Background: Microvesicles (MVs) mediate the transmission of information between cells through the miRNAs, proteins, lipids and mRNAs carried by MVs, which inhibit/promote the function of receptor cells. This study confirmed that blastocyst MVs inhibited the invasion of melanoma cells and showed a complex miRNA pedigree. Functional miRNAs were found, and the mechanism of inhibiting invasiveness was analyzed based on the miRNA lineage characteristics of mouse blastocyst MVs, combined with the miRNA bioinformatics information of miRBase, microRNA.org, NCBI and other databases.
Methods: MVs derived from D3.5 mouse blastocysts were isolated and the miRNA gene expression of MV was detected by fluorescence quantitative polymerase chain reaction (qPCR). The dynamic changes in tumor cell invasiveness in the co-culture system of blastocyst MVs and melanoma B16-F10 cells were monitored in real time. The MV miRNAs expression profile of blastocyst was identified using the qPCR array. Bioinformatics tools were used to analyze the characteristics of miRNA pedigree in blastocyst MV to screen functional miRNAs, which reduce tumor cell invasiveness and to predict functional miRNA target genes. The target genes were analyzed by KEGG and GO pathway enrichment analyses using David and FunRich software.
Results: The average diameter of round MVs extracted from mouse blastocysts was 196.4 ±7.8 nm. When B16-F10 melanoma cells were co-cultured with mouse blastocyst MVs, no significant difference in invasive ability was found between the two groups before 72 h, but increased significantly after 72 h (P < 0.05, P < 0.01). A total of 336 miRNAs were analyzed in mouse blastocyst MVs. Further analysis showed that 224 miRNAs were associated with invasion. Also, 32 invasion-related miRNAs with the strongest differential expression were verified by real-time quantitative polymerase chain reaction (qPCR). The GO analysis of the target genes showed that the 32 functional miRNAs were mainly related to 8 biological processes, including DNA binding, sequence-specific DNA binding, metal ion binding, chromatin binding, nucleotide binding, sequence specificity, DNA binding transcription factor activity and ubiquitin-protein ligase activity. The KEGG pathway analysis showed that the 32 functional miRNAs were mainly related to the PI3K-Akt signaling pathway.
Conclusion: Blastocyst MVs carrying miRNAs regulated the decrease in the invasive ability of melanoma cells through the PI3K-Akt signaling pathway.