GSE169013
Phenotypically anchored mRNA and miRNA expression profiling in zebrafish reveals flame retardant chemical toxicity networks
Purpose: The goals of this study are to examine mRNA and miR networks on exposures to flame retardant chemicals (FRCs), an ubiquitous group of chemicals in the environment Methods: Four biological replicates were created by pooling eight embryos per replicate from individual wells which were placed into an Eppendorf Safelock Tube and excess solution removed. 0.5 mM zirconium oxide beads were added along with 500 µL of RNAzol (Molecular Research Center, Inc.) and the tubes were immediately placed into a bullet blender (Next Advance), using settings recommended by the manufacturer. The RNA was purified using the Direct-zol MiniPrep kit (Zymo Research), including an optional DNase-1 digestion treatment for 15 minutes. RNA integrity (RIN) was assessed using an Agilent Bioanalyzer (Santa Clara, CA), and RNA samples with RIN values >8 were processed for library preparation and sequencing at the Oregon State University Center for Genome Research and Biocomputing. Total RNA was used as input for both mRNA and miRNA sequencing. For mRNA sequencing, mRNA was poly A selected, libraries were prepared with the PrepX™ mRNA and Illumina sequencing workflow (Wafergen Biosystems). For miRNA seq, the Illumina TruSeq Small RNA library kit was used to generate small RNA libraries from total RNA. For sequencing, an Illumina HiSeq 3000 sequencer (Illumina, San Diego) was used for mRNA and small RNA single-end sequencing at 100 and 50 base pairs, respectively. Bioinformatics analysis of sequencing data was performed on an R platform. Briefly, reads were evaluated by FastQC v0.11.3 to detect major sequencing problems, and then trimmed for quality control with Skewer v0.2.2 .  RNA-seq alignment and quantification proceeded with Bowtie2 v2.2.3  being used to build HISAT2 (Kim et al., 2013) genome index files from the Genome Reference Consortium Zebrafish Build 10 (GRCz10) genome. For miR identification and quantification, a combination of miRDeep2 v2.0.0.8, miRBase release 22 and Bowtie v1.2.1.1 were used. Differential expression between experimental and control samples was determined with functions from the Bioconductor package, edgeR; mRNAs with a log2 fold change =1.5 and Benjamini-Hochberg (BH) adjusted p = 0.05 were considered differentially expressed while an adjusted p = 0.05 was applied to miRs without any fold change cutoffs. Heatmap clustering of differentially expressed genes were generated in R based on their log2 fold changes using the ggplot2 package. Results: We found widespread disruption of mRNA and miR expression. Neurodevelopment was a key disrupted biological process across multiple FRCs and was corroborated by behavioral deficits. Several mRNAs (eg: osbpl2a) and miRs (eg- mir-125b-5p), showed differential expression common to multiple FRCs (10 and 7 respectively). These common miRs were also predicted to regulate a network of differentially expressed genes with diverse functions, including apoptosis, neurodevelopment, lipid regulation and inflammation. Commonly disrupted transcription factors (TFs) such as RXR, RAR and VDR were predicted to regulate a wide network of differentially expressed mRNAs across a majority of the FRCs. Many of the differential mRNA-TF and mRNA-miR pairs were predicted to play important roles in development as well as cancer signaling. Specific comparisons between TBBPA and its derivative TBBPA-DBPE showed contrasting gene expression patterns that corroborated with their phenotypic profiles. The newer generation FRCs such as IPP and TCEP produced distinct gene expression changes compared to the legacy FRC BDE-47. Conclusions: Our study is the first to establish a mRNA-miR-TF regulatory network across a large group of structurally diverse FRCs and diverse phenotypic responses. The purpose was to discover common and unique biological targets that will help us understand mechanisms of action for these important chemicals. Overall design: mRNA and miR profiles of zebrafish embryos exposed to 10 FRCs from 6 to 48 h post fertilization (hpf)