摘要
微小RNA(MicroRNA,miRNA)是近年新发现的一类非编码RNA;长度为19-25个核苷酸的单链RNA分子,广泛存在于从线虫到人类的真核生物中。miRNA具有高度的保守性;大多数已发现的miRNA的表达具有时序性和组织特异性;生成miRNA的基因并不是随机分布的,从同一个前体RNA加工而来的共表达miRNA存在基因簇集现象。成熟的miRNA通过其5’端的5~8个核苷酸,识别并与靶标mRNA序列进行完全互补配对或不完全互补配对,引起靶标mRNA的降解或翻译抑制,达到调控目的基因表达的作用。通过上述作用机制miRNA在基因调控网络中发挥重要的作用,并在发育、细胞增殖、凋亡、分化以及肿瘤的发生等方面起重要作用。
自从发现lin-4和let-7在线虫发育中发挥作用后,近年有大量的研究表明, miRNA及其生物合成相关的基因在着床后胚胎发育的各个阶段都有表达并对胚胎发育有影响。但是,目前尚无文献报道miRNA及其生物合成相关基因在着床前胚胎中的表达及其功能。因此,本课题拟开展miRNA及其生物合成相关基因在小鼠着床前胚胎表达的研究,以期找到miRNA对着床前胚胎生长发育、细胞分化及胚胎基因组激活等方面可能起作用的依据。目的:建立小鼠的超排、交配和胚胎收集系统,研究miRNA及其生物合成的相关基因在小鼠着床前胚胎的表达,探讨miRNA在着床前胚胎发育中的作用和意义。
方法和结果:
1.采用miRNA扩增结合miRNA芯片技术研究了小鼠的卵母细胞和着床前胚胎中miRNA表达谱。首次发现小鼠卵母细胞、受精后1.5天(2细胞)、2.5天(4-8细胞)和4.5天(囊胚)胚胎中分别表达55、53、62和72个miRNA;四个发育阶段中共筛查到表达的miRNA 94个,其中有32个miRNA在四个发育阶段均表达(包括16个在四个发育阶段均高表达的miRNA);其它的62个miRNA在不同的发育阶段特异性表达,其中在囊胚期胚胎中特异性表达一簇从小鼠干细胞克隆到的miRNA miR-290~miR-295基因簇。筛查到的miRNA中包括目前功能比较明确的miRNA miR-155和miR-181,通过对这些miRNA功能的分析,表明着床前胚胎中miRNA的表达可能对胚胎的早期发育起调控作用,并在维持胚胎细胞的分化中起作用。
2.采用qRT-PCR方法研究miR-721和let-7e在小鼠卵母细胞、4-8细胞和囊胚中的表达水平。结果显示miR-721和let-7e的表达水平在这三个发育阶段均呈下降趋势。
3.采用RT-PCR的方法研究了miRNA生物合成的相关基因Dicer1和DGCR8在卵母细胞和受精后1.5天、2.5天、3.5天和4.5天胚胎中的表达,结果表明在卵母细胞和上述各个发育阶段均表达Dicer1和DGCR8基因,他们可能是着床前胚胎miRNA合成的重要基因并对着床前胚胎发育起作用。
结论:通过成功建立小鼠的超排、交配和胚胎收集系统,发现在小鼠着床前胚胎及卵母细胞中表达miRNA及其合成相关的基因Dicer1和DGCR8。通过对着床前胚胎和卵母细胞中的miRNA表达水平研究及功能分析,表明miRNA可能通过调控在胚胎发育中的某些重要的与发育和细胞分化有关的原癌基因的表达,对胚胎发育和胚胎细胞的分化起重要作用;并可能与小鼠胚胎在2细胞后基因组的激活有关系。
MicroRNAs (miRNAs) are a newly found 19-25nt single stranded noncoding RNAs, and were distributed widely in Eukaryotes from C. elegans to human beings. MiRNAs have a characteristic of highly evolutionarily conservation and exihibit tissue-specific or developmental-stage-specific expression.Most of their genes are not randomly distributed but often closely clustered on the genome. MiRNAs act as a gene regulation molecular and function through the regulation of target genes by imperfect or perfect base-pairing with their target mRNAs, then either lead to the degradation of the target transcript (near perfect complementarity), or the inhibition of the protein translation.Through above mechnisim miRNAs play a important role 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.
Since the important roles of lin-4 and let-7 in the development of nematodes were found, many studys reported that miRNAs and genes coding for the enzymes involved in miRNA biogenesis were expressed and have influence on the development of post implantation embryos. But presently there is no report about expression and function of miRNAs in mouse preimplantation embryos. In this study, we intended to investigate the expression of miRNA and genes coding for the enzymes involved in miRNA biogenesis to find the evidence of miRNA acting on development and differentiation of preimplantation embryos
AIM: To set up a system about murine superovulation and mating and to establish a platform of embryo recovery to investigate the expression of miRNAs in mouse preimplantation embryos and to futher illustrate the possible role and significance of miRNA in the preimplantational embryonic development.
METHODS AND RESULTS:
1. We used a method of miRNA amplification system combined with miRNA microarry technique to analyze miRNA expression across mouse oocyte and preimplantation embryos. For the first time, we found that fifty-five, fifty-three, sixty-two and seventy-two miRNAs were expressed respectively in mouse oocytes, 1.5dpc(2cell), 2.5dpc(4-8cell) and 4.5dpc(blastocyst) embryos. Ninty-four miRNAs were expressed in preimplantation embryos and thirty-two were expressed commonly in each stage of development and sixty-two were expressed in the four different stages of development. Of the commonly expressed 32 miRNAs, 16 showed high levels. Of the 62 stage-specific miRNAs, cluster of miR-290~295 were expressed only in blastocyst, which were previously cloned from murine ES cells and were considered to have ES cell-specific functions. Of the identified miRNAs in microchip, the roles of miR-155 and miR-181 were clearly. Through analyzing the function of the two miRNAs suggests that miRNAs might play a role in maintenance of the embryonic cell differentiation and in the regulation of early mammalian development.
2. We used qRT-PCR to study the expression level of miR-721 and let-7e across mouse oocyte, 4-8cell and blastocyst embryos. The results of qRT-PCR show that both of them were expressed and the level of expression decreases with the development.
3. We used RT-PCR to investigate the expression of Dicer and DGCR8 across mouse oocytes, 1.5dpc (2cell), 2.5dpc (4-8cell), 3.5dpc (morula) and 4.5 dpc(blastocyst) embryos. The results indicate that the two genes are all expressed across oocyte and preimplantation embryos.They are the important factor involved in miRNA biogenesis of preimplantation embryos and might play a role in embryo development.
CONCLUSION: Through successfully setting up the system of murine ovulation and mating and the platform of embryo recovery, we identified miRNAs and genes of Dicer and DGCR8 in mouse oocyte and preimplantation embryos. In addition, we analyzed the function of the expressed miRNAs and genes coding for the enzymes involved in miRNA biogenesis. Our results suggest that miRNAs might play a role in the development and differentiation of the embryonic cells. Our analyses also indicate that there might be a correlation between miRNA and genomic activation after 2-cell development in mouse embryo.
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