Abstract
We report the first quantitative and qualitative analysis of the poly (A)+ transcriptome of two human mammary cell lines, differentially expressing (human epidermal growth factor receptor) an oncogene over-expressed in approximately 25% of human breast tumors. Full-length cDNA populations from the two cell lines were digested enzymatically, individually tagged according to a customized method for library construction, and simultaneously sequenced by the use of the Titanium 454-Roche-platform. Comprehensive bioinformatics analysis followed by experimental validation confirmed novel genes, splicing variants, single nucleotide polymorphisms, and gene fusions indicated by RNA-seq data from both samples. Moreover, comparative analysis showed enrichment in alternative events, especially in the exon usage category, in ERBB2 over-expressing cells, data indicating regulation of alternative splicing mediated by the oncogene. Alterations in expression levels of genes, such as LOX, ATP5L, GALNT3, and MME revealed by large-scale sequencing were confirmed between cell lines as well as in tumor specimens with different ERBB2 backgrounds. This approach was shown to be suitable for structural, quantitative, and qualitative assessment of complex transcriptomes and revealed new events mediated by ERBB2 overexpression, in addition to potential molecular targets for breast cancer that are driven by this oncogene.
Highlights
Global comparative analysis of transcriptomes is the most effective approach for definition of alterations in gene expression profiles and has led to the identification of key defective elements involved in complex diseases such as cancer
Adapters containing specific 4nt- barcode were designed and added to DpnII digested cDNAs from both cell lines, and samples were pooled before sequencing (Figure 1). cDNA samples were sequenced on the Titanium 454-Roche platform generating 802,214 reads, with a total of 160,223,981 high-quality nucleotides (Phred $20) (Figure 2)
Two subsets were separated according to genome alignment parameters that differed in percentage of coverage and identity: a subset of completely aligning reads and another subset of reads with partial alignment to the human genome, respectively containing 651,058 reads (89%) and 80,570 reads (11%)
Summary
Global comparative analysis of transcriptomes is the most effective approach for definition of alterations in gene expression profiles and has led to the identification of key defective elements involved in complex diseases such as cancer. Different aspects of quantitative gene expression have been investigated in breast cancer by microarray-based analysis [1,2,3,4,5,6], with important implications for prognosis [7,8]. The management of breast cancer patients takes into consideration a combination of clinical and histopathological characteristics, together with the measurement of estrogen (ER) and progesterone (PR) hormone receptors and Her2/ERBB2 overexpression/amplification. ERBB2, overexpressed in 25 to 30% of human breast cancers [11,12], is associated with metastasis [13], and ERBB2-overexpressing cells are self-sufficient with respect to, anchorage-independent growth and efficient in invasion [14]. A significant fraction (,60%) of patients with metastatic breast tumors does not respond to the treatment [15], highlighting the necessity for continued investigation of ERBB2-mediated modifications in breast cells
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