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RNA-seq analysis of SF3B2-depleted and pladienolide B-treated prostate cancer PC3 cells.

ABSTRACT: Purpose: Small interfering RNA (siRNA)-mediated knockdown of SF3B2, a component of U2 snRNP subcomplex in the spliceosome, suppresses innate immune gene expression. To examine whether pladienolide B, a pre-mRNA inhibitor, phenocopies SF3BP2 knockdown, we compared the RNA-seq profiles of SF3B2-depleted and pladienolide B-treated PC3 cells. Method: Total RNAs were extracted from the cells and treated with DNase I. After RNA-seq libraries were constructed, next-generation sequencing and Differential Expressed Gene (DEG) analysis were performed. The library was sequenced on an Illumina NextSeq 6000 as paired-end 101 bp reads. The sequence reads were aligned to the GRCh38 reference genome using HISAT2 version 2.1.0, Bowtie2 2.3.5.1. All reads were assembled with StringTie software version 2.1.3b, using annotated genes from the NCBI database. Low quality transcripts are filtered. Afterwards, TMM Normalization are performed. Statistical analysis is performed using Fold Change, exactTest using edgeR per comparison pair. The significant results are selected on conditions of |fc|>=2 & exactTest raw p-value<0.05. DEG analysis was performed on 3 comparisons pairs using edgeR. The results showed 11,306 genes which satisfied |fc|>=2 & exactTest raw p-value<0.05 conditions in at least one of comparison pairs. For Enrichment test which based on Gene Ontology (GO) DB was conducted with significant gene list using g:Profiler tool. Result: Gene signatures of SF3B2-depleted cells were compared with those of control siRNA-treated cells. GO analysis of the down-regulated genes in SF3B2-depleted cells identified GO terms related with innate immunity. Next, gene signatures of pladienolide B-treated cells were compared with those of DMSO-treated cells. GO analysis of genes altered in pladienolide B-treated cells identified GO terms related with RNA splicing. However, innate immunity-related GO terms were classified statistically not significant. Conclusion: These observations indicate that SF3B2 knockdown and pladienolide B differentially affect the gene expression signature in PC3 cells.

ORGANISM(S): Homo sapiens

PROVIDER: GSE157725 | GEO | 2021/03/10

REPOSITORIES: GEO

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Project description:The goal of this study was to investigate transcriptome remodeling induced by treatment with a camptothetin analog, Topotecan (TPT), of a primary T cell model of HIV latency. The study aimed to determine whether TPT has a global inhibitory effect on gene expression in primary CD4+ T cells, and identify mechanisms of action of this drug as an HIV “block and lock” agent. Methods: CD4+ T cells were isolated from blood of HIV seronegative study participants (N=3) and utilized to generate an in vitro model of latent HIV infection (model developed in the Spina laboratory and previously described by Spina et al., 2013 and Soto et al., 2022). Following generation of the model, cells were treated with 10 uM Topotecan or its solvent dimethyl sulfoxide (DMSO) for 24 hours. Cells were lyzed with RLT buffer containing beta-mercaptoethanol from a RNeasy micro kit (Qiagen, Inc. cat # 74004). ERCC spikes (Thermo Fisher Scientific, Inc.) were added to cell lysates based on cell number in each sample (10 ul of 1:800 dilution per million cells). RNA was extracted and subjected to deep sequencing at the Institute for Genomics Medicine (IGM) Genomics Center at the University of California San Diego. Filtering low quality reads and removal of the 3’ adapter sequences were performed using the Trim Galore tool. Reads were mapped to the human genome (build hg38) using HISAT2, and to HIV and ERCC references using bowtie. Mapped human reads were counted against the human GENCODE annotation (v37) using HT-Seq. ERCC counts were used to determine whether TPT had global inhibitory effect on transcription in primary CD4+ T cells. Because no such effect was observed, differential gene expression analysis was performed using library EdgeR in Bioconductor R without correction of expression based on ERCC spikes. Genes with false discovery rate (FDR)-corrected p-value less than 0.05 and an absolute fold change in TPT-treated samples compared to DMSO controls greater than 2, were considered significantly modulated by TPT. Pathway and gene ontology (GO) term enrichment analysis was performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) v2022q3. Pathways and GO terms with FDR-corrected p-value less than 0.05 were considered significantly enriched for differentially expressed genes. Results: An average of 19.7 million reads per sample were acquired and mapped to the human genome (build hg38). After applying filtering criteria, 9,678 human genes were identified with the HISAT2 and HTSeq workflow. Differential expression analysis was performed between TPT- and DMSO-treated samples using EdgeR. A total of 1,749 differentially expressed genes (DEGs) were identified with FDR p-value <0.05 and an absolute fold change greater than 2; of which 522 were up- and 1227 downregulated by TPT. Pathway and GO term enrichment analysis revealed that TPT interferes with gene transcription and cell signaling pathways (e.g. T cell receptor signaling, positive regulation of JNK cascade, regulation of actin cytoskeleton and positive regulation of GTPase activity were affected by TPT treatment). Conclusion: The study demonstrated that TPT induces multiple effects on transcriptome in primary CD4+ T cells. Some of these changes may represent direct and indirect mechanisms of action of TPT as an HIV “block and lock” agent.

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RNA-seq analysis of SF3B2-depleted and pladienolide B-treated prostate cancer PC3 cells.

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