mmu-miR-193在早孕小鼠子宫内膜中的表达及功能初探

发布时间：2018-09-13 09:45

【摘要】：目的: 应用Real-time RT-PCR、原位杂交对microRNAs(miRNAs)芯片筛选出的小鼠胚胎着床前后子宫内膜中差异表达最大的mmu-miR-193进行表达规律研究;运用靶基因预测数据库检索mmu-miR-193调控的靶基因;在体外通过转染实验对靶基因进行验证;通过小鼠子宫角注射mmu-miR-193激动剂研究其对胚胎着床的影响。从而探明mmu-miR-193在小鼠胚胎着床过程中的作用和机制。为研究microRNAs在生殖过程中的作用,以及阐明人类正常生殖过程机制和诊断、治疗生殖疾病打下基础。 方法: 1 1.标本的收集:建立妊娠NIH小鼠动物模型,分离孕d4、d6天小鼠的子宫内膜组织,分别冻存于-80℃备用。 2. Real-time RT-PCR检测芯片筛选出差异表达最大的mmu-miR-193,根据mmu-miR-193成熟序列设计特异性的逆转录引物和PCR上、下游引物进行real-time RT PCR。 3.原位杂交:原位杂交检测mmu-miR-193在胚胎着床前后(孕d4、d6)子宫内膜中的表达部位。 4.生物信息学分析:应用miRNAs靶基因预测数据库网站Pictar、Targetscan、miRGen来检索mmu-miR-193的预测靶基因。 5.转染实验:通过转染mmu-miR-193模拟物和抑制剂使其功能增强或者降低,Western blot检测转染后GRB7蛋白的表达变化。 6.功能实验:在小鼠孕D2天,通过子宫角注射mmu-miR-193激动剂,孕D5天检测GRB7蛋白表达,孕D7天剖腹计数胚胎着床数。 结果: 1. Real-time RT PCR结果显示mmu-miR-193在小鼠胚胎着床前后(孕D4、D6)子宫内膜中表达差异显著,孕D6与孕D4比显著上调。结果与芯片结果趋势一致。 2.原位杂交显示mmu-miR-193在小鼠子宫内膜基质细胞、腔上皮细胞和腺上皮细胞均有表达。 3.利用生物信息学,通过数据库预测得到Grb7等为mmu-miR-193所调控的靶基因。 4.在体外,我们通过转染mmu-miR-193的模拟物和抑制剂证实了GRB7的表达受mmu-miR-193调控。 5.通过子宫角注射mmu-miR-193的激动剂,导致了GRB7蛋白表达水平及胚胎着床数的明显下降。 结论: 1. mmu-miR-193在小鼠胚胎着床前后子宫内膜中存在明显差异表达,胚胎着床后较胚胎着床前显著升高。 2. mmu-miR-193在小鼠胚胎着床过程中通过调节其靶基因Grb7的表达而调控小鼠胚胎着床。
[Abstract]:Objective: to study the expression of mmu-miR-193, which was the most differentially expressed in mouse endometrium before and after implantation with microRNAs (miRNAs) microarray by Real-time RT-PCR, in situ hybridization, and to search the target genes regulated by mmu-miR-193 by using target gene prediction database. The target gene was verified by transfection experiment in vitro, and the effect of mmu-miR-193 agonist on embryo implantation was studied by injection of mmu-miR-193 agonist into mouse uterus horn in vitro. The role and mechanism of mmu-miR-193 in mouse embryo implantation were investigated. It provides a basis for studying the role of microRNAs in reproductive process, clarifying the mechanism and diagnosis of human normal reproductive process, and treating reproductive diseases. Methods: 1. Collection of specimens: the animal model of pregnant NIH mice was established and the endometrial tissues of mice on the 4th day of gestation were isolated and frozen at -80 鈩，

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