Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
Popper, U.
The Regulation of Epithelial-Mesenchymal Transition Regulators through Notch in Melanoma
[ Diplomarbeit ] Medical University of Graz; 2011. pp. 79
[OPEN ACCESS]
FullText
- Autor*innen der Med Uni Graz:
- Betreuer*innen:
- Schaider Helmut
- Altmetrics:
- Abstract:
- Abstract Background: Notch signaling pathway is a higly conserved cell signaling system, which plays a key role in modulating cell fate decisions throughout development, differentiation, apoptosis, migration and angiogenesis. Additionally Notch is one of the pathways cabable of inducing epithelial-mesenchymal transistion (EMT). EMT is an important biological event, characterized by the switch from an epithelial to a very motile, mesenchymal cell phenotype, which leads to enhanced migratory capacity, invasiveness and elevated resistance to apoptosis. Further epithelial-mesenchymal transition is involved in melanomagenesis and metastasis of melanoma. The aim of this work was to test, whether Notch is regulating epithelial-mesenchymal transition regulators (EMTRs) and to investigate, whether this effect is mediated directly or indirectly. Methods: Notch1, 2, 3 and 4 were silenced in primary melanoma cell line WM35 and metastatic melanoma cell lines WM9 and 164 using siRNA and effects on Twist, Snail, Slug and ZEB1 were detected by Western blot analyses. To cross check the results, Notch4 was overexpressed in WM35 and WM164 utilizing a retroviral vector system. To test, whether Notch is able to bind to the promoter of any of the EMTRs Electrophoretic Mobility Shift Assays (EMSA) were carried out. Results: The silencing of Notch1 does not affect any of the EMTRs. The knockdown of Notch2 decreases Twist, Snail and Slug levels in WM9. Notch3 is not expressed in WM cell lines. Silencing of Notch4 increases levels of Twist in WM35, but decreases Twist in WM164. Overexpression of Notch4 in WM35 leads according to silencing results to decreased Twist levels, but in WM164 Notch4 overexpression also showed reduction of Twist, which is inconsistent with the results described above. For the first time I was able to show direct binding of Notch via CSL to Twist and ZEB1 promoter. Conclusion: In melanoma Notch2 and Notch4 are capable of regulating EMTRs. Whereas the effect of Notch2 was only observed in one cell line, suggesting the role of Notch2 to be transient, Notch4 silencing showed differing results for Twist in different cell lines, but same results for gene silencing and overexpression, which needs to be clarified. Nevertheless the binding of Notch to the Twist promoter indicates regulation of Twist via Notch.