Verborgene bioactive Naturstoffe – miRNA in extrazellulären Vesikeln als mögliche bioaktive Wirkstoffe der Europäischen Mistel
Pflanzenzüchtung, Pflanzenpathologie
Zusammenfassung der Projektergebnisse
MiRNAs are a class of approximately 22 nucleotides single-stranded non-coding RNAs that play crucial roles in gene expression. In recent years, the cross-kingdom regulation of plant derived miRNAs has attracted great attraction. In this study, we investigated if mistletoe miRNAs play a role in the therapeutic effect of mistletoe. The bioactive effects of different mistletoe miRNAs in various tumor cell lines were evaluated by MTT assay. Val-miR218 was the most abundant miRNAs from mistletoe, and showed cytotoxicity in most tested tumor cells, especially osteosarcoma U2OS, SAOS-2 and 143B cells. What’s more, val-miR218 significantly inhibited osteosarcoma cell proliferation and induced cell apoptosis, indicating its promising potential in cancer treatment therapy. To find out the direct human targets, the RNAs associated with biotin labeled val-miR218 were pulldown and sequenced. Meanwhile, the RNAs that down regulated after val-miR218 overexpression were sequenced and analyzed. A total of 61 transcripts were considered as direct targets of val-miR218, since they were found both in the list of biotin labeled val-miR218 associated RNAs as well as the list of val-miR218 down-regulated genes. The expression of these target genes was confirmed by qPCR and western blot, and the bindings between valmiR218 and its recognition sites in the targets were confirmed by dual luciferase assay. Interestingly, functional analysis indicated that these targets were related to basic cell functions, including cell cycle; DNA replication, recombination and repair; and so on. Indeed, 11 of these 61 targets are involved in cell cycle, might explain that val-miR218 arrest osteosarcoma cells at G0/G1 phase. Naked miRNAs are extremely sensitive in the herb preparations and in the mammalian circulation system. EVs that are resistant in the enzymatic environment might offer effective protection for miRNAs. In this study, mistletoe EVs were purified and characterized. They were round or cup-shaped under SEM with average size of 119 nm. Quantitative PCR results indicated that val-miR218 was packaged inside EVs, which might explain the relative stable of val-miR218 in various mistletoe preparations. Further, with the uptake of mistletoe EVs by human tumor cells, val-miR218 could be efficiently delivered into the cells where it binds to its target genes and induce cytotoxic effects.
