ADAR1 role on transcriptome stability

Abstract: RNA editing has emerged as novel mechanisms involved in cancer progression. Despite the phenotypic implications of ADAR in several cancer models, the role of ADAR on DNA damage response (DDR) and proliferation in breast cancer (BC) has not been fully addressed. To evaluate the effect of ADAR expression or the stability of targets ZR-75-1 were transduced using a shRNA against ADAR and cultured for 16h in the presence or absence of triptolide, a robust RNA pol II inhibitor, followed to transcriptome analysis comparing vehicle and treated cells. ZR-75-1 SHC, and ZR-75-1 ADAR KD cells were treated using 100nM Triptolide - RNA pol II inhibitor (Invivogen) or 100 nM DMSO used as a vehicle. After 16 h of treatment, total RNA was extracted. RNA quality from each biological replicates (N=3) was analyzed using Experion (Biorad) system to further generate RNA-seq libraries using the TruSeq Stranded Total RNA LT kit (Illumina). Finally, RNA-seq libraries were sequencing using a Hiseq4000 (BGI, Korea). Fastq Files were aligned using STAR using hg19, counting transcripts using HT-seq software. Finally, differential expression analysis was performed using DEseq2 software according to their recommendations. Our results suggest that after ADAR knock down and triptolide treatment, DNA repair, Cell cycle progression, ERRB2 signaling among others pathways suffer a significant decrease in their mRNA stability. On the contrary, mRNAs of genes associated with rRNA processing, mRNAs of genes involved in the surveillance through the nonsense-mediated decay and pro-apoptotic related mRNAs exhibited increased stability after ADAR knock down. ZR-75-1 SHC, and ZR-75-1 ADAR KD cells were treated using 100nM Triptolide - RNA pol II inhibitor (Invivogen) or 100 nM DMSO used as a vehicle. After 16 h of treatment, total RNA was extracted using RNeasy columns following the manufacturer’s instructions, including DNAse treatment (QIAGEN). RNA quality from each biological replicates (N=3) was analyzed using Experion (Biorad) system to further generate RNA-seq libraries using the TruSeq Stranded Total RNA LT kit (Illumina). RNA-seq libraries were sequenced using a Hiseq4000 (BGI, Korea). Fastq files were aligned using STAR and hg19, counting transcripts using HT-seq software. Differential expression analysis was performed using DEseq2 software following standard recommendations.