Lu Linshan, Wang Xiaohong, Zhao Hongxi, Jiang Feng, Li Yanhong, Yao Yuanqing, Shi Changhong, Yang Yanhong
Department of Obstetrics and Gynecology, Tangdu Hospital, The Fourth Military Medical University Xi'an Shaanxi China
Department of Obstetrics and Gynecology, Chinese PLA General Hospital Beijing China
RSC Adv. 2019 Mar 22;9(16):9331-9341. doi: 10.1039/c9ra00017h. eCollection 2019 Mar 15.
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.
微小RNA-290（miR-290）簇在小鼠植入前胚胎中高度表达，但其在小鼠植入前胚胎发育中的具体作用和调控机制仍不清楚。在此，我们发现，作为miR-290簇的成熟微小RNA分子，miR-291a-5p和miR-291b-5p在小鼠植入前胚胎中动态表达。miR-291a-5p和miR-291b-5p在小鼠胚胎中的表达在2-4细胞阶段增加，并伴随着自噬相关基因Atg5和Becn1的mRNA表达降低。免疫荧光研究表明，自噬体和自噬溶酶体的形成在1细胞阶段增加，在2细胞阶段减少，并在4-8细胞阶段迅速减少。透射电子显微镜（TEM）也表明，受精卵细胞质中存在具有双层膜结构的自噬体，而在未受精卵母细胞细胞质中未观察到这种结构。此外，miR-291a/b-5p抑制了NIH/3T3细胞中Atg5和Becn1的蛋白质和mRNA表达。双荧光素酶报告基因检测证实，miR-291a/b-5p直接靶向Atg5和Becn1基因。miR-291a/b-5p抑制雷帕霉素诱导的自噬相关的LC3-I向LC3-II转化，最终抑制自噬体的形成。此外，向小鼠受精卵细胞质中显微注射miR-291a-5p抑制剂可增加小鼠胚胎中Atg5和Becn1的mRNA表达，并促进小鼠胚胎的第一次卵裂和囊胚形成。我们的结果表明miR-291a/b-5p在小鼠植入前胚胎发育过程中具有重要作用。
