Microarray data of breast cancer cell line MDA MB 468 treated with siRNA against Glutamate Pyruvate Transaminase 2(GPT2) and non targeting control.
ABSTRACT: Glutamate Pyruvate Transaminase 2(GPT2) is highly expressed in MDA MB 468 cells. Knockdown of this gene slows down cell growth. This experiment was conducted to assess the effect at gene expression level to explain the phenotypic differences seen as a result of GPT2 knockdown.
Project description:Our goal was to identify the genes, which are modulated following conditional BSP knockdown for 3 and 6 days. Thus, to elucidate the BSP relevance and to broaden the knowledge on the affected signalling pathways. Cell pellets were collected after 3 and 6 days of cultivating the cells in media with or without doxycycline. There were duplicated total RNA samples for each time interval and each condition.The mRNA expression of the in vitro miRNA treated cells was compared to the respective +dox control.
Project description:miRNAs are small noncoding RNA molecules that play an important role in post-transcriptional regulation of gene expression. Length and/or sequence variants of the same miRNA are termed isomiRs. While most isomiRs are functionally redundant compared to their canonical counterparts, so-called 5’isomiRs exhibit a shifted 5’ end and therefore a shifted seed sequence resulting in a different target spectrum. However, not much is known about the functional relevance of these isoforms. Analysis of miRNA-seq data from breast cancer cell lines identified six pairs of highly expressed miRNAs and associated 5’isomiRs. Among them, hsa-miR-140-3p was of particular interest because its 5’isomiR showed higher expression compared to the canonical miRNA annotated in miRbase. This miRNA has previously been shown to control stemness of breast cancer cells. MiRNAseq data of breast cancer patients (TCGA dataset) showed that both the canonical hsa-miR-140-3p and its 5’isomiR-140-3p were highly expressed in patients compared to normal breast tissue. In the current work, we present the functional characterization of 5’isomiR-140-3p and the cellular phenotypes associated with its overexpression in MCF10A and MDA-MB-231 cell lines in comparison to the canonical hsa-miR-140-3p. Contrary to the effect of the canonical hsa miR 140-3p, overexpression of the 5’isomiR-140-3p led to a decrease in cell viability. The latter observation was supported by cell cycle analysis, where the 5’isomiR-140-3p but not the hsa-miR-140-3p caused cell cycle arrest in G0/G1-phase. Additionally, 5’ismoiR-140-3p overexpression was found to cause a decrease in cell migration in MCF10A cells. We identified three novel direct target genes of the 5’ isomiR-140-3p; COL4A1, ITGA6 and MARCKSL1. Finally, we have shown that knocking down these genes partially phenocopied the effects of the 5’isomiR-140-4p overexpression, where COL4A1 and ITGA6 knockdown led to reduced cell viability and cell cycle arrest, while MARCKSL1 knockdown resulted in a decrease in the migratory potential of cells. In summary, this work presents evidence that there is a functional synergy between the canonical hsa-miR-140-3p and the newly identified 5’isomiR-140-3p in suppressing growth and progression of breast cancer by simultaneously targeting genes related to differentiation, proliferation, and migration. With this array, we aimed to address the question which genes are regulated by either of the two forms of the miRNA. Samples were measured in one biological replicate of cells transfected with mimic-ctrl1 and mimic-ctrl2 (Dharmacon) as control samples and two biological replicates of cells transfected with hsa-miR-140-3p and 5'isomiR-140-3p (Exiqon) in 30nM concentration using Lipofectamin 2000 as transfection reagent.
Project description:The aim was to investigate the functional role of PLA2G7 in breast cancer using PLA2G7 silencing in cultured breast cancer cells. For stable knockdown of PLA2G7, shRNAs were transduced to MDA-MB-468 cells before genome-wide gene expression analysis. Gene expression profiles of the PLA2G7 silenced samples were compared with the scrambled control treated samples and two replicate samples were studied for each treatment.
Project description:Analysis of the effect of CLL development on differentation and gene expression of splenic monocytes. C57BL/6 mice were transplanted with murine CLL cells from Eµ-TCL1 mice and after 6 weeks total RNA was isolated from splenic monocytes from leukemic mice and matched WT controls.
Project description:The aim was to investigate the functional role of PLA2G7 in breast cancer using transient PLA2G7 silencing in cultured breast cancer cells. For transient knockdown of PLA2G7, siRNA (SI00072177; siPLA2G7, Qiagen, Valencia, CA) was transfected to MDA-MB-468 cells and incubated 48 hours before genome-wide gene expression analysis. Gene expression profiles of the PLA2G7 silenced samples were compared with the scrambled control treated samples and two replicate samples were studied for each treatment.
Project description:miRNAs regulate gene expression at post-transcriptional level by targeting 3å«UTR of mRNAs. To study the changes in mRNA expression level upon miRNA overexpression, we used microarrays to analyze transcriptome of MDA-MB-231 breast cancer cell line. Cells were transfected with miRNAs and negative controls in 3 biological replicates and analyzed to study gene expression changes.
Project description:In order to identify gene expression changes associated with the deregulated of IGF2, Doxycylin inducible empty vector and IGF2 shRNA permanent cell lines were generated by lentivral transduction in the colorectal cancer (CRC) cell line SW480 and selected with puromycin. For microarray analysis, both cell lines were plated at 3 x 105 cells in 6 well plates using RPMI plus 10% FCS and penicillin/ streptomycin, either without/ with 50 ng/ ml doxycycline at the time of plating. The respective cell lines were harvested at day 1, 3, and 5. Three experiments were performed for each time point and condition and were separately analyzed. The samples were analyzed on Illumina human HT-12 v 4 expression bead chips. The microarray data was processed and normalized using the lumi pipline.
Project description:Previously, we have identified cytosolic form of the branched chain amino-acid transaminase 1 (BCAT1) as notably hypomethylated in low-malignant potential (LMP) and high-grade (HG) serous epithelial ovarian tumors, compared to normal ovarian tissues. Here we show that BCAT1 is strongly overexpressed in both LMP and HG serous EOC tumors, thus suggesting that epigenetic mechanisms might be implicated in BCAT1 overexpression in serous epithelial ovarian cancer (EOC). Knockdown of the BCAT1 expression in EOC cells led to sharp decrease of cell proliferation and induced S-phase cell cycle arrest. Additionally, BCAT1 suppression significantly inhibited EOC cell migration and invasion. Gene expression profiling and consecutive network and pathway analyses confirmed these findings, as numerous genes and pathways known previously to be implicated in ovarian tumorigenesis, including EOC tumor invasion and metastasis, were found to be downregulated upon BCAT1 suppression, while some tumor suppressor genes were induced. Taken together, our data are indicative for a strong oncogenic potential of the BCAT1 gene in advanced EOC and identify this transaminase as a novel EOC biomarker and putative EOC therapeutic target. Overall design: To better understand the molecular mechanisms of BCAT1 gene action in ovarian cancer cells, we employed the Agilent Whole Human Genome microarrays, containing ~ 44,000 genes to identify global gene expression changes upon BCAT1 suppression in SKOV3 cells. We compared the gene expression of the previously selected clone shRNA-mediated BCAT1-knockdown SKOV3 clones B1 & B2 (sh-clB1 & sh-clB2) against the corresponding control (ctrl) clone. The microarray experiments were performed in duplicates, as four hybridizations were carried out for the BCAT1-suppressing cell clones against the corresponding control, using a fluorescent dye reversal (dye-swap) technique.
Project description:MDA-MB231 cells were transfected with control vector, RAI2-overexpression vector, or vector with RAI2-4A, which has a mutated interaction domain w.r.t. CtBP/CtBP2 Three replicates each of control condition, RAI2 overexpression or RAI2-4A overexpression.