子宫内膜异位症中转录因子TCF21对雌激素合成通路的调控机制

Aromatase catalyzes the final key step of estrogen production. Steroidogenic Factor-1 (SF-1) is a transcriptional factor essential for activation of aromatase gene. These two genes and estrogen receptor-beta (ERβ) which are the three key genes in estrogen biosynthesis pathway, are highly expressed in the endometriotic stromal cells in endometriosis as compared with endometrial stromal cells. With grants support from the National Natural Science Foundation of China, it was the first time that we reported that methylation of the intron CpG islands of SF-1 gene regulated the SF-1 expression in endometriosis, PGE2 activated p38 and JNK signaling pathways, further stimulating c-Jun and ATF2 binding to aromatase and ERβ promoter regions with elevated estradiol production, and IGF-I activated the IGF1R/PI3K/AKT signaling pathway and then stimulated binding of c-Jun or CREB to the ERβ or aromatase promoter region, inhibition of IGF1R in vivo could impede the growth of ectopic lesions in nude mice. However, the key transcriptional factor that could regulate these three critical genes in estrogen biosynthesis pathway in endometriosis is unknown. Transcription factor 21 (TCF21) is a transcriptional factor that could bind to the promoter region of downstream genes, then regulate these genes’ expression. Preliminarily, we found that TCF21 was highly expressed in endometriotic stromal cells as compared with endometrial stromal cells, TCF21 siRNA knockdown downregulated aromatase, SF-1 and ERβ expression in endometriotic stromal cells. So, we suppose that TCF21 maybe a potential key transcriptional factor of these three genes. Up to now, there has been no report of this mechanism in the regulation of these three genes. Our plan is to investigate the regulation of TCF21 in aromatase, SF-1 and ERβ expression in endometriotic stromal cells by primary cell culture, siRNA knockdown, gene overexpression; find the key site of the promoter region of these three genes by deletion and site mutation, clones, and luciferase reporter gene assay; and determine the binding of TCF21 and USF2 protein to the promoter region of these three genes by chromatin immunoprecipitation assay and electrophoretic mobility shift assay. Also we will investigate the inhibition effect of TCF21 shRNA knockdown in xenograft growth and the expression of these three genes in endometriosis xenografts. Finally, our aim is to find the key point in the estrogen biosynthesis pathway in endometriosis.

类固醇基因因子1（SF-1）是芳香化酶启动激活子，两者及ERβ在异位内膜高表达，是内异症雌激素合成通路的三个关键基因。前两个基金期间我们首报了甲基化对SF-1的调控，IGF-I、PGE2对芳香化酶和ERβ的调节，但三个基因的共调机制不明。TCF21可结合到下游调控靶基因,前期我们发现转录因子TCF21在异位内膜高表达，siRNA沉默TCF21，芳香化酶、SF-1和ERβ均显著降低,提示TCF21可能为三个关键基因的核心调控因子。因机制不明，我们将在人异位内膜间质细胞，siRNA敲除和过表达TCF21，探讨其对三个基因的调控作用；构建点突变和截短体突变质粒、荧光素酶实验确定TCF21对三个基因的作用位点；应用染色质免疫沉淀、电泳迁移率确定TCF21和USF2与三个基因的结合能力；在裸鼠异位症模型沉默TCF21，论证其对三个基因的降调和内异症病灶生长的抑制作用。寻找内异症雌激素通路的关键环节。

子宫内膜异位症是一种雌激素依赖性疾病，局部的高雌环境对异位病灶的生长和存活发挥重要作用。芳香化酶是雌激素合成的关键合成酶，类固醇基因因子1（steroidogenic factor-1，SF-1）是芳香化酶的关键的启动调节子，雌激素合成后由雌激素受体（estrogen receptor, ER）介导，ERβ是内异症中介导雌激素作用的主要受体亚型,因此我们对内异症中关键因子SF-1及ERβ的共调节机制进行探讨，并以芳香化酶和ERβ为靶点，探索有潜能的内异症治疗药物或策略。. 我们研究发现，转录因子21（Transcription factor 21, TCF21）在内异症患者异位病灶中表达升高，异位内膜中异常高表达的TCF21可通过募集更多的上游刺激因子2（upstream stimulatory factors 2，USF2）结合到SF-1和ERβ基因启动子区的E-box序列从而间接上调SF-1和ERβ的表达。此外，异位内膜中高表达的TCF21参与调控异位病灶的增殖和侵袭力。特异性抑制TCF21可显著下调裸鼠内异症病灶的生长和关键致病基因SF-1、ERβ、MMP9和cyclinD1的表达，提示TCF21是内异症发生发展中的关键因子，以上研究结果发表在Biochim Biophys Acta Gene Regul Mech, 2018, 1861(8), IF=5.2。. 此外，我们发现，法尼醇X受体（Farnesoid X receptor，FXR）在异位病灶表达增加，其激动剂GW4064可通过结合并激活异位内膜中异常高表达的FXR，一方面，可通过与CREB竞争性结合芳香化酶的基因启动子区，从而下调芳香化酶的表达；另一方面，可通过激活Stat3信号通路，从而促进Stat3蛋白与ERβ基因启动子区的结合，进而对ERβ的表达发挥转录抑制作用。此外，GW4064可下调裸鼠内异症病灶的生长和关键致病基因芳香化酶和ERβ的表达，这为GW4064成为内异症的治疗药物提供初步理论依据，以上研究结果发表在Reprod Sci, 2019, 26(8), IF=2.6。