GSE157725
RNA-seq analysis of SF3B2-depleted and pladienolide B-treated prostate cancer PC3 cells.
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. Overall design: SF3B2-depleted and pladienolide B-treated prostate cancer PC3 cells