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Design, synthesis, and bio-evaluation of 9-(pyrimidin-2-yl)-9H-carbazole derivatives that disrupt mitochondrial homeostasis in human lung adenocarcinoma

ABSTRACT: Purpose:To further explore the possible molecular mechanism by which compound 5n induces A549 cell apoptosis, we analyzed global transcriptome alterations of A549 cells at certain time points (48 h) after compound 5n treatment using RNA-seq analysis. Methods: Differential expression analysis for mRNA was performed using R package edgeR. Differentially expressed RNAs with |log2(FC)| value >1 and q value <0.05, considered as significantly modulated, were retained for further analysis. This choice is motivated by the decision to maximize the sensitivity of this analysis, in order to perform a massive screening and identify candidate genes to be validated with a wider sample population with real-time PCR analysis Results: The quantity of gene expression was calculated by FPKM (Fragments Per Kilobase of transcript per Million fragments mapped). Genes with log2 (Fold change) >1 and P value < 0.05 were considered as differentially expressed genes (DEGs). There were 146 upregulated genes and 158 downregulated genes for a total of 304 DEGs that were results are shown in Fig. 8A. Furthermore, the enriched KEGG pathways were analyzed and the top 30 pathways involved in these 304 DEGs between the two groups are shown in Figure SI. Among the top 30 pathways, 9 pathways related to mitochondrial function had significant effects. 37 DEGs in KEGG pathway associated with mitochondrial function was conducted and analyzed. Conclusions: Metabolism-related pathways was affected by compound 5n according the RNA-seq analysis

ORGANISM(S): Homo sapiens

PROVIDER: GSE190682 | GEO | 2022/07/01

REPOSITORIES: GEO

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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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Design, synthesis, and bio-evaluation of 9-(pyrimidin-2-yl)-9H-carbazole derivatives that disrupt mitochondrial homeostasis in human lung adenocarcinoma

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