PHF14调控组蛋白H3第27位赖氨酸三甲基化转录抑制促肾纤维化因子的机制研究

Renal fibrosis is the final common pathological manifestation of all chronic kidney diseases progressing to ESRD, while no effective way has been found to reverse it. PHF14, a novel histone binding protein was discovered in our previous studies, which is thought to be an innate inhibitor of renal fibrosis. However, how PHF14 inhibits renal fibrosis is not unraveled. We detected that PHF14 can be induced by TGF-β1, which is thought to be the master regulator of fibrosis. TGF-β1 increases the expression of H3K4me3 in the promoters of pro-fibrotic factors by inducing SET7/9 thus to aggravate renal fibrosis. By in vitro experiments, renal fibrosis mouse models and PHF14 conditional knockout mouse, we will verify that PHF14 is induced by TGF-β1 and could recognize and bind to H3K4me3, recruit PRC2, facilitate H3K27me3 deposition, then silence the expression of pro-fibrotic factors, thus to retard the progression of renal fibrosis. We plan to illustrate PHF14, which is induced by TGF-β1, serves as an innate renal fibrosis inhibitor through regulating histone methylation to repress the over-expression of pro-fibrotic factors. This study would give a deep insight to the mechanism of renal fibrosis, and provide new strategies for the prevention of renal fibrosis in clinic.

肾脏纤维化是各型慢性肾脏病进展到终末期肾衰的共同通路，目前尚无有效治疗手段。申请者前期研究发现，一种新鉴定的组蛋白结合蛋白PHF14是重要的内源性肾纤维化抑制因子，发挥纤维化自限性抑制效应，但其抑制肾纤维化的机制尚未明瞭。有证据提示，TGF-β1增加促纤维化因子启动子区域H3K4me3水平，通过表观遗传调控加速纤维化进程；PHF14可能被TGF-β1诱导，抑制上述过程。本研究拟通过体外细胞实验、肾纤维化小鼠模型和PHF14基因诱导敲除小鼠，应用报告基因、免疫沉淀等技术，证明PHF14被TGF-β1诱导后，通过募集PRC2，促进促纤维化因子启动子区域H3K27me3聚积，沉默促纤维化因子表达，抑制肾纤维化进展，从而阐明PHF14通过调控组蛋白甲基化，发挥内源性肾纤维化抑制因子作用。此研究可加深对PHF14在肾纤维化中自限性抑制机制的认识，为临床应用表观遗传学手段干预肾纤维化提供理论依据。

肾纤维化的自限性调控机制是肾脏损伤后修复的重要环节，如促纤维化因子与抑制纤维化因子间的平衡出现紊乱，纤维化过程将持续进展，最终导致肾功能的恶化、慢性肾脏病进展。前期研究中申请者发现，一种新鉴定的组蛋白结合蛋白PHF14是重要的内源性肾纤维化抑制因子，发挥纤维化自限性抑制效应，但其抑制肾纤维化的机制尚不明晰。本研究通过构建的PHF14诱导敲除小鼠、马兜铃酸诱导的肾纤维化小鼠模型以及CRISPR-CAS9系统构建敲除PHF14的单克隆NRK-52E细胞株，通过离体与在体实验，应用报告基因、免疫沉淀、染色质免疫共沉淀等技术，发现PHF14在纤维化刺激TGF-β及缺氧情况下上调，具有保护肾功能、抑制纤维化进展的效应；缺氧通过下调促纤维化因子CTGF启动子区域H3K27me3水平促进CTGF转录，PHF14可促进H3K27me3的聚积，抑制CTGF的过度激活，从而限制纤维化进展。本研究阐明了PHF14在肾纤维化过程中的上调机制及其通过调控组蛋白甲基化抑制肾纤维化进展，揭示了PHF14在表观遗传重编程中的作用，为临床干预肾纤维化提供了理论依据及潜在靶点。