Unravelling the presence of extracellular microRNAs and their carriers in insect cell culture media and hemolymph

Non-coding small RNAs, also referred to as regulatory small RNAs, are not translated into proteins but function at several levels regulating gene expression. One class of these regulatory small RNAs are the microRNAs (miRNAs), which regulate the expression of endogenous genes. While the regulatory small RNA pathways are well-characterized at the intracellular level, extracellular small RNAs have been identified in animals and plants. In this context, studies reporting on the presence and potential function of extracellular small RNAs emerged rapidly over the past decade. In mammals, extacellular miRNAs (ex-miRNAs) have been proposed as novel non-invasive biomarkers and their functional transfer has been shown. In insects, the largest class of animals, reports on extracellular microRNAs are still very limited but starting to emerge. In this study, I have described the presence of ex-miRNAs in insects, more in particular in media of cultured Drosophila melanogaster cells and in hemolymph of the migratory locust, Locusta migratoria. First, I demonstrated the presence of ex-miRNAs in extracellular vesicles (EVs) or bound to Argonaute-1 in ribonucleoprotein complexes isolated from cell-free media of cultured D. melanogaster cells. Moreover, my data suggested the possible occurrence of selective miRNA secretion into EVs. Second, I demonstrated the presence of stable ex-miRNAs in locust hemolymph and revealed a differential abundance of specific miRNAs in hemolymph sera derived from early versus late fifth instar locusts. Moreover, I was able to affect the moulting process by modulating levels of two of these miRNAs, and revealed the importance of miRNAs for succesfull locust moulting. Third, I optimized a robust procedure for EV isolation from insect hemoymph, which is applicable for a wide range of insect species. Finally, by employing this optimized EV isolation procedure I isolated hemolymph derived EVs from early and late fifth instar locusts and revealed the differential abundance of several known, as well as newly predicted, miRNAs. Consecutive in silico target prediction and GO enrichment analyses suggested that these (putative) miRNAs might regulate developmental processes in L. migratoria. Taken together, this doctoral thesis provides new fundamental insights in the presence, identity and potential function(s) of extracellular miRNAs in insects.

Jaar van publicatie:2022

Toegankelijkheid:Open