Abstract
Purpose :
Based on the hypothesis that intercellular communication maintains the polarity of the retinal pigment epithelium (RPE), we are studying the biogenesis and secretion of exosomes from the RPE. Exosomes are nanovesicles that contain proteins, lipids and RNA. The production of these nano-vesicles and what they carry is dictated by the physiological status of the cells, which is directly modulated by the environment in which these cells grow. An important component of the genetic information carried by exosomes are the miRNAs. Because miRNAs can simultaneously impact multiple physiologies (e.g., angiogenesis, inflammation, cell cycle control and apoptosis) here we wanted to assess how serum exposure modulates the expression of these RNAs within the RPE cells and outside, in their secreted exosomes.
Methods :
RPE19 cells were cultured to 70% confluence in DMEM/HamF12 medium supplemented with 10% serum and then allowed to grow further for additional 24 hr with and without serum. Exosomes were isolated from the culture media as per our published procedures. Total cell RNA as well as exosomal RNAs were analyzed for miRNAs (800 reporter probe sets with controls, NanoString Technologies Inc., Seattle, WA). The data was analyzed using nSolver software.
Results :
RPE19 cells cultured in serum showed marginally higher expression of miRNAs (~ 13%) in comparison to cells deprived of serum. Similar increase (about 14%) was seen in miRNAs associated with exosomes. While there were qualitative differences in miRNA expression, miRNAs (such as miRNA 29b-3p, target VEGFA and 31-5p associated with inflammatory pathways), were expressed at higher levels inside the cells (+/- serum); these RNAs however, were not highly represented in the exosomes. Some miRNAs (for example 549, target HIF1α and 363-3p, target SLC12A5) were well represented in exosomes but poorly expressed within the cells exposed to serum. Importantly, miRNAs such as 1976 and 33a-5p, present at lower levels in serum-deprived cells are highly represented in exosomes, indicating serum exposure significantly influences miRNA expression profiles within the RPE19 cells and in the exosomes secreted by these cells.
Conclusions :
Differential expression levels of various miRNAs both inside the cell as well as outside (exosomes) suggests regulated and selective secretion of these RNAs by the RPE.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.