miR-18a、miR-27a作为子痫前期分子诊断指标及靶基因研究

Pre-eclampsia( PE) is a pregnancy-specific disease with high global incidence, but nowadays there still are no specifial biomarkers. In previous studies,we found that miR-18a、miR-27a were notably increased in PE placenta and maternal serum. Then, by informatics tules, we found two sepcial target genes IGF-1、LPL of miR-18a and miR-27a. Accordingly, we assumed that these two miRNAs regulated PE by targeting to IGF-1 and LPL. To verify this hypothesis, we used plenties of biomedical methods in clinical and cell experiments, such as real-time quantitative PCR, luciferase reporter system, western blot, microRNA mimic and inhibitor transfction, siRNA, et al. Firstly, we tested the expression profiles of miR-18a、miR-27a and IGF-1、LPL mRNA and protein in PE placenta tissuses. Secondly, we tested serum miR-18a、miR-27a profiles in large clincal serum samples to analyze their clinical value as PE biomarkers. Finally, we used cell expriments to verify the regulatory network of miR-18a、miR-27a and IGF-1、 LPL, and also investigated its possible machanism in PE. This progect aimed to achieve PE non-invasive dignosis by serum miRNA biomarker, and also provided theoretical and experimental basis to elucidate the pathogenesis of PE in the future.

子痫前期(PE)发病率较高，但尚无特异分子诊断指标。我们前期研究发现，miR-18a、miR-27a在PE患者胎盘组织、母体血清中表达水平显著上升；生物信息学预测其可能通过调控IGF-1、LPL参与PE的病理过程。据此提出假设：血清miR-18a、miR-27a可作为PE新的无创性分子诊断指标；它们通过调节IGF-1、LPL参与PE的发生、发展。为验证该假设，拟采用实时荧光定量PCR技术、荧光素酶报告实验、microRNA mimic和inhibitor转染、siRNA干扰等手段，开展临床、细胞实验：首先于胎盘组织中验证miRNA和靶基因的表达水平与PE相关；而后以大量临床血清标本为资料综合评判该分子诊断指标的临床应用价值；最后于细胞水平验证靶标关系，探讨功能机制。本课题旨在通过研究血清miRNA实现PE无创性诊断，并为阐明其发病机制提供实验基础和理论依据。

子痫前期(pre-eclampsia，PE)是一种妊娠期特有的多系统疾病，是导致孕产妇和新生儿死亡的主要产科并发症。而该病至今尚无特异的分子诊断指标，且机制不明。微小核糖核酸(microRNA，miRNA)作为一种转录后调控因子，参与疾病的发生、发展，血清miRNA近年来已成为一种新型的无创性疾病诊断指标，具有极高的临床应用价值。本研究假设miR-18a、miR-27a可作为PE的分子诊断指标，通过调控IGF-1、LPL参与PE的发生、发展。.本课题通过临床样本及细胞实验，得到以下结果：.1、组织结果显示：miR-18a、miR-27a在PE患者中表达水平显著高于正常孕妇，而IGF-1、LPL蛋白表达水平则显著降低，提示miR-18a、miR-27a可能调控了IGF-1、LPL的转录后表达水平。.2、血清结果显示：血清miR-18a、miR-27a在PE患者中表达水平显著高于正常孕妇，可作为一种新型的分子标志物。.3、Luciferase结果显示：梯度转染miR-18a、miR-27a mimics后Luciferase呈梯度降低，而转染mutant则无改变，结果提示IGF1为miR-18a、miR-27a的直接靶基因，LPL为miR-27a的直接靶基因。.4、细胞转染结果显示：转染miR-18a、miR-27a mimics后，IGF-1、LPL表达水平显著降低；转染miR-18a、miR-27a inhibitor后， IGF-1、LPL表达水平显著上升。结果提示：miR-18a、miR-27a共同调控IGF-1表达，miR-27a调控LPL表达 。.5、细胞功能结果显示：转染miR-18a、miR-27a mimics后细胞增殖增多；转染inhibitor后细胞增殖减少；转染IGF-1、LPL siRNA后细胞增殖增多；且同时转染miR-18a、miR-27a时呈现协同效应。.以上研究结果证实，miR-18a、miR-27a可作为PE的一种新颖的无创性分子标志物，并通过调控IGF-1、LPL的表达水平参与了PE的发生发展，为进一步的研究奠定了重要基础。