High-Throughput miRNA and mRNA Sequencing of Paired Colorectal Normal, Tumor and Metastasis Tissues and Bioinformatic Modeling of miRNA-1 Therapeutic Applications

MiRNAs are discussed as diagnostic and therapeutic molecules. However, effective miRNA drug treatments with miRNAs are, so far, hampered by the complexity of the miRNA networks. To identify potential miRNA drugs in colorectal cancer, we profiled miRNA and mRNA expression in matching normal, tumor and metastasis tissues of eight patients by Illumina sequencing. We validated six miRNAs in a large tissue screen containing 16 additional tumor entities and identified miRNA-1, miRNA-129, miRNA-497 and miRNA-215 as constantly de-regulated within the majority of cancers. Of these, we investigated miRNA-1 as representative in a systems-biology simulation of cellular cancer models implemented in PyBioS and assessed the effects of depletion as well as overexpression in terms of miRNA-1 as a potential treatment option. In this system, miRNA-1 treatment reverted the disease phenotype with different effectiveness among the patients. Scoring the gene expression changes obtained through mRNA-Seq from the same patients we show that the combination of deep sequencing and systems biological modeling can help to identify patient-specific responses to miRNA treatments. We present this data as guideline for future pre-clinical assessments of new and personalized therapeutic options

SOS1 is the second most common Noonan gene but plays no major role in cardio‐facio‐cutaneous syndrome

Background: Heterozygous gain-of-function mutations in various genes encoding proteins of the Ras-MAPK signalling cascade have been identified as the genetic basis of Noonan syndrome (NS) and cardio-facio-cutaneous syndrome (CFCS). Mutations of SOS1, the gene encoding a guanine nucleotide exchange factor for Ras, have been the most recent discoveries in patients with NS, but this gene has not been studied in patients with CFCS. Methods and results: We investigated SOS1 in a large cohort of patients with disorders of the NS–CFCS spectrum, who had previously tested negative for mutations in PTPN11, KRAS, BRAF, MEK1 and MEK2. Missense mutations of SOS1 were discovered in 28% of patients with NS. In contrast, none of the patients classified as having CFCS was found to carry a pathogenic sequence change in this gene. Conclusion: We have confirmed SOS1 as the second major gene for NS. Patients carrying mutations in this gene have a distinctive phenotype with frequent ectodermal anomalies such as keratosis pilaris and curly hair. However, the clinical picture associated with SOS1 mutations is different from that of CFCS. These findings corroborate that, despite being caused by gain-of-function mutations in molecules belonging to the same pathway, NS and CFCS scarcely overlap genotypically

Clinical parameters of the colorectal cancer patients.

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Differential expression of miRNAs in colon tumor and metastasis tissues.

<p>(<b>A</b>) Top 25 up- and down-regulated miRNAs comparing tumor (left) or metastasis (right) tissues versus normal colon samples as analyzed by Illumina sequencing. All depicted miRNAs sufficed a p-value threshold ≤0.05. A star indicates samples with p≤0.01. (<b>B</b>) Venn diagram of microRNAs expressed in colorectal cancer patients, as determined by Illumina sequencing. (Left) Numbers of detected miRNAs, specific for each tissue (normal (N) = 19, tumor (T) = 34, metastases (M) = 29) and in all tissues (559). (Middle) Venn diagram of the significantly up-regulated miRNAs (p-value ≤0.05) for all comparisons (N/T, N/M and T/M). (Right) Venn diagram of the significantly down-regulated miRNAs (p-value ≤0.05) for all comparisons (N/T, N/M and T/M).</p

Functional assays on miRNA-1 as a potential tumor-suppressor gene.

<p>(<b>A</b>) AlamarBlue cell viability assay to test the effect of miRNA-1. SW480 (primary colon cancer cell line) and SW620 (corresponding metastases cell line) cells were transfected with miRNA-1 mimics (+miR-1) or mock transfected (−miR-1) and measured using an spectrophotometer after 24 h, 48 h and 72 h. The miRNA-1 level was determined by TaqMan assays for mature microRNAs. (<b>B</b>) “Wound healing” assay for miRNA-1 in SW480 and SW620 cells. After 24 h of transfection with miRNA-1 mimics a uniform scratch was generated through each confluent cell layer and “wound” closure was documented after 24 h using a phase-contrast microscope (n = 2). (<b>C</b>) AlamarBlue cell viability assay in SW480 and SW620 cells after camptothecin treatment alone or in combination with miRNA-1. Cell viability was measured after 0 h, 24 h and 48 h of drug treatment using a spectrophotometer.</p

Translocations disrupting PHF21A in the Potocki-Shaffer-syndrome region are associated with intellectual disability and craniofacial anomalies

Contains fulltext : 110038.pdf (publisher's version ) (Closed access)Potocki-Shaffer syndrome (PSS) is a contiguous gene disorder due to the interstitial deletion of band p11.2 of chromosome 11 and is characterized by multiple exostoses, parietal foramina, intellectual disability (ID), and craniofacial anomalies (CFAs). Despite the identification of individual genes responsible for multiple exostoses and parietal foramina in PSS, the identity of the gene(s) associated with the ID and CFA phenotypes has remained elusive. Through characterization of independent subjects with balanced translocations and supportive comparative deletion mapping of PSS subjects, we have uncovered evidence that the ID and CFA phenotypes are both caused by haploinsufficiency of a single gene, PHF21A, at 11p11.2. PHF21A encodes a plant homeodomain finger protein whose murine and zebrafish orthologs are both expressed in a manner consistent with a function in neurofacial and craniofacial development, and suppression of the latter led to both craniofacial abnormalities and neuronal apoptosis. Along with lysine-specific demethylase 1 (LSD1), PHF21A, also known as BHC80, is a component of the BRAF-histone deacetylase complex that represses target-gene transcription. In lymphoblastoid cell lines from two translocation subjects in whom PHF21A was directly disrupted by the respective breakpoints, we observed derepression of the neuronal gene SCN3A and reduced LSD1 occupancy at the SCN3A promoter, supporting a direct functional consequence of PHF21A haploinsufficiency on transcriptional regulation. Our finding that disruption of PHF21A by translocations in the PSS region is associated with ID adds to the growing list of ID-associated genes that emphasize the critical role of transcriptional regulation and chromatin remodeling in normal brain development and cognitive function