Bulk transcriptomic analysis of normal, premalignant state and tumor mouse models of VAV1-MYOF gene fusion in Peripheral T-Cell Lymphoma
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ABSTRACT:
PROVIDER: PRJNA701570 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR13696651_1.fastq.gz | Fastqsanger.gz | |||
| SRR13696651_2.fastq.gz | Fastqsanger.gz | |||
| SRR13696652_1.fastq.gz | Fastqsanger.gz | |||
| SRR13696652_2.fastq.gz | Fastqsanger.gz | |||
| SRR13696653_1.fastq.gz | Fastqsanger.gz |
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Project description:The goal of this study is to identify transcriptomic effects of VAV1-MYOF expression in mouse CD4+ T lymphocytes in tumor development as well as changes in the microenvironment. To this end, mice conditionally expressing VAV1-MYOF were crossed with the CD4CreERT2 line or CD4-Cre, under the control of the mouse Cd4 promoter to generate CD4 specific VAV1-MYOF models. In inducible models, mice were treated with vehicle only or Tamoxifen to induce expression of VAV1-MYOF1 in CD4+ T cells. CD4CreERT2 mice were included as a negative control. Total RNA was extracted from whole spleen samples or CD4+ T cells isolated by single cell suspensions of spleen by negative selection using the mouse naive CD4+ T Cell Isolation Kit (Milteny#130-104-453) according to the manufacturer’s protocol. Standard Illumina poly-A RNA seq was performed.
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Project description:The goal of this study is to identify transcriptomic effects of VAV1-MYOF expression in mouse CD4+ T lymphocytes and tumor progression in TET2KO and TET2WT backgrounds. To this end, mice conditionally expressing VAV1-MYOF were crossed with the CD4CreERT2 line or CD4-Cre, under the control of the mouse Cd4 promoter to generate CD4 specific VAV1-MYOF models. In inducible models, mice were treated with vehicle only or Tamoxifen to induce expression of VAV1-MYOF1 in CD4+ T cells. Mice were crossed with CD4-CreERT2 Tet2f/f to obtain the experimental phenotypes. Whole spleen samples were processed and single cell suspensions were analyzed using scRNA-seq technologies.
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Project description:Single cell RNA-seq analysis of normal, premalignant state and tumor mouse models of VAV1-MYOF gene fusion in Peripheral T-Cell Lymphoma
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Project description:Analysis of differential gene expression profiles in VAV1-/- versus VAV+/+ BM-macrophages , respectively. A distinct expression profile in VAV1-/- versus VAV1+/+ (wild-type) BM-macrophages allows to resolve molecular mechanisms. Total RNA was obtained froms BM-macrophages of VAV1+/+ and VAV1-/- mice
2013-03-08 | E-GEOD-36123 | biostudies-arrayexpress
Project description:Analysis of differential gene expression profiles in VAV1-/- versus VAV+/+ BM-macrophages , respectively. A distinct expression profile in VAV1-/- versus VAV1+/+ (wild-type) BM-macrophages allows to resolve molecular mechanisms.
2013-03-08 | GSE36123 | GEO
Project description:Background: Even though much progress has been made in the understanding of the molecular nature of glioma, the survival rates of patients affected of this tumour have not changed significantly during these years. Thus, a deeper understanding of this malignancy is still needed in order to predict its outcome and improve patient treatment. Here, we report that VAV1, a GDP/GTP exchange factor for Rho/Rac proteins with oncogenic potential that is involved in the regulation of cytoskeletal dynamics and cell migration. Methodology/Principal Findings: VAV1 is overexpressed in 32 patients diagnosed with high-grade glioma. Such overexpression is linked to the parallel upregulation of a number of genes coding for proteins also involved in cell invasion- and migration-related processes. Unexpectedly, immunohistochemical experiments revealed that VAV1 is not expressed in glioma cells. Instead, VAV1 is found in non-tumoral astrocyte-like cells that are located either peritumoraly or perivascularly, suggesting that its expression is linked to synergistic signalling cross-talk between cancer and infiltrating cells. Conclusions/Significance: Interestingly, we show that the pattern of expression of VAV1 is a good prognostic factor to unveil populations of high-grade glioma patients with different survival and progression free survival rates.
2010-09-30 | GSE24072 | GEO
Project description:Mutations in VAV1, a multifunctional signaling protein essential for T lymphocytes, have been recently found in peripheral T cell lymphoma (PTCL) and other tumors. However, their pathobiological significance remains unsettled. Here, we show that these mutations belong to several subclasses according to the spectra of VAV1 signaling branches deregulated by them. These mutations target both known and new regulatory hotspots as well as transitional activation states of the protein. The most frequent VAV1 mutant subclass drives PTCL formation in mice by exacerbating normal VAV1- dependent signaling responses in follicular helper T cells.
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Project description:VAV1 mutations have been recently found in peripheral T cell lymphoma and non-small cell lung cancer. To understand their pathogenic potential, we generated a gene-edited mouse model that expresses a Vav1 mutant protein that recapitulates the signaling alterations present the VAV1 mutant subclass most frequently found in tumors. We could not detect any overt tumorigenic process in those mice. However, the concurrent elimination of the Trp53 tumor suppressor gene in them drives T cell lymphomagenesis. This process represents an exacerbation of the normal functions that wild-type Vav1 plays in follicular helper T cells. We also found that, in combination with an oncogenic Kras mutation, the Vav1 mutant version favors progression of non-small cell lung cancer. These data indicate that VAV1 mutations play critical, although highly cell typespecific roles in tumorigenesis. They also indicate that such functions are contingent on the mutational landscape of the tumors involved.
2022-10-12 | GSE189221 | GEO
Project description:Transcriptomic analysis of normal, premalignant and malignant lymphocytes from mouse models of Peripheral T-Cell Lymphoma
| PRJNA701567 | ENA
Project description:Background: Even though much progress has been made in the understanding of the molecular nature of glioma, the survival rates of patients affected of this tumour have not changed significantly during these years. Thus, a deeper understanding of this malignancy is still needed in order to predict its outcome and improve patient treatment. Here, we report that VAV1, a GDP/GTP exchange factor for Rho/Rac proteins with oncogenic potential that is involved in the regulation of cytoskeletal dynamics and cell migration. Methodology/Principal Findings: VAV1 is overexpressed in 32 patients diagnosed with high-grade glioma. Such overexpression is linked to the parallel upregulation of a number of genes coding for proteins also involved in cell invasion- and migration-related processes. Unexpectedly, immunohistochemical experiments revealed that VAV1 is not expressed in glioma cells. Instead, VAV1 is found in non-tumoral astrocyte-like cells that are located either peritumoraly or perivascularly, suggesting that its expression is linked to synergistic signalling cross-talk between cancer and infiltrating cells. Conclusions/Significance: Interestingly, we show that the pattern of expression of VAV1 is a good prognostic factor to unveil populations of high-grade glioma patients with different survival and progression free survival rates. 1. Oligonucleotide microarray analyses Total RNAs were extracted using the Triazol reagent (Life Technologies, Gaithersburg, MD, USA) and purified with the RNeasy Mini kit (Qiagen, Valencia, CA, USA). The integrity of RNA samples obtained was assessed using the 2100 Bioanalyzer (Agilent, Palo Alto, CA, USA). Double-stranded cDNAs and biotinylated cRNAs were synthesized using a T7-polyT primer and the BioArray RNA labelling Kit (Enzo Farningdale, NY, USA), respectively. Labelled RNAs were then fragmented and hybridised to HU-133A oligonucleotide arrays (Affymetrix, Santa Clara, CA, USA) according to standard Affymetrix protocols. After hybridization and washes, arrays were scanned using the Gene Array Scanner (Affymetrix), and the expression value for each probe set calculated using the MAS 5.0 software (Affymetrix). All examples had a scaling factor lower than threefold and 3’/5’ of GAPDH probe set <2.5. Gene levels were transformed to base two logarithms. A median normalization approach was applied. Only genes with al least three “present” calls across all samples were selected. All these steps were done at the Genomics and Proteomics Unit of the “Centro de Investigación del Cáncer, Salamanca”. 2. Microarray data analyses To visualize clusters of genes with similar expression patterns, we used a hierarchical clustering method (Cluster and TreeView software) based on the average-linkage method with the centred correlation metric [26]. A multidimensional scaling method (BRB Arrays Tools version 3.0) was also utilized by using Euclidean distance criteria [27]. Supervised learning was used to identify genes with statistically significant changes in expression among different classes by using the Significant Analysis of Microarrays (SAM) algorithm [28]. All data were permuted over 100 cycles by using the two-class (unpaired) and multi-class response format. Significant genes were selected based on the lowest false discovery ratio (between 0.6 and 0.9). In addition, nonparametric tests such as Wilcoxon rank sum test and Kruskal-Wallis test to compare more than two unpaired group were also used (SPSS 18, SPSS Inc). 3. Functional annotation of microarray data Probe sets showing significant expression change were functionally annotated and grouped according to biological function criteria using GeneOntology biological process descriptions. The functional analysis to identify the most relevant biological mechanism, pathways and functional categories in gene dada sets was generated using the Ingenuity Pathway software (Ingenuity Systems, Mountain View, CA, USA) available in the web (www.ingenuity.com) [29]. A functional network was considered significant when it fulfilled the following criteria: i) to have a minimal score of 15; ii) to have a minimum of 20 direct functional interactions among the network members. 4. Quantitative reverse transcription-PCR Total RNA was quantified in a RNA 6000 Nano Chip (Agilent Technologies) and quantitative PCR performed using the QuantiTect SYBR Green RT–PCR kit (Qiagen). To quantify VAV1 mRNA levels, we used two different sets of probes: PAIR A (5’-AAC AAC GGG AGG TTC ACC CT-3’ and 5’-GGT CCC TCA TGG CAT CCA-3’) and PAIR B (5’-AGC CAT TGG ACC CTT TCT ACG-3’ and 5’-GCC ATG GAC ATA GGG CTT CA-3’). Amplifications were performed using the iCycler apparatus (Bio-Rad Laboratories, California, USA). Analyses of data were done using the iCycler iQ Optical System Software, version 3.0a (Bio-Rad Laboratories). Primers to GAPDH were used as intersample normalizing controls. Variations in expression of VAV1 mRNA were represented as the mean value of the fold change respect the VAV1 expression levels detected in sample #19209 with both pairs of oligonucleotide primers. 5. Immunohistochemical analyses The VAV1 antibody was generated in rabbits using a synthetic peptide and purified by affinity chromatography in Bustelo’s laboratory. This antibody recognizes VAV1 proteins from humans and mice but it does not recognize other VAV family members (unpublished data). For immunostaining, tissue sections were washed thrice with Xylene and once with 100% ethanol, rehydrated by sequential changes in 80%, 70%, and 50% ethanol and a final incubation in phosphate-buffered saline (PBS). Each rehydrating step involved 3 min incubations with the indicated solutions. Endogenous peroxidases were quenched by the addition of a 3% H2O2 solution in methanol for 30 min at room temperature (RT). Tissue sections were subsequently washed twice with PBS. Antigen retrieval was performed by incubation in 1 mM EDTA for 30 min at 37°C. The slides were washed twice in PBS and blocked in blocking buffer (Zymed, CA, USA) for 30 min at RT. Specimens were then incubated with the primary antibody (1:250 dilution) in blocking buffer. After an 1 hr incubation at 37°C, slides were washed three times in PBS, incubated with a biotinylated secondary antibody for 30 min at 37°C, washed thrice in PBS, incubated with horseradish peroxidase-streptavidin for 30 min at 37°C, washed three times in PBS, and developed using the AEC substrate (Zymed). Slides were then washed twice in water, counterstained with hematoxilin (Zymed), washed again in water, and mounted with GVA (Zymed). Samples were analyzed by light microscopy and images acquired suing an Axiophot imaging system (Zeiss, Munich, Germany). 6. Fluorescence in situ hybridization analyses FISH experiments were carried out in 40 cases of glioblastoma multiforme (grade IV) positive for VAV1 expression. For this purpose, we performed dual-colour FISH analyses with locus-specific probes for centromere 7 (Abbott Molecualr, Des Plaines) exactly as previously described.[30] Polysomies were defined when more than 10% of the nuclei surveyed contained three or more CEP signals (chromosome-specific FISH probes that hybridize to highly repetitive human satellite DNA sequences, usually located near centromeres). 7. Immunohistochemistry and fluorescence in situ hybridization (FISH) in paraffin-embedded tumours Four um sections were cut from routinely processed paraffin blocks and mounted onto glass slides with a charged coating. Sections were dewaxed in Xylene and then rehydrated using increasing concentrations of alcohol before being rinsed briefly in water. Slides were heated 2 min in 1 mM EDTA (pH 9.0) in a microwavable pressure cooker. After antigen retrieval, slides were incubated 1 h at RT in a moist chamber with a primary antibody diluted in PBS supplemented with 10% foetal calf serum. Slides were incubated for 1 h with fluorochrome-conjugated antibodies to the appropriate IgG isotypes in a moist chamber in the dark. Finally, slides were washed thrice in PBS containing 0.5% Tween 20 three before FISH analysis. 8. Degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR) analyses After the staining of tissue sections with VAV1 antibodies (see above), the regions of the tumour were identified, microdissected, and collected using the PALM® microscope system (P.A.L.M. Microlaser Technologies, Munich, Germany). The genomic DNA was extracted as indicated by Isola et al [31] with modifications to small DNA amounts. Those included the resuspension of the microdissected sections in extraction buffer followed by a digestion with proteinase K (0.6 mg/ml). All samples were resuspended in 10 ul of 10 mM Tris-HCl (pH 7.4) and 0.1 mM EDTA. DOP-PCR amplification was performed in two steps. For the first, low-stringency step, 1 ul of sample was added to 4 ul of buffer A (2.5 ul of 600 uM dNTPs (Roche, Pleasanton, CA), 0.5 ul of 10 uM DOP primer 5’-CCGACTCGAGNNNNNNNATGTGG-3’, where N= A, C, G, or T) [32] and 1 ul of 5x Sequenase Reaction Buffer (Amersham, Cleveland, OH). Reactions were performed using 5 cycles of 30ºC for 5 min, 37ºC for 2 min, and 96ºC for 2 min, adding 0.65 units of Sequenase in each 30ºC step. The first phase product was then subjected to the second step usin
2010-09-30 | E-GEOD-24072 | biostudies-arrayexpress