Ectopic endometrial mesenchymal stem cells derived exosomal miR-4466 promotes angiogenesis by targeting RUNX1 in adenomyosis.

Background Abnormal angiogenesis plays a crucial role in the pathogenesis of adenomyosis (AM). Emerging evidence suggests exosomes derived from endometrial cells can boost AM progression. In this study, we aim to investigate the pro-angiogenic role and potential mechanisms of ectopic endometrial mesenchymal stem cells (eMSCs) derived exosomes (Ec-eMSCs-Exos) in adenomyotic lesions. Methods MicroRNA sequencing (miRNA-Seq) was conducted to identify differentially expressed miRNAs (DE miRNAs) in normal eMSCs derived exosomes (N-eMSCs-Exos) and Ec-eMSCs-Exos. Candidate miRNAs were selected using quantitative real-time polymerase chain reaction (qRT-PCR). The cell internalization assay were performed to confirm the internalization of exosomal miR-4466 into recipient human umbilical vein endothelial cells (HUVECs). The effects of exosomal miR-4466 on HUVEC proliferation, invasion/migration, and tube formation were analyzed in vitro using CCK8, EdU, transwell, wound healing, and µ-slide angiogenesis assays. The target gene of miR-4466 was predicted through bioinformatics analysis and further verified by qRT-PCR, western blot, dual luciferase reporter assay and rescue experiment. Results We identified 81 up-regulated and 92 down-regulated miRNAs between N-eMSCs-Exos and Ec-eMSCs-Exos. Among these DE miRNAs, miRNA-4466 was the most significantly up-regulated. The internalization assay showed that exosomal miR-4466 can be internalized by HUVECs. Overexpression or inhibition of miR-4466 can significantly promote or inhibit the proliferation, invasion/migration, and tube formation of HUVECs in vitro. By using bioinformatics predictions and luciferase reporter assay, we found that runt-related transcription factor 1 (RUNX1) was a direct targrt of miR-4466. Moreover, rescue experiment confirmed that RUNX1 overexpression reversed the pro-angiogenic effect of miR-4466 by inhibiting VEGFA expression. Conclusion Ectopic eMSCs derived exosomal miR-4466 promotes angiogenesis by targeting the RUNX1/VEGFA axis in AM. Overall design: In this study, we isolated exosomes from the culture supernatant of normal eMSCs (n=3) and ectopic eMSCs (n=3). We performed miRNA Seq to identify miRNA profiles in eMSCs-Exos, and successfully obtained the results.