Altered miRNA Expression in MSC-Derived Extracellular Vesicles Following Hydatid Antigen Stimulation
This study investigates the impact of hydatid antigens on the miRNA expression profiles within extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs). By stimulating MSCs with echinococcus granulosus protoscoleces (ESPs), hydatid cyst fluid (HCF), and particles from the laminated layer (pLL), we aim to uncover the changes in miRNA expression and their potential roles in modulating immune responses and osteogenic differentiation. Through high-throughput sequencing, differential expression analysis, and subsequent bioinformatics analyses, we identify key miRNAs and their target genes involved in these processes. Our findings provide insights into the complex interplay between parasitic infections and host cell responses, highlighting the therapeutic potential of MSC-derived EVs in treating hydatid disease. Overall design: 1. **Isolation and Culture:** Bone marrow-derived MSCs (BMSCs) are isolated and cultured in a-MEM medium. 2. **Stimulation:** BMSCs are treated with ESPs, HCF, or pLL at a concentration of 2 µg/ml for 48 hours. 3. **EV Isolation:** Extracellular vesicles are isolated from the cell culture supernatants using ultracentrifugation. 4. **RNA Extraction:** Total RNA is extracted from the isolated EVs using the Trizol method. 5. **Library Construction and Sequencing:** Small RNA libraries are constructed using the NEBNext Small RNA Library Prep Kit and sequenced on the Illumina NovaSeq platform. 6. **Data Analysis:** Sequencing data are processed for quality control, miRNA identification, and differential expression analysis. Bioinformatics analyses including GO and KEGG enrichment, PPI network construction, and transcription factor-miRNA network analysis are performed. 7. **Validation:** Differentially expressed miRNAs and their target genes are validated using quantitative PCR and Western blot analysis. 8. **Functional Assays:** The effects of ESPs, HCF, and pLL on MSC osteogenic differentiation and immune modulation are assessed.