A time course of infection of the alphavirus Sindbis virus (SINV) was used to investigate the presence of viral specific vsRNA and the changes in miRNAs profiles in human embryonic kidney 293 cells (HEK293) by high throughput DNA sequencing. Deep sequencing of small RNAs early in SINV infection (4 and 6 hpi) showed low abundance (0.8%) of viral specific RNAs (vsRNAs) , with a random uniform distribution not typical of Dicer products, suggesting they arise from non-specific degradation. Sequencing showed little variation of cellular microRNAs (miRNAs) at 4 and 6 hpi compared to uninfected cells. Twelve miRNAs exhibiting some minor differential expression by sequencing, showed insignificant modulation by Northern blot analysis. Overall design: RNA was isolated from mock infected and SINV inoculated HEK 293 cells at 4hpi and 6hpi cDNA libraries were generated for the small RNA (sRNA) content of the cells and sequenced using Illumina GA II, which yielded between 29.1M and 30.5M reads per sample
Small RNA analysis in Sindbis virus infected human HEK293 cells.
Specimen part, Cell line, Subject
View SamplesMicroRNAs (miRNAs) are short noncoding RNA molecules regulating the expression of mRNAs. Target identification of miRNAs is computationally difficult due to the relatively low homology between miRNAs and their targets. We present here an experimental approach to target identification where the cartilage-specific miR-140 was overexpressed and silenced in cells it is normally expressed in separate experiments. Expression of mRNAs was profiled in both experiments and the intersection of mRNAs repressed by miR-140 overexpression and derepressed by silencing of miR-140 was identified. The intersection contained only 49 genes, although both treatments affected the accumulation of hundreds of mRNAs. These 49 genes showed a very strong enrichment for the miR-140 seed sequence implying that the approach is efficient and specific. 21 of these 49 genes were predicted to be direct targets based on the presence of the seed sequence. Interestingly, none of these were predicted by the published target prediction methods we used. One of the potential target mRNAs, Cxcl12, was experimentally validated by Northern blot analysis and a luciferase reporter assay.
Experimental identification of microRNA-140 targets by silencing and overexpressing miR-140.
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View SamplesVitis vinifera endogenous small RNAs Overall design: Size fractionated small RNA from total RNA extracts of Vitis vinifera leaves, inflorescences, tendrils and small berries were ligated to adapters, purified again and reverse transcribed. After PCR amplification the sample was subjected to Solexa/Illumina high throughput pyrosequencing. Please see www.illumina.com for details of the sequencing technology.
Identification of grapevine microRNAs and their targets using high-throughput sequencing and degradome analysis.
Specimen part, Subject
View SamplesVitis vinifera RNA degradome Overall design: Isolated polyadenylated RNA from total RNA extracts of Vitis vinifera leaves, were ligated to 5'-adapter that include san MmeI recognition site. The ligated products were purified again, reverse transcribed and cleaved with MmeI. The 5' fragments were purified from gel and to a 3'- dsDNA adapter and PCR amplified. After PCR amplification the sample was subjected to Solexa/Illumina high throughput pyrosequencing. Please see www.illumina.com for details of the sequencing technology.
Identification of grapevine microRNAs and their targets using high-throughput sequencing and degradome analysis.
Specimen part, Subject
View SamplesGene expression from MCF7 breast cancer cells at different times of TNFa incubation:pcs2 and 14-3-3 transduced cells
Inhibition of specific NF-κB activity contributes to the tumor suppressor function of 14-3-3σ in breast cancer.
Time
View SamplesBariatric surgery, an effective treatment for obesity and diabetes, leads to profound remodeling of whole body energy homeostasis. We utilized a mouse model of vertical sleeve gastrectomy (VSG), a common bariatric surgery as a tool to identify novel secreted proteins and peptides that might act as important metabolic regulators. We analyzed gene expression in the stomach and intestines following VSG or sham surgery in diet-induced obese mice and sought to identify differentially regulated genes encoding secreted proteins/peptides. Overall design: Gene expression analysis of 5 different mouse intestinal tissues after VSG or sham surgery
LEAP2 Is an Endogenous Antagonist of the Ghrelin Receptor.
Specimen part, Treatment, Subject
View SamplesNF-kB has been linked to doxorubicin-based chemotherapy resistance in breast cancer patients. NF-kB nuclear translocation and DNA binding in doxorubicin treated-breast cancer cells have been extensively examined, however its functional consequences in terms the spectrum of NF-kB -dependent genes expressed and, thus, the impact on tumour cell behaviour are unclear.
Deficiency in p53 is required for doxorubicin induced transcriptional activation of NF-кB target genes in human breast cancer.
Cell line, Treatment
View SamplesSequencing of 5'' ends of RNA molecules from control and exosome-depleted S2 cells. Overall design: CAGE library construction from RNA extracted from control and exosome-depleted cells.
Transcription start site analysis reveals widespread divergent transcription in D. melanogaster and core promoter-encoded enhancer activities.
Subject
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Time-dependent transcriptional response of GOT1 human small intestine neuroendocrine tumor after <sup>177</sup>Lu[Lu]-octreotate therapy.
Time
View SamplesThe radiolabelled somatostatin analogue 177Lu-octreotate is a promising treatment option for malignant neuroendocrine tumors that overexpress somatostatin receptors. The human small intestine neuroendocrine tumor cell line GOT1 and Medullary thyroid carcinoma model GOT2 have shown promising treatment response to 177Lu-octreotate in xenografted mice. In clinical studies, however, only low cure rates have been achieved to date. In vitro and preclinical in vivo studies have shown that irradiation can up-regulate the expression of somatostatin receptors and thereby give an increased uptake of 177Lu-octreotate. The cellular processes that underlie positive treatment response to 177Lu-octreotate are otherwise largely unknown. Genome-wide analysis of tumor cell responses in this successful mouse model offers a venue to identify critical treatment parameters and to optimize clinical effectiveness of 177Lu-octreotate therapy. Combining 177Lu-octreotate with other anti-tumor agents has also been proposed as a strategy for optimization. Some studies have shown synergistic effects in tumor cell killing and volume reduction The hedgehog signaling pathway is involved in embryonic development and tissue regeneration and can be/is abnormally activated in various cancers. Inhibition of the hedgehog signaling pathway has yielded promising therapeutic effects on NE tumors and may potentially enhance the effects of 177Lu-octreotate treatment in patients.
Priming increases the anti-tumor effect and therapeutic window of <sup>177</sup>Lu-octreotate in nude mice bearing human small intestine neuroendocrine tumor GOT1.
Time
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