MiRNA多态性与复发性流产发病风险的关系及机制研究

Recurrent pregnancy loss (RPL) is one of the common causes of female infertility, and in 50% of these women, the cause of the preceding miscarriages is unknown. Many studies have found that genetic variants related to thrombosis and immune regulation are associated with unexplained RPL (URPL). However, important genetic susceptibility markers in the prediction of URPL is not found. MicroRNA (miRNA) are small noncoding RNAs and function as modulators of gene expression, but fewer reports were found about the association between miRNA polymorphisms and URPL. In this study, we will screen URPL related miRNA SNP using URPL samples and then further validate these SNP in massive URPL population, and build URPL associated miRNA-mRNA network database based on the distribution of miRNAs polymorphism. We will explore the role of miRNA SNP in URPL by the mice spontaneous abortion and in vitro cell model. Meantime，the possible molecular mechanisms are studied by detecting the effect of miRNA SNP on miRNA expression, processing and target gene binding capacity. In addition, we will combine clinical drug for URPL therapy to preliminary screen miRNA SNP that increase drug sensitivity in URPL treatment. Thus, this study will contribute to reveal potential causes and molecular mechanisms of URPL form the viewpoint of miRNAs polymorphism, and may also provide new targets of diagnosis and individualized treatment for URPL.

复发性流产(RPL)是常见的引起女性不育的原因之一，约有50%的RPL原因不明。已有研究发现一些基因变异与不明原因RPL(URPL)的发生相关，但尚未发现有重要价值的遗传易感标记物。MicroRNA(miRNA)是一类在基因表达调控领域起着重要作用的分子，其多态性与URPL的相关性很少见报道。本项目预利用人URPL标本筛选与URPL相关的miRNA SNP，基于miRNA多态性分布构建URPL关联的miRNA-mRNA网络数据库。通过小鼠自然流产和体外细胞模型研究miRNA SNP在复发性流产中的作用；通过研究这些SNP对miRNA表达、加工和与靶基因结合能力的影响分析其在URPL中的作用机制；并结合URPL的临床用药初步筛选可能增加URPL治疗药物敏感性的miRNA多态性位点。本研究将有助于从miRNA多态性的角度揭示引起URPL的潜在原因，为将来URPL的诊断和个体化治疗提供新的靶标。

复发性流产(RPL)是妇产科临床上难以处理的疾病之一，但是，RPL的发病原因目前仍不十分清楚，也未发现具有很高临床价值的遗传易感标志物。MicroRNA(miRNA)是一类在基因表达调控领域起着重要作用的分子，有研究报道miRNA前体的编码基因中的单个碱基突变或单核苷酸多态性(SNP)可影响成熟态miRNA的表达量从而导致疾病，但miRNA多态性与URPL的相关性很少见报道。利用Sequenom MassARRAY平台筛选复发性流产易感miRNAs SNPs，发现miR-423 rs6505162、miR-548h-3 rs9913045、miR-196a-2 rs11614913、miR-323b rs56103835等与复发性流产密切相关，进一步的人群筛查发现miR-423 rs6505162、miR-196a-2 rs11614913和miR-323b rs56103835是复发性流产的一个危险因素。利用候选基因法筛选我们前期研究发现在胚胎植入中差异表达的miRNAs多态性与复发性流产的关系，发现miR-125a AG突变、miR-21 AG突变和miR-10a rs3809783与复发性流产密切相关。利用生物信息法构建了miRNAs与mRNAs的表达调控网络。利用体外细胞模型研究了这些miRNAs多态性改变对细胞增殖、凋亡、迁移和浸润等细胞生物学行为的影响。 利用qRT-PCR等方法分析了miRNAs多态性位点对miRNA表达和加工的影响。利用targetscan、PicTar等预测了其新的尚未验证过的靶基因，利用双荧光素酶实验进行了验证，并发现miRNA序列变异可以影响miRNA与靶基因的结合能力。我们分析了miRNAs多态性位点对流产诱导药物和治疗药物的敏感性以评估miRNAs多态性位点在流产发生和治疗中的可能作用。我们的研究结果不仅有助于从miRNAs多态性的角度揭示引起RPL的潜在原因和其发病的分子机制，开发在RPL中具有诊断价值的分子标志物，也有助于为RPL个体化治疗的研发奠定理论基础。