小鼠着床前胚胎miRNA表达的实验研究

Expression of miRNA in mouse preimplantation embryos

目的研究微小RNA（miRNA）在小鼠着床前胚胎各发育阶段的表达谱，探讨miRNA在着床前胚胎发育中的作用和意义。方法建立小鼠超排卵、交配和胚胎收集系统，采用miRNA扩增、miRNA芯片和荧光实时定量逆转录聚合酶链反应（RT-PCR）的方法研究小鼠着床前胚胎miRNA表达。结果小鼠卵母细胞、2细胞胚胎、4～8细胞胚胎和囊胚分别表达55、53、62和72个miRNA；四个发育阶段共筛查到表达94个miRNA，32个在各发育阶段均表达，62个在不同发育阶段特异性表达。实时荧光定量PCR方法验证mmu-miR-721在小鼠着床前胚胎中表达。结论小鼠着床前胚胎表达大量的miRNA，其可能对胚胎发育和胚胎细胞的分化起重要调控作用。

Objectives. micro RNAs （miRNAs） are the newly found 19-25 nucleotide single stranded noncoding RNAs, which play important roles in gene regulation network and play critical roles in many biological processes including the regulation of development, cell proliferation or differentiation, apoptosis and cancer formation. In the present study, the expression of miRNAs at different stages of mouse preimplantation embryos was investigated. Methods. Mouse embryos （0. 5-4.5 days p. c） were collected after superovulation. A method of miRNA amplification system combined with miRNA microarray technique was used to analyze miRNA expression in mouse preimplantation embryos. Looped qRT-PCR was used to detect mmu-miR-721 in mouse preimplantation embryos to confirm the results of microarray. Results： Fifty-five, fifty-three, sixty-two and seventy-two miRNAs were expressed respectively in mouse embryos of 0.5 days p.c, 1.5 days p. c, 2.5 days p.c and 4.5 days p.c. A total of 94 miRNAs were found to be expressed in preimplantation embryos, of which 32 were expressed commonly in each stage of development and 62 were expressed differentially in different stages of development. To further validate reliability of the microarray, the expression of mmu-miR-721 was analyzed by looped qRT-PCR. The results of looped qRT-PCR were consistent with those of miRNA microarray. Conclusions： The present study suggests that mouse preimplantation embryos express lots of miRNAs. These miRNAs might regulate developmental and/or metabolism processes of mouse preimplantation embryos by targeting certain genes, especially transcription factors, for mRNA cleavage, translational repression, or mRNA decay mediated by miRNA-guided deadenylation.