Abstract
New Zealand has one of the highest breast cancer rates in the world. To date, all approved breast cancer drugs are based on protein targets. However, research over the last decade has shown the importance of non-coding RNA (ncRNA) in cancer development and progression. One of the largest subclasses of ncRNA is long-noncoding RNA (lncRNA), charaterised by a nucleotide length of over 200 bases. Additionally, lncRNA are expressed in a very tissue and cell specific manor making them attractive potential drug targets. The current study examines the lncRNA hMaTAR20, the human orthologue of a mouse gene associated with angiogenic tumour development and metastasis in mice. To characterise hMaTAR20 as an oncogene in breast cancer this study used two cell lines. The first was the estrogen receptor positive breast cancer cell line, T47D which had a CRISPR-Cas9 mediated hMaTAR20 knockout. The second was the triple negative breast cancer cell line, LM2i which had a CRISPRi mediated hMaTAR20 knockdown. The study was divided into three sections in-vitro, in-vivo and mechanistic investigation. The in-vitro section used 3D culture to grow tumour spheroids, which revealed that gene silencing of hMaTAR20 reduced branching, a characteristic associated with cellular proliferation and migration. The subsequent in-vivo mice trials showed that CRISPRi mediated knockdown of hMaTAR20 in triple negative breast cancer cells led to decreased tumour burden and increased necrosis. The increased tumour necrosis suggested that hMaTAR20 is mediating nutrient delivery in tumours, possibly through angiogenesis. Additionally, the mice trials showed the hMaTAR20 is not involved in the latter stages of the metastatic cascade. Subsequent targeted gene expression analysis showed a dysregulation of genes involved in the VEGF angiogenic signalling pathway in the T47D hMaTAR20 knockout cell lines. Additionally, RNA-seq was done, however results were inconclusive. Overall, this study showed the importance of hMaTAR20 in two breast cancer cell lines as a driver of cellular proliferation in-vitro and potential angiogenic driver in-vivo, further highlighting the importance of lncRNAs as possible therapeutic targets.