小睑裂综合征致病相关基因的筛选及其功能研究

Blepharophimosis ptosis epicanthus Syndrome (BPES) is 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. Gene cloning research has confirmed that about 61% to 67% BPES was caused by the gene mutation of FOXL2, which exists in the 3q23 area. The other disease genes have not been confirmed. First, we conduct a yeast two-hybrid screening, using FOXL2 as bait, to discover some new FOXL2 interaction protein. Second, we select the destination FOXL2 interaction gene to construct eukaryotic expression vector, co-transfection to human kidney epithelial cells 293T, then use co-immunoprecipitation technology verify the interaction of FOXL2 and its interaction protein in 293T cells. Third, we construct the prokaryotic expression vector. The interaction will be validated again in vitro by Pull-down experiments. Fourth, we use RNAi technology to inhibit the expression of the target gene, and to explore the relationship between FOXL2 protein and FOXL2 interaction protein by the relative level of quantitative PCR and immunoblot. Fifth, we complete gene mutation screening of FOXL2 interaction genes which have been confirmed by in vitro and in vivo tests, in BPES patients blood sample. Ultimately pathogenicity-related genes will be identified. The project will provide important experimental basis by investigating the the BPES pathogenic molecular mechanism for further genetic level, and lay a theoretical basis for the gene prevention and treatment of BPES.

小睑裂综合征（BPES）是一种常染色体显性遗传病，表现为睑裂狭小、上睑下垂、内眦赘皮等睑裂形态异常。国内外研究表明约有61%～67%的BPES是FOXL2基因突变所致，其他致病基因尚不明确。为此我们①以FOXL2为诱饵进行酵母双杂交筛选，以发现新的FOXL2互作蛋白。②选取目的FOXL2互作基因，通过构建真核表达载体，共同转染人肾上皮细胞293T，利用免疫共沉淀验证FOXL2与其互作蛋白在293T细胞中的相互作用。③构建原核表达载体，体外Pull-down实验再次验证其相互作用。④利用RNAi技术抑制目的基因表达，荧光定量PCR和免疫印迹检测基因表达的相对水平，探讨FOXL2互作蛋白对FOXL2蛋白功能的影响。⑤对筛选确认的FOXL2互作基因在BPES患者血液样本中进行突变筛查。本项目为进一步从基因水平探讨BPES发病的分子机制提供重要的实验依据，为BPES的基因预防及治疗奠定理论基础。

小睑裂综合征（BPES）是一种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。国内外研究表明约有61%～67%的BPES是FOXL2基因突变所致，其他致病基因尚不明确。为此课题组（1）通过酵母双杂交筛选实验，基因测序验证获得13个新的FOXL2互作蛋白。（2）选取目的FOXL2互作基因，通过构建真核表达载体，共同转染293T细胞，利用免疫共沉淀验证了FOXL2与其互作蛋白在293T细胞中的相互作用。（3）进行了目的基因的过表达慢病毒克隆，荧光素酶报告系统分析最终确认了目标作用基因。（4）将目的基因稳定过表达后检测对靶基因mRNA水平的调控和蛋白水平的调控。荧光定量PCR结果表明过表达has-mir-30c后，NNAT，FOXG1，TNC，RAP1GAP，ZMYND8，VCAN的表达水平都有显著上调；过表达has-mir130a后，NNAT，FOXG1，TNC，ZMYND8，VCAN的表达水平都有显著上调，POLR2G，LDHA的表达水平都有显著下调；过表达has-mir-520e后，NNAT，TSPAN7，FOXG1，TNC，RAP1GAP，ZMYND8，VCAN的表达水平都有显著上调，POLR2G的表达水平都有显著下调。免疫印迹检测结果发现：过表达has-mir-30c后，RAP1GAP，TUBB2A，FOXG1，ZMYND8的表达水平都有显著上调，VCAN，FOXL2的表达水平都有显著下调；过表达has-mir130a后，VCAN，NNAT，TNC，Foxl2，PCBP4，FOXG1，Ubqln4的表达水平都有显著下调；过表达has-mir-520e后，VCAN的表达水平都有显著上调，NNAT，TNC，Foxl2，FOXG1的表达水平都有显著下调。本项目为进一步从基因水平探讨BPES发病的分子机制提供重要的实验依据，为BPES的基因预防及治疗奠定理论基础。