RNA-seq of the human breast cancer ERα-suppressed MCF-7(MCF-7/SP10+) cells and of their internal control MCF-7 (MCF-7/C) cells
ABSTRACT: Estrogens receptor a (ERα) is essential for breast tumors,since about seventy percent of breast cancers are detected as ERα positive.Recent studies suggest that ERα is related with the epithelial cell morphology. Recently, it has demonstrated that the suppression of ERα induced epithelial-mesenchymal transition (EMT) in the MCF-7 breast cacner cells. Interestingly, the loss of ERa resulted in strong differences on the gene expression profile of a variety of genes. Therefore, the aim of the RNA-seq is to elucidate the effect of the silencing of ERα on the mRNA levels of a larger variety of genes, thus revealing possible target genes which may be implicated on the aggressive phenotype and behavior of the ERα-suppresed MCF-7/SP10+ breast cancer cells. For this reason total RNA from both MCF-7/SP10+ cells and of their internal control MCF-7/C cells was extracted in 3 biological replicates and 3 technical replicates.
Project description:TGFB2-AS1 is a long non-coding RNA which is induced by ΤGFβ signaling. In order to assess the importance of TGFB2-AS1 on the regulation of gene expression, we performed an AmpliSeq transcriptomic array in human keratinocytes (HaCaT), which stably over-express TGFB2-AS1 or control pcDNA3 empty vector. In addition, cells were stimulated with TGFβ1 for 24 hours, in order to observe the effects of TGFB2-AS1 on gene expression, downstream of TGFβ signaling. RNA from the following four conditions was used in this experiment: 1) pcDNA3, 2) pcDNA3+TGFβ1, 3) pcDNA3-TGFB2-AS1, 4) pcDNA3-TGFB2-AS1+TGFβ1. Biological triplicates were used per condition.
Project description:The aim of this work was to elucidate the role of LKB1/STK11 and PARD3 in glioblastoma multiforme cells. For this we silenced the expression of these proteins using specific siRNA in two different patient-derived glioblastoma stem cells, U3031MG and U3034MG, then we analysed how the knock-down of these genes affects gene expression using Ion torrent AmpliseqTM.
Project description:Analysis of CGTH-W-1 follicular thyroid carcinoma cells transcriptome following 48 hrs siRNA-mediated depletion of PROX1. PROX1 is a homeobox transcription factor. PROX1 depletion decreases migratory ability, motility and invasivness and induces profound cytoskeleton changes of CGTH-W-1 cells. Results provide insight into the role of PROX1 in the thyroid cancer. Three biological replicates for a given condition
Project description:To investigate the effect of indole on protection against colon injury and the role of IDO1 expression, we examined the effect of indole on hIDO1-overexpressing and empty vector control HCT116 cells by global gene expression profiling. Examination of mRNA levels from hIDO1-overexpressing and control HCT116 cell lines treated with 1mM indole or DMF for 24 hours using two replicates each.
Project description:Purpose: The goals of this study are to determine the effect of microRNA-17 overexpression on 20,803 human genes in RASFs using Ion ProtonTM System platform. Human RASFs from two RA patients were transfected with pre-miR-17 or NC-pre-miR for 48 h and total RNA was prepared using miRNeasy kit (Qiagen). Total RNA integrity was checked using an Agilent Technologies 2100 Bio analyzer (Santa Clara, CA). 10 ng of high quality RNA was used to make cDNA for amplification with the Ion AmpliSeq Transcriptome Human Gene Expression kit (ThermoFisher Scientific). The cDNA was subjected to 12 cycles of amplification with panel primers and barcoded with adapters as recommended. Resulting sequencing libraries were quantified by qPCR using SYBR FAST master mix from KapaBiosystems (Wilmington, MA). Sets of eight libraries were balanced, pooled and sequencing beads produced on an Ion Chef. Sequencing was performed on an Ion P1 semi-conductor sequencing chip using an Ion Proton™ System (ThermoFisher Scientific, Grand Island, NY). Data was collected and primary analysis performed using Torrent Suite software version 5.0.3. Reads were mapped to the panel and expression values determined. R Software version R-3.2.3 was used to generate heatmap. Among the panel of 20,803 genes, the expression of 15,067 genes as shown in the representative heat map was observed in pre-miR-17 and NC-pre-miR transfected RASFs. A total of 664 significantly modulated genes (301 upregulated and 363 downregulated) using Student ‘t’ test were further utilized for the IPA analysis. The result of IPA predicted the protein ubiquitin pathway as a major canonical pathway affected by the differentially regulated genes. Interestingly, IPA analysis generated an interactome that showed connectivity among various ubiquitin ligases, NF-ԟB family, AP-1/cJun, 20S and 26S proteasome system. Conclusion: Our results clearly shows the major pathways affected by miR-17 overexpression in RASFs were Protein ubiquitination related. mRNA profiles of pre-miR-17 and NC-pre-miR transfected RASFs were generated by AmpliSeq, in duplicate, using Ion Proton™ System.
Project description:In the present study, we used a high-throughput small RNA deep sequencing followed by a systematic computational analysis to identify genome wide mutant p53R273H regulated miRNAs in both DNA damage dependent and independent context. Several miRNA-mRNA regulatory networks have been predicted that might contribute to mutant p53 GOF properties. Differentially regulated miRNA signature profile has been validated in the lung cancer patients harboring wildtype and mutant p53. We identified specific miRNA signatures for lymph node metastasis associated with p53 mutation in lung adenocarcinoma and also predicted the possible contribution of two mutant p53 regulated miRNAs in EMT process. Furthermore, this study identified a hitherto unknown miRNA in human which might act as one of the crucial downstream targets of GOF mutant p53 to confer oncogenic properties. Determination of mutant p53R273H regulated microRNAs H1299 cells.
Project description:To test whether the addition of a peptide nucleic acid (PNA) clamp, which binds WT KRAS at codon 12, can increase the efficacy of mutation detection for KRASG12D within a targeted NGS setting. We tested the effect of clamping the wild-type KRAS sequence in a reference standard (Tru-Q 7, 1.3% Tier from Horizon Diagnostics, Cambridge, UK) with a KRAS c.35G>A mutation (KRASG12D) at an allelic frequency (AF) of 1.3% assessed by digital droplet PCR (ddPCR). We then re-tested the PNA on circulating-free DNA from a patient harbouring a KRASG12D mutation (at an AF of 3.2%, determined by ddPCR). Multiple runs were conducted using 10, 5, 2.5 and 1ng of DNA input.