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RNF5 Regulation of RBBP4 Defines Acute Myeloid Leukemia Growth and Susceptibility to Histone Deacetylase Inhibitors

ABSTRACT: To investigate a potential role for RNF5 in AML, we monitored changes in gene expression profiles in multiple AML cell lines after RNF5 knock-down. PolyA RNA was isolated using the NEBNext Poly(A) mRNA Magnetic Isolation Module, and bar-coded libraries were constructed using the NEBNext Ultra™ Directional RNA Library Prep Kit for Illumina (NEB, Ipswich, MA). Libraries were pooled and single end-sequenced (1×75) on the Illumina NextSeq 500 using the High output V2 kit (Illumina, San Diego, CA). Quality control was performed using Fastqc (v0.11.5, Andrews S. 2010)), reads were trimmed for adapters, low quality 5′ bases, and minimum length of 20 using CUTADAPT (v1.1). The number of reads per sample and the number of aligned reads suggested that read quality and number were good and that the data were valid for analysis. High-quality data were then mapped to human reference genome (hg19) using STAR mapping algorithm (version 2.5.2a). FeatureCounts implemented in Subread (v1.50) was used to count the sequencing reads from mapped BAM files. Analyses of differentially expressed genes (DEGs) were subsequently performed using a negative binomial test method (edgeR) implemented using SARTools R Package. A list of the DEGs was observed and pathway analysis was performed using IPA (http://www.ingenuity.com) based on the following criteria: |log2(fold change)| >0.4 and p value < 0.05.

ORGANISM(S): Homo sapiens

PROVIDER: GSE155929 | GEO | 2021/07/25

REPOSITORIES: GEO

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Project description:RNA-sequencing data of the prostate cancer xenograft PC346C-DCC-K model. Tumor bearing mice were treated daily with Enzalutamide (N=10, 60mg/kg) or placebo (N=5, vehicle) for a period of 7 days. Tumor were subsequently isolated and snap-frozen. Tumor tissue was lysed and homogenized in QIAzol (ref #79306, Qiagen, Hilden, Germany) using an Ultra-Turrax T25 (Janke & Kunkel, Staufen, Germany). Total RNA was isolated using the miRNA-easy mini kit (ref # 217004, Qiagen), and RNA quality was measured using the Bioanalyzer RNA 6000 Nano assay (ref #5067, Agilent, Santa Clara, California, USA). All RNA samples had a RIN value ≥7. Library Prep was performed using the NEBNext Ultra Directional RNA Kit for Illumina according to the protocol "NEBNext Ultra Directional RNA Library Prep Kit for Illumina" (NEB #E7420S/L). Briefly, mRNA was isolated from total RNA using the oligo-dT magnetic beads. After mRNA fragmentation, cDNA synthesis was performed and used for ligation with adapters and PCR amplification of the resulting product. The quality and yield after sample preparation was measured with the Fragment Analyzer. One sample was re-sequenced due high frequency of multi-mapped reads implicating inadequate depletion of ribosomal RNA. Paired-end RNA-Seq data of enzalutamide PDX samples (N = 15) was analyzed using UCSC human genome build hg38 and GENCODE annotation release v26 (GRCh38.p10) and mouse genome build mm10 (reference strain C57BL/6J) with GENCODE annotation release M15 (GRCm38.p5) for downstream disambiguation of human/mouse data. FASTQC (v0.11.5) was applied on the paired-end FASTQ files for quality control, both before and after running trimmomatic (v0.36), which removed TrueSeq adapter sequences. STAR (v2.5.3a) was used as aligner, with 2-pass mapping for each sample separately. AstraZeneca’s disambiguation algorithm (Python variant, 2013) for reads aligned to two species has been used to assess the best alignments and disambiguate the BAM files. Mapping quality plot was generated and checked based on sambamba Flagstat (v0.6.7) statistics. Count files, with the number of reads for each gene were created with subread FeatureCounts (v1.5.2) on the disambiguated BAM files. R (version 3.4.3) was used for further statistical analyses and data visualization. Differential expression analysis was performed with condition ‘enzalutamide treated’ (N = 10) versus ‘untreated’ (N = 5) using the DESeq2 package (v1.18.1) and the Wald-test. P values were adjusted using the Benjamini-Hochberg procedure. Gene set enrichment analysis (GSEA, v4.1.0) was performed using normalized gene expression values (count per million) of the individual tumor samples, with condition ‘enzalutamide treated’ versus ‘untreated’ and applied to the Molecular Signatures Database (MSigDB) hallmark gene set collection.

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RNF5 Regulation of RBBP4 Defines Acute Myeloid Leukemia Growth and Susceptibility to Histone Deacetylase Inhibitors

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