Effect of activating Wnt signalling on expression of 3' UTR spliced transcripts in colorectal carcinoma cell line HCT116
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1055037 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR27299554_1.fastq.gz | Fastqsanger.gz | |||
| SRR27299554_2.fastq.gz | Fastqsanger.gz | |||
| SRR27299555_1.fastq.gz | Fastqsanger.gz | |||
| SRR27299555_2.fastq.gz | Fastqsanger.gz | |||
| SRR27299556_1.fastq.gz | Fastqsanger.gz |
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Project description:The Wnt signalling pathway is commonly mutated or dysregulated in colorectal cancer, including approximately 93% of solid tumour samples from The Cancer Genome Atlas. Such dysregulation commonly leads to overactivation of CTNNB1, a transcription factor central to Wnt signalling. We have previously observed an increase in splicing of the 3'UTR of CTNNB1 in colon cancer samples compared to healthy samples. Additionally, we have observed isoform-specific regulation of CTNNB1 3'UTR spliced and retained isoforms in response culturing HCT116 cells with increasing concentrations of Wnt activator CHIR99021, and the opposite upon incubation with Wnt inhibitor IWR-1. By treating HCT116 cells with a high concentration of Wnt activator CHIR99021 and performing RNA sequencing, we assessed the global impact of Wnt signalling on expression of transcripts with spliced 3'UTRs. Interestingly, we observe an enrichment of alternative splicing events relating to components of the canonical Wnt signalling pathway itself, highlighting potential autoregulatory functions.
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Project description:The presence of an exon junction more than 55nt downstream of a stop codon is generally considered to be a trigger for nonsense-mediated decay (NMD). Thus, transcripts containing 3’UTR introns (3UIs) are often considered as transcriptional noise. Interestingly, upon knockdown of NMD factor UPF1 we see examples of both increased and decreased expression of the 3UI-spliced isoforms. In the oncogene HRAS, upon UPF1 knockdown, we see the 3UI-retaining isoform increases in expression where the spliced isoform decreases, suggesting that 3UI splicing protects the transcript against UPF1-mediated degradation.
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Project description:Effect of nonsense-mediated decay inhibition on expression of 3' UTR spliced transcripts in colorectal carcinoma cell line HCT116
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Project description:VEGF165 is one of the key regulators of tumor development. Using HCT116 cells transfected with full-length vegf mRNA, full-length vegf mRNA with mutations of H9D and L14E, mutated vegf mRNAs lacking untranslated regions (UTRs), and 5â??UTR mutated between nt 591 and nt 746, we found that vegf 5â??UTR resided an anti-apoptotic activity against chemotherapy independently of VEGF165. We next established HCT116 clones stably expressing vegf 5â??UTR or the mutated vegf 5â??UTR. The cells expressing 5â??UTR, but not the mutated UTR, showed the drug-resistant phenotype, anchorage-independent growth, and rapid tumor growth when implanted in athymic nude mice. Microarray and real-time PCR showed that the vegf 5â??UTR-expressing tumors up-regulated anti-apoptotic genes including mia and down-regulated pro-apoptotic genes including fas, pdcd1, nrg1, and bax. Microarray analysis also revealed specific down-regulation of IFN-inducible genes (43 genes) in the growing tumors. HCT116 cells stably transcribing vegf 5â??UTR decreased STAT1 expression and IFN alpha-STAT1 pathway. In addition to 5-fluorouracil treatment, the vegf 5â??UTR-expressing tumors did not respond to IFN alpha therapy at all. This novel tumor-promoting function in the vegf 5â??UTR may partly explain the insufficiency of the VEGF-VEGFR strategies and suggest a vegf-targeted silencing strategy as a more effective anti-tumor therapy. Experiment Overall Design: We established HCT116 clones stably expressing vegf 5â??UTR (vegf 5â??-G5) and vegf 5â??UTR mutated between nt 591 and nt 746 (vegf 5â?? mut-D5) and implanted into the left and the right flanks of the identical mouse, respectively. The generated tumors on day 14 after injection were removed and total RNA was extracted from each tumor using RNeasy mini (Qiagen). After removing contaminated DNA by DNaseI treatment, 10 micro grams each of RNA of vegf 5â??-G5-derived tumor from 3 individual mice was mixed and prepared for microarray analysis as sample RNA. The reference RNA of vegf 5â?? mut-D5-derived tumor was arranged in the same manner. The quality of both RNA samples was confirmed using an Agilent 2100 Bioanalyzer. Microarray analysis was then performed: briefly, 1 µg of sample RNA (vegf 5â??-G5) and reference RNA (vegf 5â?? mut-D5) were converted to cDNA with oligo dT conjugated T7 promoter primer and reverse transcriptase. These cDNAs were amplified using T7 RNA polymerase while labeling with either Cy5-CTP or Cy3-CTP (Low RNA Fluorescent Linear Amplification Kit, Agilent Technologies). 750 ng of Cy3 and Cy5-labeled cRNA were mixed and hybridized on an Agilent 22k human 1A array in a hybridization oven at 65oC for 17 h as rotating. Following hybridization and washing of a slide according to manufactureâ??s protocol, the arrays were scanned using the Agilent DNA Microarray Scanner (G2565BA) and microarray images were analyzed using Agilent Feature Extraction Software (v7.5). The final signal intensity was resulted from subtracting background signal from row spot signal. The gene expression level was presented as ratio of sample intensity against reference intensity.
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Project description:BACKGROUND:The rodent malaria parasite Plasmodium yoelii is an important animal model for studying host-parasite interaction and molecular basis of malaria pathogenesis. Although a draft genome of P. yoelii yoelii YM is available, and RNA sequencing (RNA-seq) data for several rodent malaria species (RMP) were reported recently, variations in coding regions and structure of mRNA transcript are likely present between different parasite strains or subspecies. Sequencing of cDNA libraries from additional parasite strains/subspecies will help improve the gene models and genome annotation. METHODS:Here two directional cDNA libraries from mixed blood stages of a subspecies of P. yoelii (P. y. nigeriensis NSM) with or without mefloquine (MQ) treatment were sequenced, and the sequence reads were compared to the genome and cDNA sequences of P. y. yoelii YM in public databases to investigate single nucleotide polymorphisms (SNPs) in coding regions, variations in intron-exon structure and differential splicing between P. yoelii subspecies, and variations in gene expression under MQ pressure. RESULTS:Approximately 56 million of 100 bp paired-end reads were obtained, providing an average of ~225-fold coverage for the coding regions. Comparison of the sequence reads to the YM genome revealed introns in 5' and 3' untranslated regions (UTRs), altered intron/exon boundaries, alternative splicing, overlapping sense-antisense reads, and potentially new transcripts. Interestingly, comparison of the NSM RNA-seq reads obtained here with those of YM discovered differentially spliced introns; e.g., spliced introns in one subspecies but not the other. Alignment of the NSM cDNA sequences to the YM genome sequence also identified ~84,000 SNPs between the two parasites. CONCLUSION:The discoveries of UTR introns and differentially spliced introns between P. yoelii subspecies raise interesting questions on the potential role of these introns in regulating gene expression and evolution of malaria parasites.
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Project description:Activating transcription factor 4 (ATF4) is activated during cellular stress through a pathway called the integrated stress response (ISR). We had previously reported that the splicing inhibitor isoginkgetin (IGG) activates ATF4 and ATF4-dependent transcripts. To determine the role of ATF4 in the transcriptional response to IGG, we used tandem CRISPR cas9 gene editing to create an ATF4 deficient HCT116 (colon cancer) cell line. We completed RNA sequencing on HCT116 parental and HCT116 ATF4 deficient cells treated with IGG, and thapsigargin (Tg), a positive control for ATF4 activation. We found that IGG led to the differential expression of 76 transcripts, and 58 of these were dependent on ATF4. Tg led to a far more robust transcriptional response, which appeared to be less ATF4 dependent.
2024-10-09 | GSE249009 | GEO
Project description:Gene expression profiles were obtained via NanoString nCounter Gene Expression Assay (PanCancer Progression Panel, XT_PGX_HuV1_CancerProg_CSO, cat. no. XT-CSO-PROG1-12, NanoString Technologies, Hamburg, Germany). We aimed to decipher the role of activating transcription factor 2 (ATF2) in colorectal cancer, so-far masked by high intratumoral heterogeneity (ITH). To overcome ITH, we generated monoclonal ATF2 knockout (KO) cells (termed F9 and E5) using CRISPR/Cas9 gene editing and analyzed alterations of their gene expression profiles in comparison to the parental HCT116 cell line (HCT116 WT).
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Project description:The 3' untranslated region (3'UTR) of mRNA plays several important roles in post-transcriptional gene regulation. Some of its functions include regulating mRNA stability by polyadenylation and microRNAs. However, the overall function of the Gata1 3’UTR in mammals has not been defined. In this study, we used CRISPR/CAS9 technology to knock out the sequence of the mouse Gata1 3’UTR. We found a defect in erythropoiesis in mutant mice, evidenced by macrocytic anemia at the baseline. Ablation of Gata1 3’UTR also resulted in a reduced number of erythroid precursors that might be associated with the cell cycle, especially G2/M disruption in fetal livers. Mechanistically, deletion of the Gata1 3’UTR destabilizes the Gata1 mRNA and ultimately reduces Gata1 protein levels. The low stability of the Gata1 mRNA is unlikely to be caused by the loss of binding of microRNAs or lack of polyadenylation; rather, in part, by the dissociation of AU-rich elements in the 3’UTR with a trans-activating factor Elavl1. Specifically, we transcribed Gata1 3’UTR in vitro and performed an RNA pulldown assay followed by mass spectrometry to profile the proteins that bind the 3’UTR. Gene Ontology analysis demonstrated that several proteins specifically targeting the 3’UTR were found to potentially bind Gata1 3’UTR, among which Elavl1 was in almost all categories related to mRNA stabilization. Western blotting and RNA immunoprecipitation confirmed the direct interaction of Gata1 3’UTR with Elavl1. Manipulation of Elavl1 activity and protein levels by the small molecule inhibitor Dihydro-tanshinone-I and Elavl1 overexpression in fetal liver erythroblasts confirmed Elavl1 as a stabilizing factor for Gata1 mRNA. Our findings shed light on the functional significance of the 3’UTR of Gata1 mRNA in the context of erythroid development. More importantly, our findings highlight the complexity and diversity of regulatory mechanisms that govern Gata1 mRNA stability and precise expression at the post-transcriptional levels. In addition, our findings prove that miRNAs are not always essential in vivo for controlling transcription factor levels to maintain body homeostasis.
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