p53 is a pivotal tumor suppressor and a major barrier against cancer. We now report that silencing of the Hippo pathway tumor suppressors LATS1 and LATS2 in nontransformed mammary epithelial cells ...reduces p53 phosphorylation and increases its association with the p52 NF-κB subunit. Moreover, it partly shifts p53's conformation and transcriptional output toward a state resembling cancer-associated p53 mutants and endows p53 with the ability to promote cell migration. Notably, LATS1 and LATS2 are frequently down-regulated in breast cancer; we propose that such down-regulation might benefit cancer by converting p53 from a tumor suppressor into a tumor facilitator.
Complex interactions between different host immune cell types can determine the outcome of pathogen infections. Advances in single cell RNA-sequencing (scRNA-seq) allow probing of these immune ...interactions, such as cell-type compositions, which are then interpreted by deconvolution algorithms using bulk RNA-seq measurements. However, not all aspects of immune surveillance are represented by current algorithms. Here, using scRNA-seq of human peripheral blood cells infected with Salmonella, we develop a deconvolution algorithm for inferring cell-type specific infection responses from bulk measurements. We apply our dynamic deconvolution algorithm to a cohort of healthy individuals challenged ex vivo with Salmonella, and to three cohorts of tuberculosis patients during different stages of disease. We reveal cell-type specific immune responses associated not only with ex vivo infection phenotype but also with clinical disease stage. We propose that our approach provides a predictive power to identify risk for disease, and human infection outcomes.
Deregulated proliferation is a hallmark of cancer cells. Here, we show that microRNA‐10b* is a master regulator of breast cancer cell proliferation and is downregulated in tumoural samples versus ...matched peritumoural counterparts. Two canonical CpG islands (5 kb) upstream from the precursor sequence are hypermethylated in the analysed breast cancer tissues. Ectopic delivery of synthetic microRNA‐10b* in breast cancer cell lines or into xenograft mouse breast tumours inhibits cell proliferation and impairs tumour growth in vivo, respectively. We identified and validated in vitro and in vivo three novel target mRNAs of miR‐10b* (BUB1, PLK1 and CCNA2), which play a remarkable role in cell cycle regulation and whose high expression in breast cancer patients is associated with reduced disease‐free survival, relapse‐free survival and metastasis‐free survival when compared to patients with low expression. This also suggests that restoration of microRNA‐10b* expression might have therapeutic promise.
Encounters between host cells and intracellular bacterial pathogens lead to complex phenotypes that determine the outcome of infection. Single-cell RNA sequencing (scRNA-seq) is increasingly used to ...study the host factors underlying diverse cellular phenotypes but has limited capacity to analyze the role of bacterial factors. Here, we developed scPAIR-seq, a single-cell approach to analyze infection with a pooled library of multiplex-tagged, barcoded bacterial mutants. Infected host cells and barcodes of intracellular bacterial mutants are both captured by scRNA-seq to functionally analyze mutant-dependent changes in host transcriptomes. We applied scPAIR-seq to macrophages infected with a library of
Typhimurium secretion system effector mutants. We analyzed redundancy between effectors and mutant-specific unique fingerprints and mapped the global virulence network of each individual effector by its impact on host immune pathways. ScPAIR-seq is a powerful tool to untangle bacterial virulence strategies and their complex interplay with host defense strategies that drive infection outcome.
The main bottleneck for genomic studies of tumors is the limited availability of fresh frozen (FF) samples collected from patients, coupled with comprehensive long-term clinical follow-up. This ...shortage could be alleviated by using existing large archives of routinely obtained and stored Formalin-Fixed Paraffin-Embedded (FFPE) tissues. However, since these samples are partially degraded, their RNA sequencing is technically challenging.
In an effort to establish a reliable and practical procedure, we compared three protocols for RNA sequencing using pairs of FF and FFPE samples, both taken from the same breast tumor. In contrast to previous studies, we compared the expression profiles obtained from the two matched sample types, using the same protocol for both. Three protocols were tested on low initial amounts of RNA, as little as 100 ng, to represent the possibly limited availability of clinical samples. For two of the three protocols tested, poly(A) selection (mRNA-seq) and ribosomal-depletion, the total gene expression profiles of matched FF and FFPE pairs were highly correlated. For both protocols, differential gene expression between two FFPE samples was in agreement with their matched FF samples. Notably, although expression levels of FFPE samples by mRNA-seq were mainly represented by the 3'-end of the transcript, they yielded very similar results to those obtained by ribosomal-depletion protocol, which produces uniform coverage across the transcript. Further, focusing on clinically relevant genes, we showed that the high correlation between expression levels persists at higher resolutions.
Using the poly(A) protocol for FFPE exhibited, unexpectedly, similar efficiency to the ribosomal-depletion protocol, with the latter requiring much higher (2-3 fold) sequencing depth to compensate for the relative low fraction of reads mapped to the transcriptome. The results indicate that standard poly(A)-based RNA sequencing of archived FFPE samples is a reliable and cost-effective alternative for measuring mRNA-seq on FF samples. Expression profiling of FFPE samples by mRNA-seq can facilitate much needed extensive retrospective clinical genomic studies.
Real time quantitative polymerase chain reaction (qPCR) is an important tool in quantitative studies of DNA and RNA molecules; especially in transcriptome studies, where different primer combinations ...allow identification of specific transcripts such as splice variants or precursor messenger RNA. Several softwares that implement various rules for optimal primer design are available. Nevertheless, as designing qPCR primers needs to be done manually, the repeated task is tedious, time consuming and prone to errors.
We used a set of rules to automatically design all possible exon-exon and intron-exon junctions in the human and mouse transcriptomes. The resulting database is included as a track in the UCSC genome browser, making it widely accessible and easy to use.
The database is available from the UCSC genome browser (http://genome.ucsc.edu/), track name 'Whole Transcriptome qPCR Primers' for the hg19 (Human) and mm10 (Mouse) genome versions. Batch query is available in the following: http://www.weizmann.ac.il/complex/compphys/software/Amit/primers/batch_query_qpcr_primers.htm
amit.zeisel@weizmann.ac.il or eytan.domany@weizmann.ac.il
Supplementary data are available at Bioinformatics online.
MicroRNAs (miRs) function primarily as post-transcriptional negative regulators of gene expression through binding to their mRNA targets. Reliable prediction of a miR's targets is a considerable ...bioinformatic challenge of great importance for inferring the miR's function. Sequence-based prediction algorithms have high false-positive rates, are not in agreement, and are not biological context specific. Here we introduce CoSMic (Context-Specific MicroRNA analysis), an algorithm that combines sequence-based prediction with miR and mRNA expression data. CoSMic differs from existing methods--it identifies miRs that play active roles in the specific biological system of interest and predicts with less false positives their functional targets. We applied CoSMic to search for miRs that regulate the migratory response of human mammary cells to epidermal growth factor (EGF) stimulation. Several such miRs, whose putative targets were significantly enriched by migration processes were identified. We tested three of these miRs experimentally, and showed that they indeed affected the migratory phenotype; we also tested three negative controls. In comparison to other algorithms CoSMic indeed filters out false positives and allows improved identification of context-specific targets. CoSMic can greatly facilitate miR research in general and, in particular, advance our understanding of individual miRs' function in a specific context.
The locus of microRNA-10b Biagioni, Francesca; Bossel Ben-Moshe, Noa; Fontemaggi, Giulia ...
Cell cycle (Georgetown, Tex.),
08/2013, Letnik:
12, Številka:
15
Journal Article
Recenzirano
Odprti dostop
Contemporary microRNA research has led to significant advances in our understanding of the process of tumorigenesis. MicroRNAs participate in different events of a cancer cell's life, through their ...ability to target hundreds of putative transcripts involved in almost every cellular function, including cell cycle, apoptosis, and differentiation. The relevance of these small molecules is even more evident in light of the emerging linkage between their expression and both prognosis and clinical outcome of many types of human cancers. This identifies microRNAs as potential therapeutic modifiers of cancer phenotypes. From this perspective, we overview here the miR-10b locus and its involvement in cancer, focusing on its role in the establishment (miR-10b*) and spreading (miR-10b) of breast cancer. We conclude that targeting the locus of microRNA 10b holds great potential for cancer treatment.
miRNAs have been implicated in the regulation of key metabolic, inflammatory, and malignant pathways; hence, they might be considered both predictors and players of cancer development.
Using a ...case-control study design nested in the ORDET prospective cohort study, we addressed the possibility that specific mRNAs can serve as early predictors of breast cancer incidence in postmenopausal women. We compared leukocyte miRNA profiles of 133 incident postmenopausal breast cancer cases and profiles of 133 women who remained healthy over a follow-up period of 20 years.
The analysis identified 20 differentially expressed miRNAs, 15 of which were downregulated. Of the 20 miRNAs, miR145-5p and miR145-3p, each derived from another arm of the respective pre-miRNA, were consistently and significantly downregulated in all the databases that we surveyed. For example, analysis of more than 1,500 patients (the UK Metabric cohort) indicated that high abundance of miR145-3p and miR145-5p was associated with longer, and for miR145-3p also statistically significant, survival. The experimental data attributed different roles to the identified miRNAs: Although the 5p isoform was associated with invasion and metastasis, the other isoform seems related to cell proliferation.
These observations and the prospective design of our study lend support to the hypothesis that downregulation of specific miRNAs constitutes an early event in cancer development. This finding might be used for breast cancer prevention.
The identification of the miRNAs as long-term biomarkers of breast cancer may have an impact on breast cancer prevention and early detection.
We present the Pareto task inference method (ParTI; http://www.weizmann.ac.il/mcb/UriAlon/download/ParTI) for inferring biological tasks from high-dimensional biological data. Data are described as a ...polytope, and features maximally enriched closest to the vertices (or archetypes) allow identification of the tasks the vertices represent. We demonstrate that human breast tumors and mouse tissues are well described by tetrahedrons in gene expression space, with specific tumor types and biological functions enriched at each of the vertices, suggesting four key tasks.