RXRA对Cx43基因的调控在法洛四联症发生机制中的作用

Tetralogy of Fallot (TOF), one of the most common complex congenital heart defects, affects the life quality of the patients and is also the leading cause of death in infants. So, it’s important to study the pathogenesis of TOF. Our previous study has suggested that the gene Cx43 regulated by RXRA, which may be involved in the pathogenesis of TOF. But this phenomenon and its mechanism should be further confirmed. We plan to launch the following research, including ① To make clear whether the RARE element of Cx43 gene is the molecular target of RXRA through the luciferase reporter gene assay, electrophoretic mobility shift assay (EMSA), shift-western blot assay and chromatin immunoprecipitation (ChIp) assay at the cellular level. ② To verify RXRA is the upstream regulatory factor for Cx43 gene by using target gene over-expression and down-regulation technology in the model organism zebrafish. ③ Create a vitamin A deficiency fetal mouse model. To study the impact of vitamin A on the expression of gene RXRA and Cx43 in model organism mouse. Based on the above serial study, we expect to elucidate the regulatory role of RXRA to Cx43 gene in the pathogenesis of TOF, and to provide experimental basis for appropriate vitamin A supplementation during early pregnancy to prevent the occurrence of TOF. The study will contribute to elucidating the genetic mechanism of TOF to some extent and finding out several molecular biomarkers associated with TOF. In addition, the research thinking and method will provide references for other birth defects.

法洛四联症（TOF）是严重影响患者生命和生活质量的复杂先心病，研究其发生机制有重要意义。我们的前期研究提示，RXRA对Cx43的调控可能参与了TOF发生，但需进一步证实。拟开展以下研究：①通过Luciferase报告基因、EMSA、Shift-Western blot、ChIp等实验在细胞水平明确Cx43基因的RARE元件是否是RXRA的分子靶点；②采用靶基因过表达和下调技术，在模式生物斑马鱼中验证RXRA是Cx43基因的上游调控因子；③建立VitA缺乏和缺乏后补充VitA胎鼠模型，在模式生物小鼠中研究VitA对RXRA和Cx43表达的影响。通过上述系列研究，探讨RXRA对Cx43的调控在TOF发生中的作用，并为在孕早期适当补充VitA预防TOF提供实验依据。本研究不仅有助于部分阐明TOF的发生机制、寻找到数个TOF相关的分子标志物，而且可为其他出生缺陷的研究提供可借鉴的研究思路和方法。

法洛四联症（TOF）是重症复杂先心病，研究其发生机制有重要意义。本课题研究结果：（1）9-cis RA处理后，Western Blot提示RXRα蛋白表达增高、Cx43蛋白表达下降，Real-time PCR提示RXRα mRNA上调，Cx43 mRNA下降；shRNA干扰细胞后，RXRα蛋白表达下调、Cx43蛋白表达上调。pGreen-RXRα-shRNA干扰细胞后，Cx43 mRNA上调。（2）经9-cis RA处理以及shRNA干扰后，免疫荧光共聚焦成像提示，RXRα定位于细胞核内，Cx43表达于细胞膜与细胞质，RXRα与Cx43的表达关系与上述结果一致。（3）荧光素酶报告基因实验提示，Cx43启动子存在功能性RARE元件，且位于转录起始位点上游-424bp至-407bp。（4）EMSA实验和Shift-Western实验提示，与Cx43启动子区RARE样序列结合的是RXRα蛋白。（5）染色质免疫共沉淀实验提示，无论9-cis RA存在与否，Cx43均可与RXRα结合；将PCR产物测序后发现，该DNA片段包含Cx43启动子区RARE样序列；定量PCR提示，对照组与9-cis RA处理组RXRɑ抗体均可富集Cx43启动子DNA片段。（6）TOF患儿Cx43启动子区SNP rs2071166测序发现，Cx43的SNP rs2071166与TOF相关，纯合子CC基因型和等位基因C是其高危因素。（7）荧光素酶报告基因实验提示，Cx43启动子区SNP rs2071166等位基因C影响RARE元件功能与启动子转录活性。（8）EMSA实验提示，RARE元件序列中，A>C影响Cx43启动子区RARE元件序列结合RXRα蛋白。（9）斑马鱼增强子验证实验提示，对照组中，35.7%斑马鱼表达GFP，19.5%心脏特异性表达。突变组中，2.3%斑马鱼微弱表达GFP，但无心脏特异性表达。（10）维生素A缺乏孕鼠模型构建后，发现VAD组成年鼠较VAN组体型稍小，VAD胎鼠心脏有室间隔缺损改变，两组Cx43和RXRα蛋白表达无明显差异。通过上述细胞水平、模式生物斑马鱼中的系列研究，探讨了RXRα对Cx43的调控在TOF发生机制中的作用，部分阐明了TOF的发生机制。