It is now clear the majority of the human genome is transcribed from both DNA strands but only 2% encodes protein. Much of this transcription is derived from DNA sequences that do not encode functional proteins. The majority of these transcripts are long non-coding RNAs (lncRNAs) defined as being >200 bp in length. While it is generally accepted lncRNA transcription is functionally significant, the scope and function of lncRNAs in cancer is still not well understood. In the last five years, genome wide association studies (GWAS) have identified 170 common variants (or SNPs) associated with an increased risk of breast cancer. Importantly, the majority of these disease-associated SNPs lie within intergenic regions and within introns of protein-coding genes, suggesting that undiscovered RNA transcripts such as lncRNAs, may be responsible for the risk in a subset of breast cancers. We have recently used a targeted RNA sequencing approach called RNA CaptureSeq to identify lncRNAs transcribed from breast cancer risk loci. This key experiment has identified hundreds of candidate breast cancer-associated lncRNAs.
In this project, we will use multiple in vitro approaches to identify lncRNAs whose expression is altered by breast cancer risk SNPs. These include eQTL analyses, chromosome conformation capture (3C)-based techniques and reporter assays. We will also generate isogenic cell lines using CRISPR/Cas9 technology, which will be used to measure lncRNA expression and identify allele-specific chromatin interactions. We expect that some of the lncRNAs will have cancer-related biological functions. We will therefore overexpress or silence lncRNAs in breast cells and examine their effects on cell proliferation, response to DNA damage, apoptosis, migration, invasion and tumour formation. We will also assess the function of lncRNAs in breast tumour formation using an explant assays in mice. The discovery of novel regulatory RNAs influencing breast cancer development may reveal entirely new avenues for breast cancer therapeutics. Students will have access to unique expertise and reagents, and will acquire skills in tissue culture, CRISPR/Cas9, RNA manipulation, and other basic molecular biology techniques.