Unknown,Transcriptomics,Genomics,Proteomics

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RNAseq of human epithelial lung carcinoma cell line A549 exposed to ceria, silver or zinc ions, micron-sized particles or nanoparticles

ABSTRACT: The toxicity of silver and zinc oxide nanoparticles is hypothesised to be mediated by dissolved metal ions and cerium dioxide nanoparticles (CeO2 NPs) are hypothesised to induce toxicity specifically by oxidative stress dependant on their surface redox state. To test these hypotheses, RNAseq was applied to characterise the molecular responses of cells to metal nanoparticle and metal ion exposures. The human epithelial lung carcinoma cell line A549 was exposed to different CeO2 NPs with different surface charges, micron-sized and nano-sized silver particles and silver ions, micron-sized and nano-sized zinc oxide particles and zinc ions, or control conditions, for 1 hour, 6 hours and 24 hours. Concentrations were the lower of either EC20 or 128 micrograms/mL. Transcriptional responses were characterised by RNAseq transcriptomics using an Illumina HiSeq2500 .

INSTRUMENT(S): Illumina HiSeq 2500

ORGANISM(S): Homo sapiens

SUBMITTER: Tim Williams

PROVIDER: E-MTAB-5734 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The A549 cell line is often used as a model for human lung adenocarcinoma, and thus, insights gained from studies using these cells can be valuable for understanding lung cancer biology. The gene of interest, GSE1, may play a crucial role in regulating various cellular functions. By knocking down GSE1 and observing the transcriptomic changes, the study aims to identify the downstream effects and pathways regulated by this gene. These could include pathways critical for cell growth, apoptosis, or other essential cellular functions. This can lead to better understanding of the role of GSE1 in lung cancer and may even identify potential therapeutic targets. The primary objectives of the experiment are: 1. To effectively knock down the expression of the GSE1 gene in A549 cells. 2. To perform a comprehensive transcriptomic analysis using RNA-Seq to identify differentially expressed genes (DEGs) post-GSE1 knockdown. 3. To perform pathway analyses to understand the functional implications of DEGs. 4. To construct interaction networks for identifying downstream targets and pathways influenced by GSE1. Experimental Workflow Overview 1. Cell Culture and Seeding: A549 cells were plated at a density of \\(2 \\times 10^5\\) cells/well in 6-well plates and cultured for 24 hours to allow for adhesion and growth. 2. Transfection: After 24 hours, cells were transfected with GSE1 siRNA or negative control siRNA. 3. Post-Transfection Incubation: Cells were incubated for an additional 48 hours post-transfection. 4. RNA Extraction: Cells were washed twice with PBS, and total RNA was extracted using the TRIzol reagent. 5. Quality Control: The quality and quantity of the extracted RNA were assessed using a NanoDrop One Microvolume UV-Vis Spectrophotometer. 6. cDNA Library Construction: cDNA libraries were generated from 500 ng of total RNA using the TruSeq RNA Sample Preparation Kit. 7. RNA Sequencing: Libraries were sequenced on a HiSeq 4000 instrument, generating approximately 20 million single-end reads (50SE) per sample. 8. Data Pre-processing: Raw sequencing data were processed to remove low-quality reads and adapters. Clean reads were obtained for downstream analyses. 9. Alignment and Mapping: Clean reads were aligned to the human reference genome using STAR software, allowing up to two mismatches. 10. Differential Expression Analysis: DEGs were identified between siGSE1 and control samples using DESeq2, applying an adjusted p-value cut-off of 0.05 and a false discovery rate (FDR) of ≤ 0.001. This structured approach allows for a comprehensive understanding of the role of GSE1 in A549 cells, potentially providing valuable insights into its role in lung cancer.

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RNAseq of human epithelial lung carcinoma cell line A549 exposed to ceria, silver or zinc ions, micron-sized particles or nanoparticles

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