MiR-291a/b-5p inhibits autophagy by targeting Atg5 and Becn1 during mouse preimplantation embryo development

microRNA-290 (miR-290) clusters are highly expressed in mouse preimplantation embryos, but their specific role and regulatory mechanisms in the development of mouse preimplantation embryos remain unclear. Here, we found that miR-291a-5p and miR-291b-5p, as mature microRNA molecules of miR-290 clusters, were dynamically expressed in mouse preimplantation embryos. The expression of miR-291a-5p and miR-291b-5p in mouse embryos increased during the 2-4-cell stages and was accompanied by the decreasing expression of the autophagy-related genes Atg5 and Becn1 in mRNA. Immunofluorescence studies showed that the formation of autophagosomes and autophagic lysosomes increased in the 1-cell stage, decreased in the 2-cell stage, and rapidly decreased during the 4-8-cell stage. Transmission electron microscopy (TEM) also demonstrated that there were autophagosomes in the cytoplasm of fertilized eggs with a double-layer membrane structure, whereas this structure was not observed in the unfertilized oocyte cytoplasm. Moreover, miR-291a/b-5p inhibited the protein and mRNA expression of Atg5 and Becn1 in NIH/3T3 cells. A dual-luciferase reporter assay confirmed that miR-291a/b-5p directly targeted the Atg5 and Becn1 genes. MiR-291a/b-5p repressed rapamycin-induced autophagy-related LC3-I to LC3-II conversion, ultimately inhibiting the formation of autophagosomes. Furthermore, the microinjection of mouse zygote cytoplasm with miR-291a-5p inhibitors increased the mRNA expression of Atg5 and Becn1 in mouse embryos and facilitated the first cleavage of mouse embryos and blastocyst formation. Our results suggest the important role of miR-291a/b-5p during mouse preimplantation embryo development.