Transcriptomic and functional analysis of fibroid extracellular vesicles

Exosomes were isolated from cultures of fibroid explants and matched myometrial explants, and their RNA cargo was analyzed by next-generation sequencing to profile both long and short RNA species. Fibroid-derived exosomes (Fib-EXO) expressed the canonical extracellular vesicle markers CD81 and CD63, and their size distribution (30-200 nm) was consistent with exosomal vesicles. The RNA cargo of Fib-EXO generally reflected that of its tissue of origin, although selective enrichment of specific transcripts, such as piR-1398740 and piR-333378, suggested active loading mechanisms. Long RNA sequencing identified differential expression of protein-coding genes and long noncoding RNAs (lncRNAs) involved in RNA binding, cytoplasmic translation, exosome pathways, and PI3K/AKT and focal adhesion signaling. qPCR validation confirmed increased IGF2, HOXA10, and decreased IGFBP6 mRNA expression in Fib-EXO. Among lncRNAs, MSC-AS1, PART1, and H19 were overexpressed in Fib-EXO. Small RNA sequencing revealed differential expression of multiple small noncoding RNA classes, including Piwi-interacting RNAs, miRNAs, snRNAs, snoRNAs, and tRNAs. KEGG analysis showed that miRNAs were primarily associated with PI3K/AKT signaling, proteoglycans in cancer, interleukin signaling, and transcriptional regulation. Functionally, Fib-EXO were internalized by myometrial cells and promoted their proliferation with no effects on apoptosis. Furthermore, Fib-EXO enhanced angiogenesis in human umbilical vein endothelial cells. Fib-EXO increased the expression of vimentin, EZH2, DNMT1, TGF-β3, and c-MYC, and phosphorylated p65 protein, reduced COL1A1 and COL3A1 expression, and decreased miR-133a, miR-29, and miR-200c while increasing miR-21 in cultured myometrial smooth muscle cells, mirroring changes observed in fibroids. These findings indicate that Fib-EXO reprogram myometrial cells toward a fibroid-like phenotype characterized by increased proliferation, inflammation, and fibrotic features, thus contributing to fibroid propagation.