Vinsamlegast notið þetta auðkenni þegar þið vitnið til verksins eða tengið í það: http://hdl.handle.net/1946/33199
Microphthalmia-associated transcription factor (MITF) is the master regulator of melanocytes and is known to directly bind to the promoter regions of the genes encoding cell cycle regulators, such as p21 and CDK2. This thesis focuses on the relationship between MITF and the cell cycle in melanoma and tests the role of novel MITF target genes. A melanoma cell line, 501mel, was synchronized, samples were taken and analyzed via flow cytometry, Western blot and qPCR. The results showed that MITF protein levels increased when the cells were mainly in G2/M phase whereas mRNA levels were unaffected, suggesting a change in protein synthesis or stability during this stage.
The relationship between MITF and several novel targets was determined using MITF knockdown and overexpression in 501mel or A375P cell lines. Knockdown of MITF showed a significant decrease on CABLES1 expression, with a trend of decreasing CDK2 expression. Overexpression of MITF did not significantly change expression of any of the cell cycle regulating genes checked, although there was a general increase of each gene checked.
To determine if the cell cycle regulators CDK2 and LZTS1 affected MITF expression, these genes were knocked down with siRNA before collecting samples for Western blot and qPCR. Knockdown of CDK2 caused a significant decrease in MITF mRNA and protein levels, whereas knockdown of LZTS1 only caused a significant decrease in MITF protein levels. This suggests that CDK2 might regulate MITF transcription in a feedback loop and LZTS1 plays a role either in translation or stability of MITF.
|Lara Stefansson MSc Thesis.pdf||1.99 MB||Opinn||Heildartexti||Skoða/Opna|