同型半胱氨酸经c-myc沉默特异性miRNAs调控肾损伤表观遗传学分子机制的研究

Homocysteine (Hcy)，which served as an independent risk factor of renal damage, the pathogenic mechanism is still unkown. Our previous study proved that DNA methylation is an important mechanism of Hcy-induced renal damage, and the pre-experimental results suggested that miRNAs is involved in the regulation of renal damage . The histone methylation plays an important role in recruiting transcription factors to regulate gene transcription cooperating with DNA methylation .Then，the hypothesis we will propose like this: Hcy via transcription factor c-myc which coordinate with H3K27 methylation to silence specific miRNAs is the significant mechanism in renal damage. In order to vertify this hypothesis , to define the role of c-myc in renal damage induced by Hcy; To clarify the interrelation of c-myc and miR-92a, screene and identify the specific miRNAs(miR-92a) by the channel of the microarray technology, construct the recombinant and inference plasmid of transcription factor c-myc to transfecte the podocyte ; Silence and overexpress EZH2 ,c-myc, analysis the level of methylation of miR-92a promoter region, to confirm the epigenetic mechanisms of c-myc combine the promoter region of miR-92a then catalysis H3K27me3 by recruiting EZH2 to silence miR-92a .In this way, we will seek the essential target, and provide a new strategy for the prevention and curement of Hcy-induced renal damage.

同型半胱氨酸(Hcy)是终末期肾病的独立危险因子，但其致病机制未清。课题组前期观察到DNA甲基化是Hcy致肾损伤的重要机制，且预实验结果提示miRNAs参与了肾损伤的调控。而组蛋白甲基化具有招募转录因子协同DNA甲基化调控基因转录的作用，故我们推测：Hcy经转录因子c-myc协同H3K27甲基化沉默特异性miRNAs是肾损伤的重要机制。为了验证该假说，首先探讨c-myc在Hcy致肾损伤中的作用；运用微阵列等技术筛选并确定其特异性miRNAs(miR-92a)，构建c-myc重组和干扰质粒转染足细胞，明确c-myc与miR-92a的相互调控关系；使EZH2和c-myc沉默和过表达载体并转染，分析miR-92a启动子区甲基化水平，阐明c-myc结合到miR-92a启动子区并募集EZH2催化H3K27me3致miR-92a沉默的表观遗传学分子机制，寻找关键靶点，为防治Hcy致肾损伤提供新策略。

高同型半胱氨酸血症（Hyperhomocysteinemia, HHcy）是心血管疾病和肾小球基底膜屏障结构受损、肾功能障碍的重要独立危险因素。尽管在实验研究和临床治疗中已经使用了多种降低同型半胱氨酸（Homocysteine, Hcy）水平的方法，但是仍然缺乏有效的方法可以用于完全预防HHcy诱导的损伤。因此，寻找致病过程中的关键靶点可能为开发治疗HHcy相关损伤的新治疗方法提供理论依据。本课题拟用Cbs小鼠复制HHcy致肾损伤模型，观察肾损伤过程中miR-30a和c-Myc表达的改变，探讨H3K27甲基化和DNA甲基化是否参与miR-30a的表达调控，阐明c-Myc通过招募DNMT1和EZH2调控miR-30a转录在肾损伤中的机制。