Thesis (M.Sc.(Biochemistry)) -- University of Limpopo, 2019. / Retinoblastoma binding protein 6 (RBBP6) is the protein encoded by the Retinoblastoma Binding Protein 6 (RBBP6) gene that is located in chromosome 16p12.2. There is a growing list of newly discovered RBBP6 hypothetical splice variants but there are only three RBBP6 splice variants that are well documented. RBBP6 has been previously implicated in the regulation of cell cycle and apoptosis but little is known about the expression and regulation of the human RBBP6 splice variants during cell cycle progression and breast cancer development. This study was aimed at determining the expression pattern of RBBP6 alternatively spliced variants during arsenic trioxide-induced cell cycle arrest and apoptosis in breast cancer MCF-7 cells. It was also aimed at determining RBBP6 specific microRNAs and how they are regulated in MCF-7 breast cancer cells. MCF-7 cells were maintained and subjected to arsenic trioxide-induced cell cycle arrest and apoptosis. The MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) and the Muse™ Count & Viability assays were used to evaluate the effect of arsenic trioxide on the viability of MCF-7 cells. Cell cycle arrest using 11 μM arsenic trioxide and apoptosis using 32 μM arsenic trioxide were analysed using the MUSE® Cell Analyzer, light and fluorescence microscopy. Arsenic triode-induced apoptosis was analysed using the Muse™ Annexin V & Dead Cell Kit, MultiCaspase and MitoPotential assays using the Muse™ MultiCaspase Kit and Muse™ MitoPotential Kit. Arsenic trioxide-induced cell cycle arrest was analysed using the Muse™ Cell Cycle Kit. Semi-quantitative analysis of RBBP6 variants was carried out using the conventional Polymerase Chain Reaction (PCR), while the quantitative analysis was done using the Real-Time Quantitative PCR. The localization of RBBP6 isoforms was done using Immunocytochemistry (ICC). Web based Bioinformatics tools were used to identify RBBP6-specific microRNAs. The MTT results showed that arsenic trioxide decreased the viability of the MCF-7 cells in a dose-dependent manner. The Muse™ Cell Cycle analysis showed that 11 μM of arsenic trioxide induced G2/M cell cycle arrest in MCF-7 cells, while the Muse™ Annexin V & Dead Cell assay showed that 32 μM of arsenic trioxide induced the extrinsic apoptotic pathway in MCF-7 breast cancer cells. Using the conventional PCR, the MCF-7 cells were found to express the RBBP6 variant 1 transcript but lacks the expression of variant 2 and 3 transcripts, contrary to the kidney embryonic Hek 293 cells that exhibited the expression of RBBP6 variant 1, 2 and 3. Additionally, arsenic trioxide downregulated RBBP6 variant 1 in breast cancer cells during cell cycle arrest and apoptosis. The Real-Time PCR confirmed that MCF-7 cells lowly express RBBP6 variant 3. On the other hand, the ICC analysis showed that RBBP6 isoform 1 is localized and highly expressed in MCF-7 breast cancer cells. The Web based Bioinformatics tools showed that RBBP6 variant 1 specific microRNAs are down regulated in MCF-7 breast cancer cells. These results together showed that As2O3 is effective against MCF-7 cells and also regulated the expression of RBBP6 variants, especially, variant 1.
This study showed that there are RBBP6 variants that are involved in breast cancer progression and there are those that may be involved in breast cancer suppression. Targeting these RBBP6 variants for therapeutic development is a promising strategy. In conjunction with RBBP6 expression, arsenic trioxide should be further explored as a breast cancer drug.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ul/oai:ulspace.ul.ac.za:10386/2864 |
| Date | January 2019 |
| Creators | Makgoo, Lilian |
| Contributors | Mbita, Z. |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | xix, 110 leaves |
| Relation |
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