Illumina Microarray of T47D cell with or without KDM7A-DT silencing under normal and oxidative stress conditions
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ABSTRACT: KDM7A Divergent Transcript (KDM7A-DT) is a stress-induced lncRNAs. In our previous studies, KDM7A-DT showed the most robust cellular phenotype alteration and a significant TP53-dependency upon oxidative and oncogenic stress induction. Since about 50% of human breast cancers have mutant p53 and associated genetic alterations, it is essential to determine what effect a mutant p53 would have on acquired pro-oncogenic functions of KDM7A-DT. In these experiments, we used four antisense LNA Gapmer sequences designed by Exiqon (Vedbaek, Denmark). One of these was a negative control, while the other 3 were designed to target the end of the transcript representing the full-length lncRNA KDM7A-DT (lncRNA; GRCh38/hg38, chr7: 140,178,951-140,179,640). To test the effect of KDM7-DT in the context of mutant p53, we selected the p53-negative luminal BC cells T47D. For each of the LNA gapmers, we utilized 3 technical replicates across two main experimental conditions (normal vs. oxidative stress induced via 30 mins of 0.2 mM H2O2 treatment). Overall, we measured the signals for 47,323 Illumina array probes across 24 individual experiments (4 LNA gapmers * 3 technical replicates * 2 main experimental conditions).
Project description:KDM7A Divergent Transcript (KDM7A-DT) is a stress-induced lncRNAs. In our previous studies, KDM7A-DT showed the most robust cellular phenotype alteration and a significant TP53-dependency upon oxidative and oncogenic stress induction. To investigate the functional roles of KDM7A-DT, we first performed overexpression experiments using fibroblasts. We found that MRC5 cells overexpressing KDM7A-DT escaped cell cycle proliferation and long-term survival ability and were associated with a distinct stress-related morphology (enlarged and flattened cells) compared to cells expressing just the vehicle control. To examine the effects of KDM7A-DT overexpression on the transcriptome, we performed a differential expression gene (DEG) analysis comparing a group of four independent biological replicates of MRC5 overexpressing KDM7A-DT to a group of samples from control cells.
Project description:We describe here the proteome of the human breast cancer cell line MDA-MB-231 expressing normal levels of the lncRNA NORAD, or after knock-down NORAD with siRNA and LNA gapmers.
Project description:Development of LNA gapmers, antisense oligonucleotides used for efficient inhibition of target RNA expression, is limited by non-target mediated hepatotoxicity issues. In the present study, we investigated hepatic transcription profiles of mice receiving non-toxic and toxic LNA gapmers after a single and repeat administration.
Project description:Development of LNA gapmers, antisense oligonucleotides used for efficient inhibition of target RNA expression, is limited by non-target mediated hepatotoxicity issues. In the present study, we investigated hepatic transcription profiles of mice receiving non-toxic and toxic LNA gapmers after a single and repeat administration. To understand the mechanism of LNA gapmer-induced heptotoxicity in mice, we investigated the transcription profiles of liver RNA isolated from mice receiving non-toxic sequence (NTS-1), toxic sequence (TS-2), or severely toxic sequence (HTS-3) of LNA gapmers at 25 mg/kg (dose volume of 10 mL/kg) at 8, 16, or 72 hrs after a single administration (by subcutaneous injection ) using microarray analysis. We also investigated the transcription profiles of liver RNA isolated from mice receiving non-toxic sequence (NTS-1) or toxic sequence (TS-2) of LNA gapmers at 25 mg/kg (dose volume of 10 mL/kg) at 2 weeks after repeated administration (by subcutaneous injection ) using microarray analysis.
Project description:We immunoprecipitated EZH2, together with associated chromatin isolated from the HUVECs (2 x 10^6) that were transfected with MEG3 GapmeRs (10 nM, 48 h) or a scrambled control GapmeRs (Ctr). For transfection, we used 10 nM scrambled LNA (locked nucleic acids) GapmeR control (Cat. No. 339515) or phosphorothioate antisense standard GapmeRs MEG3-lncRNA (Cat No. 339511, Qiagen). On beads crosslinked chromatin complexes were reversed, and DNA purified using QIAquick® PCR Purification Kit and quantified by Qubit HS assay (Q33230)
Project description:Identification of transcriptional program influenced by the expression of lncRNA MAAT, a novel lncRNA highly expressed and strongly correlated with ALK-negative anaplastic large cell lymphomas. LncRNA MAAT was silenced through Antisense LNA GapmeRs strategy.
Project description:We have performed a transcriptomics study in which we first transfected LNA GapmeRs against DR5-AS lncRNA to silence it in HeLa cells. Total RNA was isolated from control as well as transfected cells and silencing was confirmed by qPCR. Total RNAs were subjected to deep-sequencing to identify differentially expressed mRNAs. We then took advantage of bioinformatic tools to identify which pathways are affected by DR5-AS knock-down.