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RNA sequencing of wild-type and mutant KRAS human lung cancer cell lines with of without FASN inhibition.

ABSTRACT: The goal of this study is to compare the RNA-seq derived transcriptome of mutant KRAS and wild-type KRAS lung cancer cell lines under fatty acid synthase (FASN) inhibition. We extracted total RNA with TRIzol from cells treated with 0.2 μM TVB-3664 (FASN inhibitor) or 0.2% DMSO for 4 days. One µg of total DNAse-treated RNA was used for library. cDNA was ligated to adapters and PCR amplified. Before being normalized and pooled, samples were quantified by Qubit then run on the Illumina NextSeq 500. Reads were aligned to the human hg38 reference genome. Only uniquely mapped reads were retained for further analyses. Gencode annotation for human (version v37) was used as reference alignment annotation and downstream quantification. Gene level expression was calculated using featureCount (v2.0.1) using intersection-strict mode by exon. Counts were calculated based on protein-coding genes from the annotation file. Low expressed genes were filtered using a per time-point approach with RPKM >= 0.5 in all samples in one or the other group.

ORGANISM(S): Homo sapiens

PROVIDER: GSE168782 | GEO | 2022/06/29

REPOSITORIES: GEO

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Project description:We examined FASN knockdown LNCaP cells obtained by shRNA transduction with Mission lentiviral transduction particles (SHCLNV-NM 00410, TRCN3128, Sigma) (FASN-RNAi cells). In this study, we used cells transfected with non-targeting shRNA as a control (control-RNAi cells). The expression of genes related to cellular proliferation (phospholipase A2, group IVA, PLA2G4A; tensin 3, TNS3; glypican 4 GPC4), cell adhesion and extracellular matrix organization [peroxidasin homolog (Drosophila) PXDN; sarcoglycan epsilon, SGCE; von Willebrand factor, VWF; hydroxysteroid (17-beta) dehydrogenase 12, HSD17B12; cysteine-rich secretory protein LCCL domain containing 2, CRISPLD2], and cell motility (TNS3, RAP2B member of RAS oncogene family, RAP2B) were shown to be down-regulated by FASN inhibition with RNAi. FASN inhibition led to down-regulation of the PLA2G4A and HSD17B12 genes encoding phospholipase A2 and 17-beta hydroxysteroid dehydrogenase, respectively, which are the key enzymes related to production of an intracellular second messenger arachidonic acid and androgen hormones, both playing roles in promotion of tumor progression. We also found that the genes related to arachidonic acid signalling, including RGS2, SPAG16, VWF and RAP2B, were also suppressed with FASN inhibition. Gene expression profiling therefore demonstrated that FASN inhibition induces down-regulation of genes related to cell proliferation, cell adhesion, migration, and invasion, as well as the production of arachidonic acid and androgen hormones, both of which drive tumor progression. Total RNA isolation was performed with a Micro-to-Midi total RNA purification system (Invitrogen). The integrity of total RNAs was evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies). Low Input Quick Amp Labeling Kit, one-color (Agilent Technologies) was used to prepare Cy3-labelled target cRNA according to the manufacturer's instructions. Labeled cRNAs were hybridized with a SurePrint G3 Human GE 8M-CM-^W60K Microarrays (Agilent Technologies). Two separate hybridizations were performed for each sample. Array images were captured using a DNA Microarray Scanner (Agilent Technologies), and data were analyzed using Feature Extraction Software (Agilent Technologies) to obtain background-corrected signal intensities. The data were further analysed with GeneSpring GX Software (Version 11.0, Agilent Technologies). After filtering of data, mRNAs differentially expressed in target versus control were considered using the Fisher exact test, followed by multiple corrections using the Benjamini and Hochberg false discovery rate (FDR) method. Gene sets with a FDR q-value < 0.05 were considered significant.

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RNA sequencing of wild-type and mutant KRAS human lung cancer cell lines with of without FASN inhibition.

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