【作者】 沈炼桔；

【导师】 王应雄；

【作者基本信息】 重庆医科大学 ， 临床检验诊断学， 2013， 博士

【摘要】 第一部分雌孕激素对miRNAs调控的初探目的：妊娠的成功依赖于一个健康的子宫，能接受胚泡的植入并支持胎儿的发育。子宫是一个内分泌器官，它能对卵巢所分泌的雌孕激素作出响应，雌孕激素通过与其相应的受体ER和PR结合从而激活下游的靶基因。雌孕激素信号已被证实对于胚泡植入至关重要。MicroRNAs(miRNAs)可以调控基因的表达，并有研究证实miRNAs参与了胚泡植入的过程，但是对于谁来调控miRNAs的表达还知之甚少。由于许多参与植入的关键基因受到雌孕激素的调控，那么雌孕激素是否可以通过调控miRNAs表达进而影响胚泡植入的过程，这是本研究的目的。方法：收取小鼠妊娠第1天（D1）和第6天（D6）子宫内膜组织以模拟雌孕激素调节模型（妊娠D1是E2的高峰期，P4水平低；妊娠D6是P4的高峰期，E2水平低）。提取总RNA后进行microRNA测序，所得到的结果进行生物信息学分析。结果：miRNAs测序发现，妊娠D6与D1相比，有6个miRNA表达上调2倍以上，73个miRNA表达下调2倍以上。通过生物信息学分析，筛选出差异表达miRNAs的靶基因及其所参与的信号通路，这些miRNAs所调控的靶基因多参与代谢、MAPK、紧密连接等信号通路。结论：本研究结果初步反映了在小鼠子宫内膜组织中，miRNAs的表达受到雌孕激素的调控。通过生物信息学分析发现，在妊娠D1和D6中存在差异表达的miRNAs，其调控的靶基因所参与的信号通路均在植入过程中发挥重要作用，提示miRNAs在胚胎着床过程中起一定的调控作用。第二部分mmu-miR-200a过表达对胚胎着床的影响目的：胚泡成功植入需要子宫进入接受态以及激活的囊胚，大量的基因、细胞因子以及其他分子都参与了这一过程。众所周知，MicroRNAs(miRNAs)可以调控基因的表达，而且有研究表明miRNAs参与了调控胚泡植入的过程，但调控此过程的具体miRNA还研究尚少，这值得我们去探索。通过前期研究结果发现，mmu-miR-200a在妊娠D6，即孕激素高峰时表达下调，而且在植入前后具有表达差异，也有研究证实miR-200a在多种子宫内膜疾病（如子宫内膜癌和子宫内膜异位）的表达差异明显。因此，本研究旨在探索mmu-miR-200a在胚泡植入过程中所起的作用及其靶基因，为深入了解胚泡植入的分子机制提供基础，并有可能为治疗女性不孕不育以及开发避孕药提供新的思路。方法：Real Time-PCR检测mmu-miR-200a在妊娠D4、D5和D6子宫内膜以及D5着床点和着床点旁的表达水平，原位杂交检测mmu-miR-200a在妊娠D4、D5以及D6子宫中的表达模式。通过生物信息学分析、基质细胞原代培养及转染和荧光强度分析确认其靶基因为PTEN，免疫组化确定PTEN在小鼠植入过程子宫中的表达定位，宫角注射mmu-miR-200a过表达慢病毒观察植入情况。最后，利用流式细胞术检测转染mmu-miR-200a的对照、模拟物、抑制物后，基质细胞的凋亡状况。结果:应用Real Time-PCR发现在妊娠D5子宫内膜着床点及其旁组织表达差异明显，且在着床点的表达明显低于着床点旁。mmu-miR-200a的mRNA表达水平在妊娠D4、D5、D6子宫内膜中依次下降，且主要表达在基质细胞中，腔上皮及腺上皮细胞表达较少。mmu-miR-200a的靶基因PTEN的表达定位与其相同，但表达水平模式正好相反。宫角注射mmu-miR-200a过表达慢病毒发现胚胎着床率明显下降。流式细胞术检测到转染mmu-miR-200a抑制物后，基质细胞凋亡明显增加，此时PTEN蛋白表达上调；转染mmu-miR-200a模拟物后，基质细胞凋亡明显减少，PTEN蛋白表达下调。结论：研究发现mmu-miR-200a过表达会导致小鼠胚泡植入率的下降。根据mmu-miR-200a与其靶基因PTEN的表达模式以及转染mmu-miR-200a模拟物和抑制物后基质细胞凋亡率以及PTEN的蛋白表达水平，我们相信mmu-miR-200a是通过PTEN调控基质细胞的适度增殖以确保植入正常进行。更多还原

【Abstract】 PART IPRELIMINARY STUDY OF ESTROGEN ANDPROGESTERONE ON MICRORNAS REGULATIONBackground: Successful pregnancy is dependent on a healthy uterus thatis fit to receive and support a fertilized embryo. The uterus is an endocrineorgan, responsive to the presence of the ovarian steroid hormones, estrogenand progesterone, which activate transcription of target genes through thebinding of their cognate receptors, the estrogen receptor and theprogesterone receptor. Estrogen and progesterone signaling have beendemonstrated to be critical for the initiation and maintainance of pregnancy.MicroRNAs(miRNAs) regulate the expression of many genes, and previousstudies have demonstrated the relationship between miRNAs expressionand embryo implantation. However, there is little report about whoregulates miRNAs expression. Because many crucial genes which areinvolved with implantation are regulated by E2and P4. Then, the purpose of this study is to explore whether E2and P4affect implantation throughregulating miRNAs expression.Methods: Collected mouse uterus endometria tissues on pregnancyD1and D6to mimic estradiol and progesterone regulate model (E2levelreaches a peak on D1, and P4is very low; instead, P4level reaches a peakon D6, and E2is very low). Then extracted total RNA to performmicroRNA sequencing. At last, the results were analyzed by Bioinformatics.Results: The results of miRNA sequencing showed that6miRNAexpression was up-regulated more than2fold and73miRNA expressionwas down-regulated more than2fold when D6versus D1, and screen forthe target genes of these differential miRNAs are involved in whichsignaling pathways. The target genes of these differential miRNAs areinvolved in metabolism, MAPK, tight junction signaling throughBioinformatics analysis.Conclusions: These results preliminarily reflect in mouseendometrium, the expression of miRNAs is regulated by estrogen andprogesterone. Some miRNAs are differentially expression in endometriumon pregnancy D1, and D6and the target genes of these miRNAs areinvolved in several signaling pathways which are very important during theimplantation process through Bioinformatics analysis. This suggested thatmiRNAs have a role in mouse implantation. PART IIEFFECT OF MMU-MICRORNA-200A OEREXPRESSION ONMOUSE EMBRYO IMPLANTATIONBackground: Successful mouse embryo implantation requires areceptive uterus and an activated blastocyst. A large number of genes,cytokines and other factors are involved with the process.MicroRNAs(miRNAs) regulate the expression of many genes, and previousstudies have demonstrated that miRNAs regulate embryo implantationthrough their target genes. However, there is much less report about specificmiRNA involved in this process. According to previous results, we foundthat mmu-miR-200a expression was down-regulated by P4and expresseddifferentially before and after implantation. Moreover, studies have alsoshown that miR-200a was differentially expression in many endometrialdeseases (such as endometrial carcinoma and endometriosis). Thus, thisstudy is to explore the role of mmu-miR-200a during embryo implantationand its target gene, and clarifying the physiological functions of uterinemiRNAs will elucidate the embryo implantation process, and may evencontribute to curing infertility and inventing new contraceptives.Methods: Real Time-PCR examined mmu-miR-200a expression levelon pregnancy D4, D5and D6, and in the implantation sites andinter-implantation sites in the uterine endometria on pregnancy D5. Then we performed in situ hybridization to detect the expression pattern ofmmu-miR-200a. The target gene of mmu-miR-200a was verified bybioinformatics analysis, primary stromal cell culture, transfection andfluorescence intensiy assay. The implantation rate was examined byinjecting lentivirus which was designed to promote overexpression ofmmu-miR-200a in the area of injection. Finally, we examined the apoptosisrate of primary stromal cells after tranfection with mmu-miR-200a control,mimic or inhibitor through flow cytometry.Results: Here it showed that the expression of mmu-miR-200a wasmuch lower at implantation sites than that at inter-implantation sites onpregnancy D5. Mmu-miR-200a had the highest level of expression on D4,and the expression levels decreased gradually on D5and D6, and it wasmainly expressed in stromal cells and weakly expressed in epithelial andglandular cells. PTEN, as the target gene of mmu-miR-200a, had the sameexpression location and the opposite expression level with mmu-miR-200a.Moreover, the implantation rate was decreased by injecting mmu-miR-200aoverexpression lentivirus. Additonally, when transfection withmmu-miR-200a inhibitor, the apoptosis rate of stromal cells was increaseobviously and PTEN protein expression level was up-regulated. Instead,When transfection with mmu-miR-200a mimic, the apoptosis rate ofstromal cells was decrease and PTEN protein expression level wasdown-regulated. Conclusions: This study demonstrated that overexpression ofmmu-miR200a leads to decreased implantation rate. According to theexpression pattern of mmu-miR-200a and its target gene PTEN, and therate of apoptosis of primary stromal cells by transfection withmmu-miR-200a mimic and inhibitor, we believed that mmu-miR-200aregulate moderate proliferation of stromal cells to ensure normal embryoimplantation process through PTEN.更多还原