含有微卫星的EST序列约占序列总数的15%。此外,还发现桉树EST序列所含微卫星长度的变异速率与重复单元长度呈负相关;微卫星的丰度与重复单元长度也呈负相关（三碱基重复微卫星除外）。在桉树EST序列中,重复单元长度为三碱基的微卫星最为丰富。三碱基重复单元微卫星的过度富集可能是由于遗传密码选择所致。在微卫星的丰度及长度变异方面,桉树EST序列与杨树（Populus trichocarpa）基因组注释的转录序列随重复单元长度的变化呈现出相同的规律,但桉树EST序列中微卫星频率及三碱基重复微卫星的含量显著偏低,推测含微卫星的基因表达丰度极有可能低于不含微卫星的基因。通过对发现的所有微卫星位点进行引物设计,并对设计的引物进行PCR检测,结果表明所设计的引物具有极高的扩增成功率。     

桉树EST序列中微卫星含量及相关特征

通过对桉树属(Eucalyptus)的10 000条EST序列进行分析, 在其中的1 499条序列上共发现1 775个微卫星重复序列。含有微卫星的EST序列约占序列总数的15%。此外, 还发现桉树EST序列所含微卫星长度的变异速率与重复单元长度呈负相关; 微卫星的丰度与重复单元长度也呈负相关(三碱基重复微卫星除外)。在桉树EST序列中, 重复单元长度为三碱基的微卫星最为丰富。三碱基重复单元微卫星的过度富集可能是由于遗传密码选择所致。在微卫星的丰度及长度变异方面, 桉树EST序列与杨树(Populus trichocarpa)基因组注释的转录序列随重复单元长度的变化呈现出相同的规律, 但桉树EST序列中微卫星频率及三碱基重复微卫星的含量显著偏低, 推测含微卫星的基因表达丰度极有可能低于不含微卫星的基因。通过对发现的所有微卫星位点进行引物设计, 并对设计的引物进行PCR检测, 结果表明所设计的引物具有极高的扩增成功率。     

A survey of tandem repeat instabilities and associated gene expression changes in 35 colorectal cancers

Background

Colorectal cancer is a major contributor to cancer morbidity and mortality. Tandem repeat instability and its effect on cancer phenotypes remain so far poorly studied on a genome-wide scale.

Results

Here we analyze the genomes of 35 colorectal tumors and their matched normal (healthy) tissues for two types of tandem repeat instability, de-novo repeat gain or loss and repeat copy number variation. Specifically, we study for the first time genome-wide repeat instability in the promoters and exons of 18,439 genes, and examine the association of repeat instability with genome-scale gene expression levels. We find that tumors with a microsatellite instable (MSI) phenotype are enriched in genes with repeat instability, and that tumor genomes have significantly more genes with repeat instability compared to healthy tissues. Genes in tumor genomes with repeat instability in their promoters are significantly less expressed and show slightly higher levels of methylation. Genes in well-studied cancer-associated signaling pathways also contain significantly more unstable repeats in tumor genomes. Genes with such unstable repeats in the tumor-suppressor p53 pathway have lower expression levels, whereas genes with repeat instability in the MAPK and Wnt signaling pathways are expressed at higher levels, consistent with the oncogenic role they play in cancer.

Conclusions

Our results suggest that repeat instability in gene promoters and associated differential gene expression may play an important role in colorectal tumors, which is a first step towards the development of more effective molecular diagnostic approaches centered on repeat instability.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1902-9) contains supplementary material, which is available to authorized users.     

Nonsense-mediated mRNA decay impacts MSI-driven carcinogenesis and anti-tumor immunity in colorectal cancers

Nonsense-mediated mRNA Decay (NMD) degrades mutant mRNAs containing premature termination codon (PTC-mRNAs). Here we evaluate the consequence of NMD activity in colorectal cancers (CRCs) showing microsatellite instability (MSI) whose progression is associated with the accumulation of PTC-mRNAs encoding immunogenic proteins due to frameshift mutations in coding repeat sequences. Inhibition of UPF1, one of the major NMD factors, was achieved by siRNA in the HCT116 MSI CRC cell line and the resulting changes in gene expression were studied using expression microarrays. The impact of NMD activity was also investigated in primary MSI CRCs by quantifying the expression of several mRNAs relative to their mutational status and to endogenous UPF1 and UPF2 expression. Host immunity developed against MSI cancer cells was appreciated by quantifying the number of CD3epsilon-positive tumor-infiltrating lymphocytes (TILs). UPF1 silencing led to the up-regulation of 1251 genes in HCT116, among which a proportion of them (i.e. 38%) significantly higher than expected by chance contained a coding microsatellite (P<2x10(-16)). In MSI primary CRCs, UPF1 was significantly over-expressed compared to normal adjacent mucosa (P<0.002). Our data provided evidence for differential decay of PTC-mRNAs compared to wild-type that was positively correlated to UPF1 endogenous expression level (P = 0.02). A negative effect of UPF1 and UPF2 expression on the host's anti-tumor response was observed (P<0.01). Overall, our results show that NMD deeply influences MSI-driven tumorigenesis at the molecular level and indicate a functional negative impact of this system on anti-tumor immunity whose intensity has been recurrently shown to be an independent factor of favorable outcome in CRCs.     

Ligase-based detection of mononucleotide repeat sequences

Up to 15% of all colorectal cancers are considered to be replication error positive (RER(+)) and contain mutations at hundreds of thousands of microsatellite repeat sequences. Recently, a number of intragenic mononucleotide repeat sequences have been demonstrated to be targets for inactivating genes in RER(+)colorectal tumors. In this study, thermostable DNA ligases were tested for the ability to detect alterations in microsatellite sequences in colon tumor samples. Ligation profiles on mononucleotide repeat sequences were determined for four related thermostable DNA ligases, Thermus thermophilus ( Tth ) ligase, Thermus sp. AK16D ligase, Aquifex aeolicus ligase and the K294R mutant of the Tth ligase. While the limit of detection for point mutations was one mutation in 1000 wild-type sequences, the ability to detect a single base deletion in a 10 base mononucleotide repeat was one mutation in 100 wild-type sequences. Furthermore, the misligation error increased exponentially as the length of the mono-nucleotide repeat increased, and was 10% of the correct signal for a 19 base mononucleotide repeat. A fluorescent ligase-based assay [polymerase chain reaction/ligase detection reaction (PCR/LDR)] correlated with results obtained using a radioactive assay to detect instability within the TGF-beta Type II receptor gene. PCR/LDR was also used to detect the APCI1307K mononucleotide repeat allele which has a carrier frequency of 6.1% in Ashkenazi Jewish individuals. In a blind study, 30 samples that had been typed for the presence of the APCI1307K allele were tested. The PCR/LDR results correlated with those obtained using sequencing and allele-specific oligonucleotide hybridization for 16 samples carrying the mutation and 13 wild-type samples. Ligation assays that characterize mononucleotide repeats can be used to rapidly detect somatic mutations in tumors, and to screen for individuals who have a hereditary predisposition to develop colon cancer.     

Use of haploid mixtures and heteroduplex analysis enhance polymorphisms revealed by denaturing gradient gel electrophoresis

PCR-based codominant genetic markers were developed by using primer sequences designed from cDNA clones of loblolly pine (Pinus taeda L.). Such markers offer certain advantages relative to simple-sequence repeat (SSR), also known as short-tandem repeat (STR) markers, and include the ability to quantify and map DNA polymorphisms in expressed genes. However, detecting these DNA polymorphisms is more problematic because many DNA polymorphisms in genes involve base substitutions rather than insertions or deletions. Denaturing gradient gel electrophoresis (DGGE) is a sensitive and efficient method for detecting sequence differences among PCR fragments. This paper demonstrates the application of DGGE to genetically map expressed genes in loblolly pine. Also, heteroduplex DNA fragments, formed during the amplification of DNA from heterozygotes and from mixes of haploid DNAs from megagametophytes, enhanced and strengthened genetic interpretations and genotypic classifications.     

Isolation and enrichment of abundant microsatellites from a channel catfish (Ictalurus punctatus) brain cDNA library

Efforts to construct a genetic linkage map of channel catfish have involved identification of random genomic microsatellite markers, as well as anchored Type I loci (expressed genes) from channel catfish. To identify Type I markers we constructed a directional cDNA library from brain tissue to obtain expressed catfish sequences that could be used for single nucleotide polymorphism (SNP) marker development. These cDNA sequences surprisingly contained a high proportion of microsatellites (about 14%) in noncoding regions of expressed sequence tags (ESTs), many of which were not associated with known sequences. To further identify cDNAs with microsatellites and reduce the number of sequencing reactions needed for marker development, we enriched this library for repeat sequences and sequenced clones from both directions. A total of 1644 clones from seven repeat-enriched captures (CA, GT, CT, GA, MTT, TAG, and TAC) were sequenced from both ends, and 795 nonredundant clones were assembled. Thirty-seven percent of the clones contained microsatellites in the trimmed sequence. After assembly in the TIGR Catfish Gene Index (CfGI), 154 contigs matched known vertebrate genes and 92 contigs contained microsatellites. When BLAST-matched orthologues were available for similarity alignments, 28% of these contigs contained repeats in the 5'-UTR, 72% contained repeats in the 3'-UTR, and 8% contained repeats at both ends. Using biotinylated repeat oligonucleotides coupled with streptavidin-coated magnetic beads, and rapid; single-pass hybridization, we were able to enrich our plasmid library greater than two-fold for repeat sequences and increase the ability to link these ESTs with known sequences greater than six-fold.     

Isolation and Enrichment of Abundant Microsatellites from a Channel Catfish (Ictalurus punctatus) Brain cDNA Library

Efforts to construct a genetic linkage map of channel catfish have involved identification of random genomic microsatellite markers, as well as anchored Type I loci (expressed genes) from channel catfish. To identify Type I markers we constructed a directional cDNA library from brain tissue to obtain expressed catfish sequences that could be used for single nucleotide polymorphism (SNP) marker development. These cDNA sequences surprisingly contained a high proportion of microsatellites (about 14%) in noncoding regions of expressed sequence tags (ESTs), many of which were not associated with known sequences. To further identify cDNAs with microsatellites and reduce the number of sequencing reactions needed for marker development, we enriched this library for repeat sequences and sequenced clones from both directions. A total of 1644 clones from seven repeat-enriched captures (CA, GT, CT, GA, MTT, TAG, and TAC) were sequenced from both ends, and 795 nonredundant clones were assembled. Thirty-seven percent of the clones contained microsatellites in the trimmed sequence. After assembly in the TIGR Catfish Gene Index (CfGI), 154 contigs matched known vertebrate genes and 92 contigs contained microsatellites. When BLAST-matched orthologues were available for similarity alignments, 28% of these contigs contained repeats in the 5'-UTR, 72% contained repeats in the 3'-UTR, and 8% contained repeats at both ends. Using biotinylated repeat oligonucleotides coupled with streptavidin-coated magnetic beads, and rapid, single-pass hybridization, we were able to enrich our plasmid library greater than two-fold for repeat sequences and increase the ability to link these ESTs with known sequences greater than six-fold.     

Cis-elements governing trinucleotide repeat instability in Saccharomyces cerevisiae

Trinucleotide repeat (TNR) instability in humans is governed by unique cis-elements. One element is a threshold, or minimal repeat length, conferring frequent mutations. Since thresholds have not been directly demonstrated in model systems, their molecular nature remains uncertain. Another element is sequence specificity. Unstable TNR sequences are almost always CNG, whose hairpin-forming ability is thought to promote instability by inhibiting DNA repair. To understand these cis-elements further, TNR expansions and contractions were monitored by yeast genetic assays. A threshold of approximately 15--17 repeats was observed for CTG expansions and contractions, indicating that thresholds function in organisms besides humans. Mutants lacking the flap endonuclease Rad27p showed little change in the expansion threshold, suggesting that this element is not altered by the presence or absence of flap processing. CNG or GNC sequences yielded frequent mutations, whereas A-T rich sequences were substantially more stable. This sequence analysis further supports a hairpin-mediated mechanism of TNR instability. Expansions and contractions occurred at comparable rates for CTG tract lengths between 15 and 25 repeats, indicating that expansions can comprise a significant fraction of mutations in yeast. These results indicate that several unique cis-elements of human TNR instability are functional in yeast.     

A TGFβRII frameshift-mutation-derived CTL epitope recognised by HLA-A2-restricted CD8+ T cells

Microsatellite instability (MSI) is recognised as genome-wide alterations in repetitive DNA sequences caused by defects in the DNA mismatch repair machinery. Such mutation patterns have been found in almost all analysed malignancies from patients with hereditary non-polyposis colorectal cancer, and in approximately 15% of sporadic colorectal cancers. In cancers with the MSI phenotype, microsatellite-like sequences in coding regions of various cancer-related genes, including transforming growth factor beta receptor type II (TGF betaRII), are targets for mutations. The TGF betaRII gene harbours a 10-bp polyadenine tract, and mutations within this region are found in 90% of colorectal cancers with MSI. The frameshift mutations result in new amino acid sequences in the C-terminal part of the proteins, prematurely terminating where a novel stop codon appears. In this study we have defined new cytotoxic T lymphocyte (CTL) epitope (RLSSCVPVA), carrying a good HLA-A*0201 binding motif, and resulting from the most common frameshift mutation in TGF betaRII. A CTL line and several CTL clones were generated from an HLA-A2+ normal donor by repeated stimulation of T cells with dendritic cells pulsed with the peptide. One of the CTL clones was able to kill an HLA-A2+ colon cancer cell line harbouring mutant TGF betaRII. This epitope may be a valuable component in cancer vaccines directed at MSI-positive carcinomas.     

Cloning of region-specific genetic markers of planaria using a new method--ordered differential display

A new method for finding differentially expressed genes, termed ordered differential display of mRNAs (ODD), was used in the search for region-specific molecular markers of freshwater planarian Dugesia tigrina. In this method, the effect of selective suppression of a polymerase chain reaction (PCR) is used for the differential amplification of a pool of 3'-terminal cDNA fragments generated by digestion of cDNAs with a restriction endonuclease. In the resulting amplified cDNAs, every mRNA is represented by a cDNA fragment whose length is determined by the position of the restriction site nearest to the 3'-terminus. Subsequent PCR with primers 3'-extended by two random nucleotides allowed the amplification of 1/192 part of all cDNA molecules present in the sample. The comparison of the generated pools of cDNA molecules separated by PAGE leads to the identification of differentially expressed sequences. The systematic study of the total mRNA pool is achieved by the successive use of all possible combinations of extended primers. Some sequences preferentially expressed along the anterior-posterior axis of planarian were identified using ODD.