BioProject

The goals of this study were to identify LIN28 downstream gene targets in breast cancer cells. We use a subclone of the MCF-7 breast cancer cell line, MCF-7M as our model system. Methods: mRNA profiles from MCF-7M breast cancer cells treated with siRNA against non-targeting control (NT), LIN28, hnRNP A1, LIN28 and hnRNPA1 (LIN28A1) for 72 hrs were generated by deep sequencing, in duplicate, using Illumina HiSeq 2000. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: Burrows–Wheeler Aligner (BWA) followed by ANOVA (ANOVA) and TopHat followed by Cufflinks. qRT–PCR validation was performed using TaqMan and SYBR Green assays Results: Using an optimized data analysis workflow, we mapped over 200 million sequence reads per sample to the human genome (build h19). Each of the four groups had two biological replicates. We developed a custom method to identify alternative splicing events and identified 111 genes with significant (FDR<0.05) differential splicing for LIN28 depleted cells compared to non-targeting siRNA control, as well as 249 and 182 genes for hnRNP A1 and LIN28A1 respectively. RNA-seq data were validated with by qRT–PCR analysis of a subset of genes. Conclusions: Results reveal that LIN28 regulates alternative splicing and steady state mRNA expression of genes implicated in aspects of breast cancer biology. Notably, cells lacking LIN28 undergo significant isoform switching of the ENAH gene, resulting in a decrease in the expression of ENAH exon 11a isoform. Expression of ENAH isoform 11a has been shown to be elevated in breast cancers that express HER2. Overall design: mRNA profiles of MCF-7M cells treated with siRNA for NT control, LIN28, hnRNP A1, and LIN28 plus hnRNP A1 (A1) (LIN28A1) were generated by deep sequencing, in duplicate, using Illumina HiSeq 2000