孕期地塞米松暴露所致子代足细胞发育毒性的miRNA编程机制及干预靶标研究

Prenatal dexamethasone exposure could induce glomerulosclerosis of the offspring. Podocyte injury, which is induced by the inhibition of the podocyte phenotype, is the onset of glomerulosclerosis. Kruppel-like factor 4 (KLF4) contributes to maintain the podocyte phenotype, and our previous study found that dexamethasone could reduce the expression of KLF4 of the offspring as well as induced podocyte developmental toxicity. miR-135a inhibits the expression of KLF4 at post-transcription level. Moreover, the expression of miR-135a is tightly regulated via epigenetics modification, and we predicted that the promoter of miR-135a might bind to glucocorticoid receptor (GR). According to the results of our pre-study, we hypothesis that, dexamethasone/GR binds to the promoter of miR-135a, induces DNA hypo-methylation and histone hyper-acetylation, and causes the high expression programming change of miR-135a, in turn reduces the expression of KLF4 and induces podocyte developmental toxicity. This project aims to clarify the miR-135a related molecular mechanism of podocyte developmental toxicity induced by dexamethasone, and to prove that miR-135a could be the potent interference target for the treatment of glomerulosclerosis by prenatal dexamethasone exposure. The data of this research will provide the clues for guiding the medication during pregnancy as well as for establishing the early therapy strategy of dexamethasone induced kidney developmental toxicity.

孕期地塞米松暴露可致子代肾小球硬化。足细胞损伤是肾小球硬化的始动因素，其发生与足细胞表型抑制有关。Kruppel样因子4（KLF4）参与维持足细胞表型。我们发现，地塞米松抑制子代肾脏KLF4表达，导致足细胞发育毒性。miR-135a是KLF4上游抑制因子，易受表遗传修饰调控，且可能与糖皮质激素受体（GR）结合。依据预实验结果，我们推测：地塞米松/GR结合于miR-135a启动子区，并通过影响表遗传酶的表达，诱导miR-135a的组蛋白高乙酰化/DNA低甲基化，导致miR-135a高表达编程改变，进而抑制KLF4表达，造成足细胞发育毒性。本项目拟系统阐明地塞米松通过GR诱导miR-135a编程效应导致足细胞发育毒性的分子机制，并证实miR-135a可能作为孕期地塞米松暴露所致子代肾小球硬化的潜在干预靶标。本项目研究结果将为指导孕期合理用药和探寻地塞米松肾脏发育毒性的防治策略提供实验依据。

地塞米松（DEX）在临床上广泛用于早产相关疾病的治疗。我们前期研究发现，孕期地塞米松暴露（PDE）导致子代肾小球硬化，但机制不明。本项目中，我们证实孕期DEX暴露激活胎肾GR，诱导乙酰化酶p300的表达，结合于miR-135a启动子区，上调其组蛋白乙酰化水平和表达，进而抑制KLF4和足细胞功能基因表达，造成足细胞发育毒性，导致子代成年后肾小球硬化。而出生后早期给予antagomiR-135a干预可以缓解子代足细胞和肾功能损伤，证实miR-135a是PDE所致足细胞发育毒性的有效干预靶标。此外，我们还观察到PDE导致雌性子代肾小球硬化的多代遗传，其潜在的毒性机制为：DEX通过激活胎肾糖皮质激素受体，抑制DNMT3a表达，引起MEG3低甲基化及高表达编程改变，进而抑制了Notch信号通路，导致肾脏发育不良并引发成年后肾小球硬化的发生。MEG3低甲基化/高表达可经卵母细胞传递至 F2/F3代，这可能是PDE导致子代肾脏发育毒性经母系多代遗传的原因。上述研究结果为阐明PDE致子代肾脏发育毒性及多代遗传的宫内编程机制、探寻肾脏发育毒性的早期预警和干预靶标提供了重要的实验和理论依据。