小睑裂综合征MicroRNA表达谱和功能研究

Blepharophimosis syndrome (BPES), an autosomal dominant genetic disease, manifests as blepharophimosis, ptosis, epicanthus, etc. In addition, female patients with type I suffer from diseases such as infertility and ovarian atrophy, which have been only confirmed as a series of diseases related to autosomal disorders and ovarian development. Consequently, it is essential and valuable to research on the causative genes and the control mechanism of BPES...A large number of domestic and foreign studies confirmed that 61% of the BPES patients could be associated with the gene mutation of FOX L2. Except this, there exists some unknown regulatory mechanisms involved in pathogenesis of BPES...MicroRNA is a class of non-coding, endogenous small RNA which is discovered recently. It widely participates in the control process of development, growth, senescence, apoptosis and it is a great breakthrough in functional genomics. ..Based on the long-term research, the research group has obtained the accurate experimental data by several experiments and confirmed that three microRNAs involved in the BPES gene expression...This project intends to make a research on the difference of microRNA expression spectrum according to the blood system acquired from 312 patients and 520 family relatives of 67 pedigrees to filter and verify the key microRNA that are involved in the regulation of BPES disease. Research on the mechanism of the key microRNA when it controls the disease genes, such as FOX L2, in the BPES molecular signaling pathway. Confirm the BPES biomarker. This research will provide new scientific basis for the study of BPES regulation mechanism and key genes.

小睑裂综合征（BPES）为常染色体显性遗传病，表现为睑裂狭小、上睑下垂、内眦赘皮等，是目前唯一获得确证的常染色体失调与女性卵巢发育相关的疾病。因此BPES致病基因及调控机制研究具有极为重要的价值。国内外研究证实，61%BPES患者发病与FOXL2基因突变相关，除此之外尚存在其他的调控机制参与BPES的发病。microRNA(miRNA)是最近发现的一类非编码、内源性的小RNA。它广泛参与发育、生长、凋亡等调控过程，是功能基因组学研究的重大突破。课题先期研究通过多个实验获得了确切的数据证实了3个miRNA参与了BPES基因的表达。课题拟对67个家系中312例患者及520例亲属血液样本系统开展miRNA表达差异谱的研究。研究关键miRNA参与调控FOXL2等致病基因在BPES相关分子信号通路中的作用机理，确认BPES的生物标志物。课题研究将为BPES发生的调控机制及关键基因研究提供新科学依据。

小睑裂综合征（BPES）为常染色体显性遗传病，表现为睑裂狭小、上睑下垂、内眦赘皮等，是目前唯一获得确证的常染色体失调与女性卵巢发育相关的疾病。课题组通过Affymetrix microRNA 芯片实验，获得一系列差异表达的miRNAs。研究结果表明：（1）荧光定量PCR检测不同microRNA的mimics转染细胞后目的基因的变化发现，has-miR-30c、has-miR-20a、hsa-miR-133b 、has-miR-130a转染后FOXL2的表达量明显升高， has-miR-520e 、has-miR-29c-3p mimics、has-miR-148a转染后FOXL2的表达量明显降低；（2）Western印迹法检测不同microRNA的mimics转染细胞后目的蛋白的变化，发现转染细胞48h后microRNA mimics组FOXL2蛋白表达水平较其他各组均有显著降低。（3）双荧光素酶检测和免疫印迹结果发现miR-30a通过抑制FOXL2基因来上调BCL2A1、IER3和cyclin D2的表达。（4）FOXL2基因在Hela细胞中高表达，过度表达FOXL2基因会抑制Hela细胞的增殖和促进Hela细胞的凋亡。沉默FOXL2基因会促进Siha细胞的侵袭，但不会影响它的黏附。过度表达FOXL2基因会降低Hela细胞中Ki67的表达。（4）sox14可通过激活βWnt /β-catenin信号通路来增强Hela细胞的增殖与侵袭能力。.研究关键miRNA参与调控FOXL2等致病基因在BPES相关分子信号通路中的作用机理，确认BPES的生物标志物。课题研究将为BPES发生的调控机制及关键基因研究提供新科学依据。