生物钟核心基因Clock/Bmal1通过调控KiSS-1转录抑制滋养细胞浸润及参与子痫前期发病的研究

Abnormal function of circadian clock can affect placental function and may be related to preeclampsia. Clock/Bmal1, the core genes of the circadian clock, have been found expression in placental tissue, and increased with the progression of pregnancy, especially higher in women with preeclampsia. After transfected by Clock/Bmal1, the expression of KiSS-1 in cytotrophoblast increased with decreased of the invasion ability. The protein dimer of CLOCK /BMAL1 could identify and bind three E-box sequences which located in the promoter of KiSS-1, We make a hypothesis: Clock/Bmal1could regulate KiSS-1 transcription and then play an important role in stage-specific regulation of trophoblast invasion. The aim of this project ① To investigate the role of Clock/Bmal1 in stage-specific regulation of trophoblast invasion during normal pregnancy and preeclampsia. ② To explore the mechanism of Clock/Bmal1 regulate transcription of KiSS-1 , by electrophoretic mobility shift assay(EMSA), chromatin immunoprecipitation assay(ChIP), dual luciferase reporter assay, E-box mutation and DNA affinity chromatography. ③ Introduce siRNA of Clock/Bmal1 to preeclampsia animal models characterized with shallow invasion of trophoblast ,which built by over-expressing KiSS-1 and KiSS-1-E-box mutant and detecte the rescure effect of siRNA of Clock/Bmal1 on trophoblast shallow invasion. This Study will elucidate the pathogenesis of preeclamsia and to provide the new target of prevention and treatment.

生物钟功能异常可影响胎盘功能并与子痫前期发生可能有关。前期研究发现生物钟核心基因Clock/Bmal1在胎盘中表达随妊娠进展而增加，而在子痫前期时呈持续性高表达；转染Clock/Bmal1后，浸润抑制基因KiSS-1表达增加，滋养细胞浸润能力下降。Clock/Bmal1形成的蛋白二聚体可识别并与KiSS-1启动子的三个E-box结合，推测Clock/Bmal1通过调控KiSS-1转录参与滋养细胞浸润时序调控。本项目拟采用凝胶迁移实验、染色质免疫共沉淀、双荧光素酶报告基因实验、E-box突变和DNA亲和层析等方法探讨Clock/Bmal1在正常妊娠和子痫前期滋养细胞浸润的调控及对KiSS-1转录的调控机制；利用 KiSS-1和KiSS-1启动子E-box突变过表达构建的子痫前期动物模型，验证Clock/Bmal1 siRNA对滋养细胞浸润不良的逆转作用，阐明子痫前期发病机制,提供防治靶点。

生物钟功能异常可影响胎盘功能并与子痫前期发生可能有关。胎盘组织生物钟核心基因Clock和Bmal1翻译的蛋白CLOCK蛋白和BMAL1蛋白相互结合形成异二聚体CLOCK/BMAL1入核后可以调控KiSS-1基因的转录和表达；生物钟核心基因Clock/Bmal1通过调控KiSS-1转录实现对滋养细胞浸润行为的时序化调控，维持正常妊娠的进展，一旦在妊娠早期Clock/Bmal1 基因异常上调，或者提前出现上调，则会导致靶基因KiSS-1表达增加，滋养细胞浸润障碍，螺旋小动脉重铸受阻，最终导致子痫前期等病理妊娠。而在子痫前期发生的过程中，改变生物钟基因的表达，则有可能改善子痫前期的病理改变和临床症状，为子痫前期发病机制的研究和诊治提供新的理论依据和思路。