Autophagy and EVs in cancer cells. Effect of ATG8 knockdown on the biogenesis of CD81+ extracellular vesicles.

Dysregulation of both the autophagy pathway and the biogenesis of extracellular vesicles (EVs) in cells is involved in cancer development. Evidence is now emerging that these pathways may somehow be interlinked via ATG proteins. Further elucidation of the relationship between these pathways will lead to increased understanding of the intricacies of the cancer progression and possibly lead to the discovery of new chemotherapeutics or improved early detection of cancer. Thus, this study aimed to investigate EV secretion in cancer cells with or without autophagy disruption and determine if ATG8 gene knockout, or knockout of its sub-families LC3 and GABARAP, impacts the number and size of EVs. The sub-objective was to positively identify EVs as the isolated particles by examining protein markers specific to EVs and imaging the vesicles with electron microscopy. HeLa cancer cell lines were used with or without gene disruption of the six ATG8s, the three LC3s, and the three GABARAP genes. EVs from the cell lines' conditioned medium were immunocaptured with CD81 Fab-Strep. After isolation, the particles were analyzed with NanoSight for particle concentration and size, immunoblotted for protein markers with SW and imaged with TEM. This study determined that the EV secretion from ATG8 hexa-KO cell lines was significantly reduced. First data from the TKO cell lines indicate that EV secretion is also decreased in those. The identification of EVs with EV-specific protein markers was not completed successfully. However, electron microscopy images and analysis of numbers of particles with NTA indicate the positive isolation of EVs, but this cannot be asserted. Understanding the function of autophagy and EVs in health and illness is a worthy research topic that requires further exploration of these mechanisms and their relationship.

Author dc:creator

• Ingibjörg Steinunn Sigurbjörnsdóttir 1996-

Contributors dc:contributor

• Háskóli Íslands

• Lyfjafræði
• Krabbameinsrannsóknir

Language dc:language.iso

en

Handle dc:identifier.uri

http://hdl.handle.net/1946/40688

OAI identifier oai:identifier

oai:skemman.is:1946/40688