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Title: Role of microRNAs in determining the cancer-associated fibroblast phenotype
Author: Inche Zainal Abidin, Siti Amalina
ISNI:       0000 0004 6424 1487
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2017
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Background: Transforming growth factor beta 1 (TGF-β1), a pro-fibrotic tumour- derived factor, promotes myofibroblast differentiation in the tumour microenvironment and is thought to contribute to the development of pro-tumourigenic cancer associated fibroblasts (CAF). To date, the molecular mechanisms underlying CAF differentiation are poorly characterised. Here, the contribution of a class of small non-coding RNA, miRNA, to myofibroblastic CAF differentiation was examined. The expression profile of miRNA in experimentally-derived CAF (eCAF) was determined compared to normal fibroblasts and CAF derived from oral cancers, and the functionality of selected miRNAs in the reprogramming of normal fibroblasts to eCAF or in tumour-derived CAF was investigated. Methods: NOF (normal oral fibroblasts) were treated with a range concentration of TGF-β1 (0.05-5 ng/ml) for 24 h and 48 h. The expression of myofibroblast markers, a- SMA and FN-EDA1, were determined using qRT-PCR and western blot. The formation of stress fibres was assessed by fluorescence microscopy, and associated changes in contractility assessed using collagen contraction assays. miRNA expression profiling in NOF, eCAF, and tumour-derived CAF was carried out using tiling low density array (TLDA) cards, with two distinct methods of analysis employed. Candidate miRNAs were validated using qRT-PCR. miRNA inhibitors used in the loss-of-function experiments; anti-miR-424-3p and anti-miR-145-5p while a pre-miR-424-3p used to overexpress miR- 424-3p. Results: TGF-β1 induced a myofibroblastic, CAF-like, phenotype in NOF (termed eCAF) as assessed by expression of molecular markers, the formation of stress fibres and increased contractility. TLDA analysis and qPCR validation demonstrated that miR-424- 3p and miR-145-5p were upregulated in eCAF. Overexpression of miR-424-3p inhibited TGF-β1-induced eCAF phenotype in NOF. Conversely, knockdown of miR-424-3p or miR-145-5p had no effect on eCAF formation. Overexpression of miR-424-3p did not inhibit basal myofibroblast phenotype of CAF or further TGF-β1-induced myofibroblast phenotype in CAF. Neither inhibition of miR-424-3p nor miR-145-5p increased basal myofibroblast phenotype of CAF. Inhibition of miR-424-3p decreased TGF-β1-induced a-SMA stress fibre formation and TGF-β1-induced contractility in CAF. Inhibition of miR-145-5p increased TGF-β1-induced gel contraction, but not other myofibroblast phenotype, in CAF. Reanalysis of TLDA data using a more stringent approach identified other potential miRNA candidates differentially expressed in eCAF and CAF, compared to NOF. The miRNA expression profiles of tumour-derived CAF (two subtypes of OSCC) was distinctly different from that observed in eCAF, suggesting a difference between CAF and fibroblasts artificially induced to become CAF-like cells in vitro. Conclusions: The findings suggest that miR-424-3p might partially participate in TGF- β1-induced CAF-like myofibroblast differentiation. Other miRNA was identified which showed differences in expression between NOF, eCAF and CAF which could be functionally analysed in future studies. Collectively the data suggest that miRNA may play a role in CAF development, and could provide translational benefits in cancer therapy.
Supervisor: Lambert, Daniel W. ; Hunt, Stuart Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available