环状RNA介导高雌二醇调控卵巢过度刺激综合症子代心肌功能的机制研究

Cardiovascular diseases origin from adverse exposure during embryonic and fetal development. Our previous study showed that cardiac diastolic dysfunction in offspring of ovarian hyperstimulation syndrome was associated with high prenatal estradiol exposure. But the underlying mechanism is unclear. Our preliminary results showed cardiovascular diastolic dysfunction in offspring of prenatal high estradiol exposure mice, and EACR (Estradiol Associated Circular RNA) was significantly down-regulated in cardiac tissue. To test the hypothesis that high estradiol impair cardiac function of offspring via EACR, we plan to build mouse model of prenatal high estradiol exposure, and evaluate the structure and function of the mice using ultrasound and Langendorff perfusion system. We will also construct cardiomyocyte model differentiated from embryonic stem cell, and evaluate the effect of high estradiol on expression of EACR and CACNA1C, shortening/re-lengthening of cardiomyocyte. After overexpression and knock-down of EACR, re-evaluate the above parameters. Assess the bind capability of EACR and biotin-marked miR-29 with ChIP, and test the inhibition of miR-29 by EACR with luciferase reporting system. With the above studies, we hope to provide support for the regulation of cardiac function by prenatal high estradiol.

心血管疾病的源头可追溯到胚胎期不良因素暴露。前期研究提示卵巢过度刺激综合症子代出现心脏舒张功能异常，原因可能与高雌二醇暴露相关，但具体机制不明。本课题在预实验发现孕期高雌二醇子代小鼠心脏舒张功能异常、心肌EACR（雌二醇相关环状RNA）表达下调的基础上，拟深入研究EACR介导高雌二醇调控子代心肌功能的机制。课题拟构建高雌二醇子代小鼠模型，通过超声和心脏灌流技术评估其心脏功能，并检测心肌EACR及CACNA1C（电压门控钙离子通道）表达；构建高雌二醇处理的胚胎干细胞-心肌细胞模型，检测EACR、CACNA1C的表达和心肌收缩/舒张功能；过表达及敲减EACR后，检测上述指标是否改变；染色质免疫共沉淀/荧光素酶报告基因系统探究EACR对miR-29及其靶基因Cacna1c表达的调控作用。本研究将为阐明高雌二醇对卵巢过度刺激综合症子代心脏功能的调节机制提供理论依据。

心血管疾病的源头可追溯到胚胎期不良因素暴露。本研究前期研究提示卵巢过度刺激综合症子代出现心脏舒张功能异常，原因可能与高雌二醇暴露相关，但具体机制不明。为了研究该问题，本课题构建孕期高雌激素子代小鼠模型，对心肌功能进行了超声功能测定；对小鼠心肌样本进行了Circular RNA及MicroRNA的测序分析；对上述测序分析进行了针对性生物信息学分析，筛选出最有可能影响心肌的circRNA-MicroRNA配对；构建体外高雌激素心肌细胞模，验证候选CircRNA-MicoRNA配对在高雌激素处理下的表达情况;构建了目标CircRNA，即Estradiol Associated Circular RNA 的siRNA敲减系统，利用TUNEL实验、流式细胞术研究评估心肌细胞凋亡情况；构建EACR/miR-129-5P的荧光素酶报告系统，探索EACR与miR-129-5P的结合。我们发现，高雌激素子代小鼠心脏舒张功能受损，符合前期队列研究中人类子代的表型；根据生物信息学挑选出了CircRNA-MicroRNA配对（EACR-miR129-5p）；高雌激素心肌细胞模型提示EACR表达上升；敲减EACR后，心肌细胞凋亡增多；荧光素酶实验提示miR-129-5p与EACR存在有效结合。本课题在前期坚实的流行病学证据基础上，从CircRNA角度探索孕期高雌二醇对子代心脏功能的影响，研究高雌激素是否以及如何通过环状RNA的介导使OHSS子代心脏舒张功能受损，为阐明高雌激素对卵巢过度刺激综合症子代心脏功能的调节机制提供理论依据。