Bridge, GEM; (2013) Using Kaposi's Sarcoma-Associated Herpesvirus to Elucidate the Role of Cellular MicroRNAs in Endothelial Biology. Doctoral thesis , UCL (University College London).

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Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic γ-herpesvirus that is the etiologic agent of Kaposi sarcoma (KS). KS is an angioproliferative neoplasm composed of cells of endothelial origin. For the work described in this thesis, KSHV infection of endothelial cells was used as a tractable model to study the role of microRNAs (miRNAs) in endothelial cell biology. Previous work in the laboratory had identified miRNAs which are either upregulated or downregulated upon KSHV infection of lymphatic endothelial cells (LEC). Target prediction analysis of these miRNAs and cross-comparison of predicted targets with expression levels of pro- and anti-angiogenic genes following KSHV infection, revealed the predicted targeting of Delta-like 4 (DLL4) by the miR-30 family. DLL4 is significantly upregulated in KSHV-infected lymphatic endothelial cells (KLEC) whereas the miR-30 family is significantly downregulated. DLL4 is a membrane-bound ligand belonging to the Notch signalling family that plays a fundamental role in vascular development and angiogenesis. Targeting of DLL4 by miR-30b and miR-30c was confirmed by examining mRNA and protein expression following transfection of endothelial cells with miR-30 mimics and inhibitors or infection with miR-30-expressing lentiviruses. The exact target site within the DLL4 3’UTR was identified using a luciferase reporter assay and site-directed mutagenesis. Overexpression of miR-30b in endothelial cells led to increased vessel number and length in an in vitro model of sprouting angiogenesis. Microinjection of miR-30 into zebrafish embryos resulted in suppression of dll4 and subsequent excessive sprouting of intersegmental vessels and reduction in dorsal aorta diameter. Use of a target protector against the miR-30 site within the dll4 3’UTR upregulated dll4 and synergised with Vegfa signalling knockdown to inhibit angiogenesis. Furthermore, restoration of miR-30b or miR-30c expression during KSHV infection attenuated viral induction of DLL4. Overall, the work presented in this thesis demonstrates that the miR-30 family targets DLL4 to regulate angiogenesis.

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