We transfected PC3 cells with 100nM of a scrambled antisense oligonucleotide (AON) and an AON directed against MBNL1 exon 7 (36 basepairs) in order to skip the latter. Cells were harvested at 72h post-transfection and RNAseq was performed with ribozero depletion. Overall design: For RNA-Seq library preparation we followed the Illumina TruSeq RNA Sample Preparation Kit v2 manual. At least 70 million, 75bp long paired end reads were mapped to the GRCh37/hg19 version of the human genome per replicate using STAR 2.4.2a (doi: 10.1093/bioinformatics/bts635) using the default parameters.
MBNL1 alternative splicing isoforms play opposing roles in cancer.
Specimen part, Cell line, Subject, Time
View SamplesThis SuperSeries is composed of the SubSeries listed below.
MicroRNA-10b pleiotropically regulates invasion, angiogenicity and apoptosis of tumor cells resembling mesenchymal subtype of glioblastoma multiforme.
Specimen part, Cell line
View SamplesDownregulation of EZH2 Leads to Cellular Senescence with Features of SASP Overall design: Cells were infected with a lentivirus vector expressing shRNA against EZH2 and harvested at 4 and 8 days after infection. Total RNA was harvested from cells using Trizol reagent (Invitrogen) and further purified using the Purelink RNA Mini kit (Invitrogen) with DNase I digestion. RNA library preparation with polyA selection and Illumina HiSeq 2x150bp sequencing was performed by GeneWiz Inc. Paired-end reads were quality trimmed using Trim galore v0.4.0 and subsequently aligned to the human reference genome, hg19, using HISAT2 v2.1.0. Reads mapping to annotated genes were quantified using featureCounts (Liao et al., 2014). Differential gene expression was determined using DESeq2 v1.12.4 (Love et al., 2014) and significance was defined as FDR-corrected p-values of <0.05. The log2 fold change for each gene was used to rank the list of genes for GSEAPreranked analysis (Subramanian et al., 2005). FPKM values were calculated using DESeq2 and Z-scores were generated from FPKMs
Regulation of Cellular Senescence by Polycomb Chromatin Modifiers through Distinct DNA Damage- and Histone Methylation-Dependent Pathways.
Subject, Time
View SamplesAn experimental lung metastasis assay was used to derive an invasive subline of U87 that is metastatic in mice.
MicroRNA-10b pleiotropically regulates invasion, angiogenicity and apoptosis of tumor cells resembling mesenchymal subtype of glioblastoma multiforme.
Specimen part, Cell line
View SamplesMicroRNA-10b may target numerous genes in gliomagenesis. The target genes of miR-10b may differ according to the cellular context.
MicroRNA-10b pleiotropically regulates invasion, angiogenicity and apoptosis of tumor cells resembling mesenchymal subtype of glioblastoma multiforme.
Specimen part, Cell line
View SamplesA doxycyline-inducible INS-1 insulinoma cell line expressing proinsulin (C96Y)-GFP was engineered. Addition of doxycyline causes the production of the proinsulin (C96Y)-GFP, which is retained in the endoplasmic reticulum. This study analyzes the gene expression changes that occur after doxycyline-induced expression of proinsulin (C96Y)-GFP for 24h, 48h and 5 days. Expression changes were compared between control un-induced cells and cells treated with doxycyline. Three replicates (experiments) were performed for each time point.
Endoplasmic reticulum stress response in an INS-1 pancreatic beta-cell line with inducible expression of a folding-deficient proinsulin.
Cell line
View SamplesHepatocellular carcinoma (HCC) is a deadly disease, often unnoticed till the late stages, where treatment options become limited. Thus, there is a critical need to identify early biomarkers for detection of the developing HCC, as well as molecular pathways that would be amenable to therapeutic intervention. While efforts using human serum and tissues from late stage patients have been undertaken, progress has been limited. We have therefore explored the possibility of utilizing established mouse models for the discovery of biomarkers, as well as to understand in a systematic manner the molecular pathways that are progressively deregulated by the various etiological factors in contributing to HCC formation. As an initial effort, we have used the Hepatitis B surface antigen (HBsAg) transgenic mice as a hepatitis model, which have been exposed to aflatoxin B1 (AFB1). In this report, we present the initial findings from a extensive longitudinal study, which confirms the synergistic effect of both these etiological factors, with a gender bias towards male mice. Tumors from the mouse models were validated both histologically as well as by molecular transcriptome analysis by comparison with human HCCs. In addition, using these models, we have identified carnitine as a novel biomarker for HCC development, which was again validated using human HCC samples. Conclusion: This study therefore highlights the utility of these mouse models in identifying biomarkers for detection of human HCCs, as well as for the systematic analysis of molecular pathways that are affected by various etiological agents during the progression of HCC from an untransformed hepatocyte, which could provide novel options for targeted therapy.
Molecular characterization of hepatocarcinogenesis using mouse models.
Specimen part
View SamplesThis SuperSeries is composed of the SubSeries listed below.
Regulation of constitutive and alternative splicing by PRMT5 reveals a role for Mdm4 pre-mRNA in sensing defects in the spliceosomal machinery.
Specimen part, Treatment
View SamplesProtein Arginine MethylTransferase 5 (PRMT5) is known to mediate epigenetic control on chromatin and to functionally regulate components of the splicing machinery. In this study we show that selective deletion of PRMT5 in different organs leads to cell cycle arrest and apoptosis. At the molecular level, PRMT5 depletion results in reduced methylation of Sm proteins, aberrant constitutive splicing and in the Alternative Splicing (AS) of specific mRNAs. We identify Mdm4 as one of these mRNAs, which due to its weak 5-Donor site, acts as a sensor of splicing defects and transduces the signal to activate the p53 response, providing a mechanistic explanation of the phenotype observed in PRMT5 conditional knockout mice. Our data demonstrate a key role of PRMT5, together with p53, as guardians of the transcriptome. This will have fundamental implications in our understanding of PRMT5 activity, both in physiological conditions, as well as pathological conditions, including cancer and neurological diseases.
Regulation of constitutive and alternative splicing by PRMT5 reveals a role for Mdm4 pre-mRNA in sensing defects in the spliceosomal machinery.
Specimen part, Treatment
View SamplesA phase I trial of a SRC kinase Inhibitor, dasatinib, in combination with paclitaxel and carboplatin in patients with advanced or recurrent ovarian cancer. Background: We conducted a phase I study of dasatinib, an oral SRC tyrosine kinase inhibitor, in combination with paclitaxel and carboplatin in advanced and recurrent epithelial ovarian cancer (EOC). Methods: The primary objective was to determine the maximum tolerated dose (MTD). Secondary objectives included toxicity, response rate (RR), pharmacokinetics and pharmacodynamics. Based on the 3+3 design, cohorts of 3-6 pts received paclitaxel 175 mg/m2 and carboplatin AUC 6 every three weeks with escalating doses of dasatinib (100, 120, 150 mg daily), followed by an 8 patient expansion cohort. Results: Twenty patients were enrolled between 06/07 and 12/09. The median age was 61 yrs (42-82) with a median of 2 prior regimens (0-6), and 71% had platinum-sensitive disease. There were 3-6 pts in each cohort, and 8 in the expansion cohort. Pharmacokinetics were observed over the first 2 cycles of therapy. One DLT was observed in the 100 mg dasatinib cohort (grade 3 myalgia. Other toxicities in all cycles included neutropenia (95% grade 3-4), thrombocytopenia (35% grade 3-4), and fatigue (10% grade 3). The RR was 45% (complete responses, 3/18(17%); partial responses, 5/18(28%)) and 56% (10/18) had stable disease. The PFS6-month actuarial estimate was 86%. The median PFS and OS were 7.8 and 16.2 months, respectively. Conclusions: Due to the high incidence of myelosuppression with subsequent cycles the recommended phase II dose is 150 mg daily of dasatinib in combination with paclitaxel and carboplatin. The combination was safe with evidence of clinical activity in advanced EOC.
A phase I trial of dasatinib, an SRC-family kinase inhibitor, in combination with paclitaxel and carboplatin in patients with advanced or recurrent ovarian cancer.
Subject
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