Project description:We depleted TIMM13 using short interfering RNA (siRNA) in A549 lung cancer cells, and found that silencing of TIMM13 could significantly inhibit A549 cell proliferation. Thus, we conducted RNA-seq in the A549 cells transduced with either nontargeting siRNA or two distinct TIMM13-specific siRNAs. We observed widespread gene expression change in A549 cells upon TIMM13 knockdown.
Project description:CNOT2 depletion partially decreased number of colony in colony forming assay and cell number in proliferation assay for NSCLC cells. Co-treatment with TRAIL and depletion of CNOT2 dramatically declined survival rate and increased apoptosis compared with TRAIL only in TRAIL resistant NSCLC cells. So, to find major signaling molecules in CNOT2 depleted condition of A549 cells, gene expression analysis was designed.
Project description:Background: Non-Small Cell Lung Cancer (NSCLC) presents as a highly metastatic disease with Kras and P53 as prevalent oncogenic driver mutations. Endocytosis, through its role in receptor recycling and enrichment, is important for cancer cell proliferation and metastasis. Huntingtin Interacting Protein 1 (HIP1) is a clathrin mediated endocytic adapter protein found overexpressed in different cancers. However, conflicting roles both as a tumour promoter and suppressor are reported. HIP1 expression is found repressed at advanced stages and some HIP1-ALK fusions are reported in NSCLC patients. However, the molecular mechanisms and implications of HIP1 depletion are not completely understood. Methods: HIP1 depletion was performed using siRNA transient transfection and validated using immunoblotting for each experiment. HIP1 depleted A549 cells were analysed for deregulated global proteome using label-free LC-MS. Gene expression dataset was analysed using TCGA, GTeX and GEO to explore HIP1 expression in Lung cancer patients. Kaplan–Meier Plotter database was used to analyse the survival correlation between HIP1 mRNA expression in lung cancer patients. Various functional assays such as matrigel based invasion, trans-well migration, soft agar colony, tube formation are performed after HIP1 depletion. NRF2 inhibitor was used after HIP1 knockdown to assess its effect on invasion and soft agar colony formation. Results: In silico analysis of HIP1 transcript expression reveals that it is reduced in high-grade and metastatic lung cancer patients correlating with poor survival. Global proteome profiling reveals that HIP1 depleted A549 cells are enriched in pathways associated with metabolism, proliferation and survival. Molecular and functional analysis indicate higher invasive ability of HIP1 depleted cells. The secretome from HIP1 depleted cells also increases the angiogenic potential of HUVEC cells. NRF2 inhibition in HIP1 depleted cells reduces invasion of NSCLC cells with different driver mutations. Conclusion: Our study shows that HIP1 depletion leads to activation of various molecular pathways responsible for cell proliferation and survival. Additionally, enhancement of invasion in HIP1 depleted subsets of NSCLC cells is via upregulation of NRF2 and can be reversed by its inhibitor.
Project description:To investigate the difference of miRNA expression between lung cancer cell A549 and its DDP-resistant cell strain A549/DDP, we have employed miRNA microarray expression to discover the difference expression of miRNAs of A549 cells and A549/DDP. We conducted RT-qPCR to examine the expression levels of top differential expressed miRNAs, namely, miR-197-5p, miR-4443, miR-642a-3p, miR-27b-3p and miR-100-5p, confirming low variability between two methods. The A549/DDP was established from A549 in our laboratory, by exposing A549 to gradually increasing DDP concentrations, until the final concentration at 1μg/ml. To avoid the influence of drug to the A549/DDP cells, they were cultured in a drug-free medium for at least two weeks before gene expression analysis. miRNA expression of A549 and A549/DDP was then analzyed.
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:Interactions between the gene products encoded by the mitochondrial and nuclear genomes play critical roles in normal eukaryotic cellular function. Here, we characterized the metabolic and transcriptional properties of A549 lung cancer cells and their isogenic mitochondrial DNA (mtDNA)-depleted rho zero counterparts grown in cell culture and as tumor xenografts in immune-deficient mice. A manuscript summarizing our conclusions is under review. Experiment Overall Design: A549 rho zero and their parental A549 cells were grown in culture and as xenografts in nude mice. All experiments conducted in culture were performed in triplicate (6 experiments total) and all experiments conducted in xenografts were performed in quadruplicate (8 experiments total). A manuscript summarizing our experimental design is under review.
Project description:Resveratrol, a natural phytoestrogen found in red wine and a variety of plants, is reported to have protective effects against lung cancer, however there is very little work directed towards the understanding of the mechanism of action of resveratrol in lung cancer. In this study we used an experimental approach to understand the biological activity and molecular mechanisms of resveratrol in A549 lung cancer cells. Gene expression profiles were compiled using an oligonucleotide microarray to determine altered expression levels in resveratrol treated cells. Keywords: Genetic modification of A549 cells in response to resveratrol
Project description:To investigate the difference of miRNA expression between lung cancer cell A549 and its DDP-resistant cell strain A549/DDP, we have employed miRNA microarray expression to discover the difference expression of miRNAs of A549 cells and A549/DDP. We conducted RT-qPCR to examine the expression levels of top differential expressed miRNAs, namely, miR-197-5p, miR-4443, miR-642a-3p, miR-27b-3p and miR-100-5p, confirming low variability between two methods.
Project description:Embelin (15 uM) induced alterations in the gene expression profile was studied in A549 lung cancer cells during the initial stages of apoptosis (4h following treatment)