MiRNA profiling of triple breast cancer; an in vitro and in silicon study of potential diagnostic markers and drug targets
Date
2016-02-12
Authors
Crawford, Nicole
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Abstract
Triple-negative breast cancer (TNBC) represents approximately 20% of breast cancers and is characterised by a lack of expression of oestrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor 2 (HER2). TNBC is particularly aggressive and affects younger pre-menopausal women, and is associated with a poor prognosis, particularly in African females. Currently the only treatment available for TNBC is chemotherapy, which tends to result in resistance as well as recurrence after 5 years and ultimately death. Hormone therapy is not a viable treatment strategy due to the tumours lacking surface hormone receptors. The deficit in knowledge relating to this breast cancer type has resulted in a deficiency of effective targeted therapies. In this regard, there is a great need for TNB tumours to be subclassified in order to optimise treatment plans for individual patients. While at present there is no reliable method for categorising TNBC, a method based on gene expression profiles has yielded 6 different subtypes. In the present study a novel approach was taken to evaluate the utility of microRNAs (miRNAs) as markers of hormone insensitive breast cancer. miRNAs are short, single-stranded non-coding RNAs with a regulatory function in cells. The expression of these molecules is often dysregulated in cancer, and their involvement in disease development and progression has been proven in many different cancers. The aim was thus to identify miRNAs which play a role in TNBC pathology and to determine their gene targets in order to discover novel targets for therapy. An in vitro cell culture approach was taken to investigate the utility of miRNA analysis in TNBC targeted therapy development and subclassification. Extracted RNA from each cell line was shipped to Seattle, USA for miRNA profiling by two different storage methods. One set of duplicate samples was transported in the conventional frozen, dry ice mode, while the other set of duplicates was transported in a fairly novel “dry” RNAstable mode. The miRNA expression results were compared between the frozen and dried duplicate samples with a standard two-tailed Student’s t-test. The test yielded no significant difference between the two storage strategies (p-values < 0.05). These results indicate that RNAstable is a reliable, inexpensive mode of transporting sensitive biological samples. To profile the expressed miRNAs, Nanostring nCounter analysis was performed on two different TNBC cell lines (MDA-MB-231 and MDA-MB-436), as well as on a non-cancerous epithelial breast cell line (MCF-10A) and on an oestrogen-sensitive breast cancer
cell line (MCF-7). The expression results were compared to identify aberrantly expressed miRNAs in the two TNBC cell lines, relative to the two control cell lines. A comparison between the two TNBC cell lines was also considered to appraise a novel method for subclassifying TNBC. A total of 12 significantly altered miRNAs, 9 overexpressed, and 3 underexpressed within the two TNBC cell lines were identified based on selection criteria. miRNAs expressed in the two TNBC cell lines with a fold change of at least 2 relative to the two control cell lines, were considered significant and were selected for bioinformatic analysis. Following this, in silico analysis using the DIANA microT v.4 software was used to ascertain significant gene targets of the aberrant miRNAs. Another selection process for miRNA target analysis was carried out to determine the top three most significant gene targets. Subsequently, further analyses of targets were carried out through a comprehensive literature review. The comparison between the two TNBC cell lines yielded a 25% difference in the miRNA expression patterns, suggesting that miRNA expression profiles may well be a novel and reliable method for subclassifying TNBCs. Ultimately, this could elucidate more effective and personalised treatment plans.
Description
Degree of Master of Science by research only:
A Dissertation submitted to the Faculty of Health Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science in Medicine. August 2015