Background and aims: It has been shown that membrane-presented CD40 ligand (mCD40L) is highly pro-apoptotic to carcinoma cells but not normal cells. This effect occurs even when mCD40L is presented to carcinoma cells by mCD40L-expressing cells.MicroRNAs (miRNAs) are small non-coding RNAs with a length of about 22 nt that play various roles in cell biology including apoptosis. MiRNA usually regulates apoptosis in two ways (i): by upregulating apoptosis pathway or (ii) by downregulating it. It is not known what miRNAs are involved in CD40-mCD40L-induced apoptosis. This study aims to find the most significant miRNAs responsible for apoptosis in CD40 after its ligation with mCD40L.Material and methods: CD40 activation in human EJ (bladder carcinoma) cells was performed by co-incubation with mCD40L expressing murine fibroblasts for 6 hours. Control co-cultures of EJ cells with murine fibroblasts that do not express mCD40L were also performed. Induction of apoptosis was determined using DNA fragmentation, cytotoxic glo, cascade 3-7 assays, and western blot. RNA was isolated from cells and submitted to NovoGene for RNAseq analysis. Bioinformatic analysis of RNAseq data was performed using adapter sequence trimming, low-quality read removal plus alignment, and annotation techniques.Results: Apoptosis was triggered in EJ carcinoma cells after incubation with mCD40L-expressing cells, while no such effect was seen with 3T3Neo cells. Western blot and SDS-PAGE analyses showed TRAF1 expression, confirming that mCD40L-CD40 interaction initiates apoptosis in EJ cells. Additionally, 23 miRNAs were found to play significant roles in this apoptosis process, with 90% of them linked to pathways that either promote or inhibit apoptosis, based on target gene analysis.Discussion and Conclusion: The results demonstrate apoptosis is induced in bladder carcinoma cells through CD40-CD40L ligation and the identification of microRNAs associated with the induction of apoptosis. Several microRNAs likely to regulate apoptosis are identified through RNA sequencing and bioinformatic analysis. These findings provide new insights into the molecular mechanisms of mCD40L, induced cell death in carcinoma cells, and highlight key microRNAs involved in this process.
| Date of Award | 9 Jun 2025 |
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| Original language | English |
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| Supervisor | Roger Phillips (Main Supervisor) |
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