Generation of Patient-Matched Malignant and Normal Primary Cell Cultures from Clear Cell Renal Cell Carcinoma Patients
ABSTRACT: Transcriptome profiling of de novo-derived ccRCC cell cultures and their matching parental tumours. VHL-mutant and VHL wild-type cultures were established by isolating CA9+ and CA9- cells from tumor samples using FACS. RNASeq expression profiling of 18 renal cell carcinoma samples, including 6 patient tumours, 6 VHL mutant and 6 VHL WT derivative cell cultures
Project description:Interferons (IFNs) are key players in the antiviral response. IFN sensing by the cell activates transcription of IFN-stimulated genes (ISGs) able to induce an antiviral state by affecting viral replication and release. IFN also induces the expression of ISGs that function as negative regulators to limit the strength and duration of IFN response. The ISGs identified so far belong to coding genes. However, only a small proportion of the transcriptome corresponds to coding transcripts and it has been estimated that there could be as many coding as long non-coding RNAs (lncRNAs). To address whether IFN can also regulate the expression of lncRNAs, we analyzed the transcriptome of HuH7 cells treated or not with IFNα2 by expression arrays. Analysis of the arrays showed increased levels of several well-characterized coding genes that respond to IFN both at early or late times. Furthermore, we identified several IFN-stimulated or -downregulated lncRNAs (ISRs and IDRs). Further validation showed that ISR2, 8, and 12 expression mimics that of their neighboring genes GBP1, IRF1, and IL6, respectively, all related to the IFN response. These genes are induced in response to different doses of IFNα2 in different cell lines at early (ISR2 or 8) or later (ISR12) time points. IFNβ also induced the expression of these lncRNAs. ISR2 and 8 were also induced by an influenza virus unable to block the IFN response but not by other wild-type lytic viruses tested. Surprisingly, both ISR2 and 8 were significantly upregulated in cultured cells and livers from patients infected with HCV. Increased levels of ISR2 were also detected in patients chronically infected with HIV. This is relevant as genome-wide guilt-by-association studies predict that ISR2, 8, and 12 may function in viral processes, in the IFN pathway and the antiviral response. Therefore, we propose that these lncRNAs could be induced by IFN to function as positive or negative regulators of the antiviral response. HuH7 cells were treated with 10000 units/ml of IFN a2 and RNA was isolated 3 days post-treatment
Project description:Long noncoding RNAs (lncRNAs) have appeared to be involved in the most diverse cellular processes through multiple mechanisms. Here we describe a previously uncharacterized human lncRNA, CONCR (cohesion regulator noncoding RNA), transcriptionally activated by MYC, which is upregulated in multiple cancer types. The expression of CONCR is cell cycle-regulated, and it is required for cell cycle progression and DNA replication. Moreover, cells depleted of CONCR show severe defects in sister chromatid cohesion, suggesting an essential role for CONCR in cohesion establishment during cell division. CONCR interacts with and regulates the activity of DDX11, a DNA-dependent ATPase and helicase involved in DNA replication. These findings suggest a novel mechanism of action for CONCR in the modulation of DDX11 enzymatic activity, unveiling the direct involvement of a lncRNA in the establishment of sister chromatid cohesion. Characterization of the function of the long noncoding RNA CONCR. HCT116 p53-/- cells were left untreated (0h) or treated with the DNA damaging drug 5-FU for 4h and 12h.
Project description:RNA-seq profiling was conducted on clinically-annotated human pancreatic adenocarcinoma cancer tissues We measured the transcriptome in 51 clinically-annotated human pancreatic adenocarcinoma cancer tissues
Project description:Knowledge of the expression profile and spatial landscape of the transcriptome in individual cells is essential for understanding the rich repertoire of cellular behaviors. Here we report multiplexed error-robust fluorescence in situ hybridization (MERFISH), a single-molecule imaging approach that allows the copy numbers and spatial localizations of thousands of RNA species to be determined in single cells. Using error-robust encoding schemes to combat single-molecule labeling and detection errors, we demonstrated the imaging of 100 – 1000 unique RNA species in hundreds of individual cells. Correlation analysis of the ~10^4 – 10^6 pairs of genes allowed us to constrain gene regulatory networks, predict novel functions for many unannotated genes, and identify distinct spatial distribution patterns of RNAs that correlate with properties of the encoded proteins. A single sample is analyzed
Project description:Prostate cancer is the most common cancer in men and androgen receptor (AR) downstream signalings promote prostate cancer cell proliferation. To investigate the AR signaling, we performed directional RNA sequence analysis in AR positive prostate cancer cell line, LNCaP and VCaP. Using Noncode and GENCODE data sets. We identified androgen-regulated long non-coding RNAs (lncRNAs) in prostate cancer cells. Directional RNA sequence analysis of androgen-regulated lncRNAs in prostate cancer cells
Project description:Long non-coding RNAs (lncRNAs) exhibit a poor interspecies conservation and often show spatial- and temporal-specific expression patterns. What, if any, role they have in oxidative stress remains unknown. To identify potential roles for lncRNAs, we examined their expression in normal and H2O2-treated human umbilical vein endothelial cells. Oxidative stress related lncRNAs were generated by deep sequencing, using Illumina HiSeq 2000 or 2500 platform. Sequencing of the cDNA libraries from H2O2-treated HUVECs generated 12.5 million uniquely valid reads, meanwhile, 10.2 million valid fragments were obtained from control group in our experiment. A total of 10, 765 known and 30, 629 novel putative lncRNAs were identified according to RNA-Seq. Among them, 2, 091 of known and 25, 800 of novel lncRNAs were differentially expressed in H2O2-treated HUVECs compared with control HUVECs, and 12 of these were validated with qRT–PCR. Taken together, our findings provide evidence differentially expressed lncRNAs were mediated by oxidative stress in HUVECs, it is, therefore, likely that aberrant expression of lncRNAs, at least in part, participate in the process of endothelial injury caused by oxidative stress. Examination of lncRNAs in the oxidative-stressed human umbilical vein endothelial cells
Project description:C2H2 zinc finger proteins represent the largest and most enigmatic class of human transcription factors. Their C2H2 arrays are highly variable, indicating that most will have unique DNA binding motifs. However, most of the binding motifs have not been directly determined. We have determined the binding sites and motifs of 119 C2H2 zinc finger proteins and the expression pattern of 80 cell lines overexpressing C2H2 zinc finger proteins in order to study the role of C2H2 zinc finger proteins in gene regulation. We expressed GFP-tagged C2H2-ZF proteins in stable transgenic HEK293 cells. Total RNA was isolated using Trizol and sequencing libraries were constructed using TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero Gold or TruSeq RNA Library Preparation Kit v2.
Project description:Chromatin immunoprecipitation followed by next-generation DNA sequencing (ChIP-seq) is a widely used technique for identifying transcription factor (TF) binding events throughout an entire genome. However, ChIP-seq is limited by the availability of suitable ChIP-seq grade antibodies, and the vast majority of commercially available antibodies fail to generate usable datasets. To ameliorate these technical obstacles, we present a robust methodological approach for performing ChIP-seq through epitope tagging of endogenous TFs. We used Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-based genome editing technology to develop CRISPR Epitope Tagging ChIP-seq (CETCh-seq) of DNA-binding proteins. We assessed the feasibility of CETCh-seq by tagging several TFs spanning a wide range of endogenous expression levels in the hepatocellular carcinoma cell line HepG2. Our data exhibit strong correlations between both replicate types as well as with standard ChIP-seq approaches that use TF antibodies. Notably, we also observed minimal changes to the cellular transcriptome and to the expression of the tagged TF. To examine the robustness of our technique, we further performed CETCh-seq in the breast adenocarcinoma cell line MCF7 as well as mouse embryonic stem cells and observed similarly high correlations. Collectively, these data highlight the applicability of CETCh-seq to accurately define the genome-wide binding profiles of DNA-binding proteins, allowing for a straightforward methodology to potentially assay the complete repertoire of TFs, including the large fraction for which ChIPquality antibodies are not available. CRISPR/Cas9 mediated epitope tagging of transcription factors in human cell types
Project description:The goal of this study was to determine the similarity between human dermal microvascular endothelial cells, induced endothelial cells from fibroblasts, and fibroblasts through RNA-seq expression analysis. RNA samples from independently induced cultures, plus fibroblast and human dermal microvascular endothelial cultures were converted into individual cDNA libraries using Illumina TruSeq methods and subjected to single-end 50 base-sequence analysis at 20-30 million read depths. Examination of one fibroblast culture, one human dermal mibrovascular endothelial cell culture, and two induced endothelial cell cultures.