Comparison of gene expression profiles of A549 cell treated with NRF2 siRNA and control siRNA
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ABSTRACT: To examine the role of NRF2 in accelerating cell proliferation and to identify the target genes responsible for this function, transcriptome analysis was performed using A549 cells, in which NRF2 is constitutively activated. NRF2 was knocked down by siRNA against NRF2, and the gene expression profile was compared with that of A549 cells treated with control siRNA. To exclude off-target effects, three different siRNAs against NRF2 was independently applied.
Project description:To examine the role of NRF2 in accelerating cell proliferation and to identify the target genes responsible for this function, transcriptome analysis was performed using A549 cells, in which NRF2 is constitutively activated. NRF2 was knocked down by siRNA against NRF2, and the gene expression profile was compared with that of A549 cells treated with control siRNA. To exclude off-target effects, three different siRNAs against NRF2 was independently applied. NRF2 siRNA or control siRNA was transfected into A549 cells. Cells were harvested 24 hours after transfection, and total RNA was purified.
Project description:KEAP1 overexpressed and NRF2 siRNA knockdown A549 NSCLC cells were used to identify downstream genes of NRF2 pathway separately and by combinatorial analysis. We used triplicate microarrays of transfected A549 cells with mKeap1-GFP for overexpression, siRNAs targeting NRF2 for knockdown and siGFP as control respectively. As a result, we identified several genes which are involved in cancer metabolic functions in these cells. We used microarrays to identify the gene downregulated in both KEAP1 overexpressed and NRF2 siRNA knockdown A549 NSCLC cells and found a subset of downregulated genes which are involved in metabolic functions.
Project description:To elucidate the mechanisms by which Nrf2 regulates cell growth, we performed global gene expression profiling of A549 lung cancer cells with knockdown of Nrf2. Gene networks associated with carbohydrate metabolism and drug metabolism were significantly downregulated in Nrf2-depleted A549 cells. Gene Set Enrichment Analysis revealed significant enrichment of genes associated with carbohydrate catabolic processes, positive regulation of metabolic processes, PPP, and arachidonic acid metabolism. In summary, this analysis revealed that Nrf2 positively regulates transcription of genes that play key roles in central carbon metabolism. A549 cells were transfected with non targeting NS siRNA or siRNA targeting Nrf2. Mock transfected A549 cells were treated with transfection reagent alone. We had 3 biological replicates for each of the 3 groups. Ninty six hours post transfection, cells were lysed and total RNA was isolated.
Project description:Aldehyde dehydrogenase isozymes ALDH1A1 and ALDH3A1 are highly expressed in non small cell cell lung cancer. Neither the mechanism nor the biological significance for such over expression have been studied. We used microarrays to analyze changes in A549 lung cancer cell line in which ALDH activity was reduced using lentiviral mediated expression of siRNA against both isozymes (Lenti 1+3) Experiment Overall Design: A549 lung cancer cell lines were transduced with lentiviral vectors containing specific siRNA sequences against ALDH1A1, ALDH3A1, both vectors (Lenti 1+3 cells), and against the green flourescent protein (GFP) gene (GFP cells, used as control).
Project description:This study aimed to elucidate the relationships between NRF2 and disease progression and provide insight into NRF2-mediated cancer progression/tumorigenesis by identifying novel genes and pathways regulated by NRF2 in A549 NSCLC cells
Project description:To elucidate the mechanisms by which Nrf2 regulates cell growth, we performed global gene expression profiling of A549 lung cancer cells with knockdown of Nrf2. Gene networks associated with carbohydrate metabolism and drug metabolism were significantly downregulated in Nrf2-depleted A549 cells. Gene Set Enrichment Analysis revealed significant enrichment of genes associated with carbohydrate catabolic processes, positive regulation of metabolic processes, PPP, and arachidonic acid metabolism. In summary, this analysis revealed that Nrf2 positively regulates transcription of genes that play key roles in central carbon metabolism.
Project description:We identified RNA binding motif protein 47 (RBM47) as a target gene of transforming growth factor (TGF)-beta in mammary gland epithelial cells (NMuMG cells) that have undergone the epithelial-to-mesenchymal transition (EMT). TGF-beta repressed RBM47 expression in NMuMG cells and lung cancer cell lines. Expression of RBM47 correlated with good prognosis in patients with lung, breast, and gastric cancer. RBM47 suppressed the expression of cell metabolism-related genes, which were the direct targets of nuclear factor erythroid 2-related factor 2 (Nrf2; also known as NFE2L2). RBM47 bound to KEAP1 and Cullin3 mRNAs, and knockdown of RBM47 inhibited their protein expression, which led to enhanced binding of Nrf2 to target genomic regions. Knockdown of RBM47 also enhanced the expression of some Nrf2 activators, p21/CDKN1A and MafK induced by TGF-beta. Both mitochondrial respiration rates and the side population cells in lung cancer cells increased in the absence of RBM47. Our findings, together with the enhanced tumor formation and metastasis of xenografted mice by knockdown of the RBM47 expression, suggested tumor suppressive roles for RBM47 through the inhibition of Nrf2 activity. Effect of shRNA for RBM47 and TGF-beta on gene expression was evaluated by RNA-seq and RBM47-bound RNAs were identified by RIP-seq in A549 cells.
Project description:In this study we transfected A549 cells with siRNA against TNFAIP2, infected them with L. pneumophila and performed transcriptional profiling. We found enrichment of genes in pro-inflammatory pathways by Pathway Over-respresentation analysis upon infection. There was no significant change in gene expression that we could attribute specifically to the knockdown of TNFAIP2. Examination of the transcriptional response of A549 cells to Legionella infection with concomitant TNFAIP2 knockdown