绵羊克隆囊胚差异表达microRNA 的筛选及其功能研究

Somatic cell nucleus transfer (SCNT) technology show a good application prospect in agriculture, species conservation and medicine. However, developmental abnormalities of cloned embryo always restricted development and application of SCNT. MicroRNA (miRNA)，a new non-coding RNA，play an important role in cell proliferation and differentiation during mammalian development. But the associations between miRNA and developmental abnormalities of cloned embryo are lacking. In our earlier study, we found that some miRNAs were upregulated in cloned blastula compared to in-vivo fertilized blastula. Thus, we speculate that the aberrated expression of miRNA result in developmental abnormalities of cloned embryo, which may be a mechanism of aberrated development of cloned embryo. To confirm our speculation, we want to study microRNA expression profiling of cloned blastula and in-vivo fertilized blastula by SCNT, superovulation, embryo flushing and Illumina technology. Differential expression miRNA will be determined by bioinformatics. Theirs target gene and function will be analyzed in development of cloned embryo.The study will determined the role of miRNA in developmental abnormalities of cloned embryo,which will provide a new method for increase cloned animal rate.

体细胞核移植（克隆）技术已在农业、物种保护、医疗等领域显示出诱人的应用前景。然而，克隆胚胎发育异常及效率低一直是制约克隆技术发展应用的瓶颈。microRNA（miRNA）是新发现的一类非编码RNA，具有调控动物胚胎发育、细胞增殖和分化的功能。然而miRNA与核移植克隆胚胎发育异常的关系，迄今仍然不清楚。前期研究中，本课题组发现与绵羊体内受精囊胚相比，核移植克隆囊胚中部分miRNA表达量显著上调。因此推测异常表达的miRNA可能导致克隆胚胎发育异常，这可能是一种导致克隆胚胎发育异常的新机制。为证实这一假说，本研究拟采用体细胞核移植，绵羊超排冲胚，Illumina高通量测序等技术研究绵羊克隆囊胚与体内受精囊胚miRNA表达谱差异；并分析这些差异表达miRNA的靶基因及其在胚胎发育中的功能，从miRNA角度阐明绵羊体细胞克隆胚胎发育异常的分子机制，为提高体细胞克隆效率提供新手段和依据。

microRNA（miRNA）是一种内源性非编码RNA分子，大约由21-25个核苷酸组成，约占动物基因组总量的1%。miRNA是新发现的一类非编码RNA，具有调控动物胚胎发育、细胞增殖和分化的功能。然而，miRNA在核移植克隆胚胎中的表达和功能尚不清楚。本研究收集了202枚绵羊克隆囊胚和150枚受精囊胚，利用Illumina测序平台进行miRNA表达谱测序，共获得76个差异表达的miRNA，其中21个miRNA表达量显著上调，54个miRNA表达量显著下调。选取19个表达显著差异的miRNA利用荧光定量RT-PCR进行分析，发现其中7个miRNA在绵羊克隆囊胚中表达量上调，12个miRNA在绵羊克隆囊胚中表达量下调。选取表达上调的miR-127为后续的研究对象，利用 Target Scan软件预测miR-127的靶基因为RTL1基因。利用EGFP基因报告系统验证miR-127的靶基因，证实miR-127的靶基因为RTL1。利用荧光定量PCR证实miR-127与RTL1表达水平的负相关，表明在囊胚中miR-127的靶基因为RTL1。将miR-127在绵羊克隆胚胎中沉默，发现miR-127沉默对克隆囊胚的发育率无显著影响，但是可显著提高克隆囊胚的细胞数，说明miR-127对克隆囊胚发育有一定的影响。利用荧光定量RT-PCR证实miR-127基因沉默后RTL1表达量升高，进一步表明RTL1是miR-127的靶基因。本项目研究结果说明克隆胚胎中的miR-127被异常重编程，miR-127在克隆囊胚中异常表达导致胎盘发育相关基因RTL1的表达下调，RTL1在克隆囊胚中的异常表达可能是克隆胚胎发育异常的主要原因之一。本研究为从miRNA角度理解克隆胚胎发育异常的分子机制提供了理论基础。