Abstract 1987: RNA-seq of the bronchial airway transcriptome identifies novel gene and microRNA expression changes associated with smoking and lung cancer

Abstract

Abstract Prior studies have shown that cigarette smoke creates a field of molecular injury in the airway epithelium. Using microarrays, our group has previously identified gene and microRNA expression differences in bronchial airway epithelial cells that are associated with smoking and lung cancer. We hypothesize that RNA-seq of the airway transcriptome will enhance our understanding of the response to tobacco smoke exposure and lung cancer pathogenesis by identifying mRNA splice variants, non-coding RNA, and novel RNA not interrogated by microarrays. We profiled pooled bronchial airway epithelial cell brushings (n=3 patients/pool) obtained during bronchoscopy from healthy never and current smoker volunteers and smokers with and without lung cancer undergoing surgery for suspicion of lung cancer. The high MW fraction (>200 bp) was amplified using a combination of oligo d(T) and random hexamers (NuGEN, San Carlos, CA) followed by library preparation with Illumina's mRNA Seq Library Prep Kit and sequencing using Illumina's Genome Analyzer generating ∼27-30 million 36 bp reads/sample. The low MW fraction (15-40 bp) was processed using ABI's Small RNA Library Sequencing Kit and sequenced using ABI's SOLiD system generating ∼50-90 million 35 bp reads/sample. The high MW reads were aligned to rRNA (5-13%), the human genome (33-40%), and to computationally generated splice junctions (1%) using BowTie. Differential gene and isoform expression was evaluated using Cufflinks and the R package Genominator. Low MW reads were aligned using both RNA2MAP and Geospiza to a filter containing rRNA, tRNA, and repeats (4-14%), to miRBase (4-10%), and the human genome minus miRBase sequences (4-8%). Expression values were calculated for microRNAs. We compared Affymetrix Exon 1.0 ST, HGU133A2, and Invitrogen miRNA microarrays to RNA-seq and found strong correlation for genes/miRNAs interrogated by microarrays, with most smoking- and cancer related changes in transcript expression being identified by sequencing and not by arrays. Genes found differentially expressed only by sequencing were validated by RT-PCR. RNA-seq also reveals differentially expressed isoforms, potential novel miRNAs, and several isoforms of known miRNAs which are being further investigated. RNA-seq provides a comprehensive and high-resolution view of the airway transcriptome and will provide insights into the molecular field of injury induced by smoking and the pathogenesis of smoking-related lung disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1987.